http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10052 Description, recognition and analysis biological images plays an important role for human to describe and understand the related biological information. The color images are separated by color reduction. A new and efficient linearization algorithm is introduced based on some criteria of difference chain code. A series of critical points is got based on the linearized lines. The series of curvature angle, linearity, maximum linearity, convexity, concavity and bend angle of linearized lines are calculated from the starting line to the end line along all smoothed contours. The useful method can be used for shape description and recognition. The analysis, decision, classification of the biological images are based on the description of morphological structures, color information and prior knowledge, which are associated each other. The efficiency of the algorithms is described based on two applications. One application is the description, recognition and analysis of color flower images. Another one is related to the dynamic description, recognition and analysis of cellcycle images. 2013-04-07T22:38:08.978Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10081 This paper presents an efficient and innovative method for the automated counting of cells in a microscopic image. The performance of watershed-based algorithms for the segmentation of clustered cells has been well demonstrated. The strength of our algorithm lies in the fact that it incorporates knowledge of color in the image. Our method uses the watershed transform with iterative shape alignment and is shown to be more accurate in retaining cell shape. We report a sensitivity of 97% and specificity of 96% when all color bands are used. Our methods could be of value to computer-based systems designed to objectively interpret microscopic images, since they provide a means for accurate cell segmentation. 2013-04-05T01:03:54.331Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:4672 One approach to investigating neural death is through systematic studies of the changing morphology of cultured brain neurons in response to cellular challenges. Image segmentation and neuron skeleton reconstruction methods developed to date to analyze such changes have been limited by the low contrast of cells. In this paper we present new algorithms that successfully circumvent these problems. The binary method is based on logical analysis of grey and distance difference of images. The spurious regions are detected and removed through use of a hierarchical window filter. The skeletons of binary cell images are extracted. The extension direction and connection points of broken cell skeletons are automatically determined, and broken neural skeletons are reconstructed. The spurious strokes are deleted based on cell prior knowledge. The reconstructed skeletons are processed furthermore by filling holes, smoothing and extracting new skeletons. The final constructed neuron skeletons are analyzed and calculated to find the length and morphology of skeleton branches automatically. The efficacy of the developed algorithms is demonstrated here through a test of cultured brain neurons from newborn mice. 2013-04-03T03:34:50.809Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9067 In 2002, the 45 year old Sorell Causeway Bridge in Tasmania’s south east was demolished due to fears surrounding the serviceability of the prestressed concrete beams, in relation to the corrosion of the post-tensioning strands. Attempts were made to determine the extent and severity of the corrosion prior to its demolition by employing a number of conventional non-destructive and diagnostic techniques, yet a firm conclusion could not be reached regarding the structure’s condition. To further investigate the degree of correlation between these conventional assessment techniques and the physical condition of the embedded steel in relation to corrosion risk guidelines recommended in the literature, a number of beams of varying condition were salvaged from the bridge demolition and subjected to further detailed investigations. The current paper focuses on the half-cell potential and chloride profile results obtained for two such beams in good and poor condition relating to the reinforcing and prestressing steel condition. In summary, the comparison of results yielded inconsistencies with the literature guidelines. Survey areas showing highly negative potentials and elevated chloride concentrations did not necessarily indicate corrosion activity or severity. In contrast, instances of severe corrosion were found on both reinforcing and prestressing steel where literature guidelines for these tests recommend that the risk of corrosion is low. 2013-03-14T04:12:41.382Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12628 Research Doctorate - Doctor of Philosophy (PhD) 2013-03-12T05:57:04.420Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7732 Research Doctorate - Doctor of Philosophy (PhD) 2013-02-26T03:40:18.972Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12520 UVB radiation is the most mutagenic component of the UV spectrum that reaches the earth’s surface and causes the development of DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts. UV radiation usually results in cellular death, but if left unchecked, it can affect DNA integrity, cell and tissue homeostasis and cause mutations in oncogenes and tumour-suppressor genes. These mutations, if unrepaired, can lead to abnormal cell growth, increasing the risk of cancer development. Epidemiological data strongly associates UV exposure as a major factor in melanoma development, but the exact biological mechanisms involved in this process are yet to be fully elucidated. The nucleotide excision repair (NER) pathway is responsible for the repair of UV-induced lesions. Patients with the genetic disorder Xeroderma Pigmentosum have a mutation in one of eight NER genes associated with the XP complementation groups XP-A to XP-G and XP variant (XP-V). XP is characterized by diminished repair capacity, as well as a 1000-fold increase in the incidence of skin cancers, including melanoma. This has suggested a significant role for NER in melanoma development as a result of UVB exposure. This review discusses the current research surrounding UVB radiation and NER capacity and how further investigation of NER could elucidate the role of NER in avoiding UV-induced cellular death resulting in melanomagenesis. 2013-02-04T05:10:08.411Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12462 This paper investigates the initial yield surface of a new type of hollow sphere structure (HSS). For this new type, the sphere shell is perforated by several holes in order to open the inner sphere volume and surface. Multi-axial tensile loading is applied to investigate the initial yield surface of perforated HSS with ideal plastic base material properties. The influence of the hole diameters and different geometries of linking elements on the initial yield surface are shown. The results are compared to classical configurations without perforation. It is shown that the initial yield surface can be represented as a cone in the principal stress coordinate system. Increasing of the hole diameter (decreasing of the average density) decreases the diameter of this cone. Compared to the changes for different hole diameters, the shape of the initial yield surface is less sensitive to the geometry of the link between two spheres. In addition, the elastic properties of PHSS, i.e. Young's modulus and Poisson's ratio, are investigated. To this end, three-dimensional finite element analysis is used to investigate primitive cubic unit cell models. 2013-01-22T04:54:45.165Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12449 The mode of synaptic transmission in the vestibular periphery, between type I hair cells and their associated calyx terminal, has been the subject of much debate. The close and extensive apposition of pre- and post-synaptic elements has led some to suggest potassium (K⁺) accumulates in the intercellular space and even plays a role in synaptic transmission. During patch clamp recordings from isolated and embedded hair cells in a semi-intact preparation of the mouse cristae, we noted marked differences in whole-cell currents. Embedded type I hair cells show a prominent droop during steady-state activation as well as a dramatic collapse in tail currents. Responses to a depolarizing voltage step (-124 to +16 mV) in embedded, but not isolated, hair cells resulted in a>40 mV shift of the K⁺ equilibrium potential and a rise in effective K⁺ concentration (>50 mM) in the intercellular space. Together these data suggest K⁺ accumulation in the intercellular space accounts for the different responses in isolated and embedded type I hair cells. To test this notion, we exposed the preparation to hyperosmotic solutions to enlarge the intercellular space. As predicted, the K⁺ accumulation effects were reduced; however, a fit of our data with a classic diffusion model suggested K⁺ permeability, rather than the intercellular space, had been altered by the hyperosmotic change. These results support the notion that under depolarizing conditions Substantial K⁺ accumulation occurs in the space between type I hair cells and calyx. The extent of K⁺ accumulation during normal synaptic transmission, however, remains to be determined. 2013-01-22T01:20:05.041Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12441 Introduction: Acanthophis genus (i.e. death adders) and the Naja genus (i.e. cobras) belong to the family elapidae. The current study compared the in vitro cytotoxicity of venoms from four Acanthophis spp. and three Naja spp. on rat aortic smooth muscle cells, A7r5, and rat skeletal muscle cells, L6. The ability of CSL death adder antivenom and SAIMR antivenom, for Acanthophis spp. and Naja spp. venom respectively, to negate the cytotoxicity was also examined. Methods: A cell proliferation assay was used to determine cell viability following treatment with venom in the presence or absence of antivenom. Sigmoidal growth curves were obtained, and IC₅₀ values were determined. Results: Acanthophis spp. and Naja spp. venoms produced concentration-dependent inhibition of cell proliferation in both cell lines. Naja spp. venoms were significantly more cytotoxic than the most potent Acanthophis venom (i.e. A. antarcticus) in both cell lines. Naja spp. venoms also displayed higher sensitivity in L6 cells. SAIMR antivenom significantly inhibited the cytotoxic actions of all Naja spp. venoms in both A7r5 and L6 cells. However, death adder antivenom (CSL Ltd) was unable to negate the cytotoxic effects of Acanthophis spp. venoms. Discussion: Concentrations of the predominantly cytotoxic Naja spp. venoms used were approximately three times less than the predominantly neurotoxic Acanthophis spp. venoms. SAIMR antivenom was partially effective in neutralising the effects of Naja spp. venoms. Death adder antivenom(CSL Ltd) was not effective in negating the cytotoxic effects of venom from Acanthophis spp. These results indicate that the cell-based assay is suited to the examination of cytotoxic snake venoms and may be used in conjunction with organ bath experiments to pharmacologically characterise snake venoms. Furthermore, the results suggest that the use of a skeletalmuscle cell line is likely to bemore clinically relevant for the examination of cytotoxic snake venoms. 2013-01-17T02:20:06.777Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12382 Functional gastrointestinal disorders (FGIDs) are common and currently defined by a symptom-based classification with no discernable pathology. In functional dyspepsia (FD), the duodenum is now implicated as a key area where symptoms originate. This is attributed to immune activation with increasing evidence indicating a role for duodenal eosinophilia. In irritable bowel syndrome (IBS), mastocytosis has been documented throughout the small and large intestine. Eosinophils and mast cells are an important link between innate and adaptive immunity, and are important in allergic type TH2 inflammation. Eosinophils may give rise to symptoms due to release of preformed cytokine proteins, which trigger neural excitation, muscle spasm, and pain. The close relationship of mast cells to nerves in IBS may similarly give rise to symptoms. Genetic studies also support of the role of innate immunity in FGIDs. The data supporting a prime role for eosinophils and mast cells in subsets of FD and IBS has become credible, and these data should be used to implement advances in diagnosis and therapeutic trials. 2013-01-10T01:40:04.399Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12308 In spinal cord injury (SCI) research, axon regeneration across spinal lesions is most often assessed using anatomical methods. It would be extremely advantageous, however, to examine the functional synaptic connectivity of regenerating fibres, using high-resolution electrophysiological methods. We have therefore developed a mouse horizontal spinal cord slice preparation that permits detailed analysis of evoked dorsal column (DCol) synaptic inputs on spinal neurons, using whole-cell patch clamp electrophysiology. This preparation allows us to characterise postsynaptic currents and potentials in response to electrical stimulation of DCol fibres, along with the intrinsic properties of spinal neurons. In addition, we demonstrate that low magnification calcium imaging can be used effectively to survey the spread of excitation from DCol stimulation in horizontal slices. This preparation is a potentially valuable tool for SCI research where confirmation of regenerated, functional synapses across a spinal lesion is critical. 2012-12-18T23:25:34.941Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12304 Purpose: Oxaliplatin (OHP) in combination with 5-Xuorouracil/leucovorin (FOLFOX) is clinically used as frontline therapy in patients with advanced colorectal carcinoma (CRC), with response rates ranging from 46 to 71%. This combination is now considered a standard treatment for metastatic CRC and also in the post-operative adjuvant setting. Reversible, cumulative, peripheral sensory neuropathy is the principal dose-limiting toxicity of OHP therapy. Pyridoxine (vitamin B6) has been shown to reduce cisplatin and Xuoropyrimidine-related neurotoxicity but its administration with OHP has not yet been studied. Low doses of pyridoxine are free of side effects; it can be given orally. If pyridoxine administration with oxaliplatin has no adverse effect on OHP cytotoxicity effects, it will be a simple and cost-effective way to minimise OHP-induced neurotoxicity. Methods: In vitro simultaneous combination of OHP and pyridoxine was studied in 6 CRC cell lines (HT29, Widr, SW480, HCT116, H630 and SW1116), in an ovarian cancer cell line (A2780) and its cisplatin-resistant subline (ADDP) and in an oestrogen-dependent breast cancer cell line (MCF-7). Three fixed concentrations of pyridoxine: 1, 10 and 25 μM were combined with varying concentrations of OHP, and the growth inhibitory effects were evaluated using the MTT cell growth assay. Results: Oxaliplatin induced consistent cytotoxicity in all cell lines with GI₅₀ values between 0.23 and 7.6 μM. Addition of pyridoxine at concentrations of 1–25 μM does not affect OHP cytotoxicity. Conclusions: Administration of pyridoxine, at concentrations extending across possible therapeutic plasma levels in humans, does not antagonise OHP antitumour effects in a range of relevant tumour cell lines. This study provides a foundation for clinical studies to test whether pyridoxine can minimise OHP-related neurotoxicity, and clinicians can be confident that pyridoxine is very unlikely to reverse the antitumour effects of OHP, as seems to be the case with Ca/Mg infusions. This could prove to be a cost-effective way to minimise OHP-related neurotoxicity, allowing more effective less toxic treatment and better outcomes in patients. 2012-12-18T22:38:01.006Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12039 Research Doctorate - Doctor of Philosophy (PhD) 2012-11-20T00:41:42.269Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11888 Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize. 2012-11-01T04:26:24.449Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11556 Cell expansion is a major component of plant cell development and plays a key role in organ growth, hence realization of crop productivity. Thus, unraveling mechanisms controlling plant cell expansion is essential not only for understanding fundamental plant biology but also for designing innovative approaches to increase crop yield and quality. The multicellular plant tissues, however, impose enormous technique challenges to assess the contribution of molecular or cellular events to the expansion of given cell types as they often deeply embedded within the tissues, thus are not readily accessible for sampling or measurement. In this context, cotton fibers, single celled hairs developed from the seed coat epidermis represent an ideal system for studying regulation of cell expansion, owing to their rapid and synchronized elongation (up to 3~5 cm long) and high accessibility for experimentation. Recently, we demonstrated the essential role of vacuolar invertase (VIN) in early fiber elongation. Remarkably, we discovered that VIN controls cotton fiber and Arabidopsis root elongating through osmotic dependent and independent pathways, respectively. This shows mechanistic complexity of cell expansion. Here, we evaluate the coordinated actions of multiple pathways in regulating cotton fiber elongation linking solute transport and metabolism with plasmodesmatal gating, water flow and cell wall dynamics and we outline future directions for deepening our understanding of plant cell expansion. 2012-09-19T05:03:12.397Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9196 The giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less commonly FAT2, FAT3, and FAT4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that, like Drosophila FAT, human FAT1 is cleaved into a non-covalent heterodimer before achieving cell surface expression. The use of inhibitors also established that such cleavage requires the proprotein convertase furin. However, in melanoma cells, the non-cleaved proform of FAT1 is also expressed at the cell surface together with the furin-cleaved heterodimer. Moreover, furin-independent processing generates a potentially functional proteolytic product in melanoma cells, a persistent 65-kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. In vitro localization studies of FAT1 showed that melanoma cells display high levels of cytosolic FAT1 protein, whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. Such differences in protein distribution appear to reconcile with the different protein products generated by dual FAT1 processing. We suggest that the uncleaved FAT1 could promote altered signaling, and the novel products of alternate processing provide a dominant negative function in melanoma. 2012-09-18T04:40:05.567Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11495 Regulatory T cells (Tregs) play an essential role in maintaining the homeostatic balance of immune responses. Asthma is an inflammatory condition of the airways that is driven by dysregulated immune responses toward normally innocuous antigens. Individuals with asthma have fewer and less functional Tregs, which may lead to uncontrolled effector cell responses and promote proasthmatic responses of T helper type 2, T helper 17, natural killer T, antigenpresenting, and B cells. Tregs have the capacity to either directly or indirectly suppress these responses. Hence, the induced expansion of functional Tregs in predisposed or individuals with asthma is a potential approach for the prevention and treatment of asthma. Infection by a number of micro-organisms has been associated with reduced prevalence of asthma, and many infectious agents have been shown to induce Tregs and reduce allergic airways disease in mouse models. The translation of the regulatory and therapeutic properties of infectious agents for use in asthma requires the identification of key modulatory components and the development and trial of effective immunoregulatory therapies. Further translational and clinical research is required for the induction of Tregs to be harnessed as a therapeutic strategy for asthma. 2012-09-10T05:00:12.455Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11418 Plasmid pBaSysBioII was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type event and is non-mutagenic, placing the fusion at the homologous chromosomal locus. Using phoA, murAA, gapB, ptsG and cggR promoters that are responsive to phosphate availability, growth rate and carbon source, we show that detailed profiles of promoter activity can be established, with responses to changing conditions being measurable within 1 min of the stimulus. This makes pBaSysBioII a highly versatile tool for real-time gene expression analysis in growing cells of B. subtilis. 2012-08-29T05:51:43.971Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11216 Vacuolar invertase (VIN) has long been considered as a major player in cell expansion. However, direct evidence for this view is lacking due, in part, to the complexity of multicellular plant tissues. Here, we used cotton (Gossypium spp.) fibers, fast-growing single-celled seed trichomes, to address this issue. VIN activity in elongating fibers was approximately 4-6-fold higher than that in leaves, stems, and roots. It was undetectable in fiberless cotton seed epidermis but became evident in initiating fibers and remained high during their fast elongation and dropped when elongation slowed. Furthermore, a genotype with faster fiber elongation had significantly higher fiber VIN activity and hexose levels than a slow-elongating genotype. By contrast, cell wall or cytoplasmic invertase activities did not show correlation with fiber elongation. To unravel the molecular basis of VIN-mediated fiber elongation, we cloned GhVIN1, which displayed VIN sequence features and localized to the vacuole. Once introduced to Arabidopsis (Arabidopsis thaliana), GhVIN1 complemented the short-root phenotype of a VIN T-DNA mutant and enhanced the elongation of root cells in the wild type. This demonstrates that GhVIN1 functions as VIN in vivo. In cotton fiber, GhVIN1 expression level matched closely with VIN activity and fiber elongation rate. Indeed, transformation of cotton fiber with GhVIN1 RNA interference or overexpression constructs reduced or enhanced fiber elongation, respectively. Together, these analyses provide evidence on the role of VIN in cotton fiber elongation mediated by GhVIN1. Based on the relative contributions of sugars to sap osmolality in cotton fiber and Arabidopsis root, we conclude that VIN regulates their elongation in an osmotic dependent and independent manner, respectively. 2012-08-10T01:05:10.803Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11166 Germ cell sex differentiation in the mouse embryo is denoted by meiosis entry in females and mitotic arrest in males. Because p38 mitogen-activated protein kinase (MAPK) signaling initiates mitotic arrest in other differentiating cell types, we investigated its potential role in XY germ cell differentiation in mice. We report that p38 MAPK is phosphorylated and therefore activated only in XY germ cells around the time of sex differentiation. Quantitative RT-PCR analysis showed that 14 known targets of p38 MAPK signaling are expressed in the embryonic gonads at this time and that five of these targets (Mapkapk5, Max, Myc, Hbp1, and Cebpa) have expression profiles similar to that of activated p38 MAPK. Inhibition of p38 MAPK signaling in XY germ cells ex vivo reduced expression of the pluripotency marker POU5F1 and increased the expression of Stra8 and SYCP3, premeiosis and meiosis markers, respectively, to levels approaching those observed in XX germ cells. These data suggest that p38 MAPK signaling antagonizes entry into meiosis in XY germ cells, instead directing them toward mitotic quiescence and a spermatogenic fate. 2012-07-31T05:16:13.840Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11164 Background: Asthma typically originates in early-life, and the impact of infection during immunologic maturation is a critical factor in disease pathogenesis. The progression of aberrant TH2 cell responses and disease development has been attributed to a lack of infections. However, exposure to specific pathogens such as Chlamydia may alter immunologic programming and predispose to asthma. Objective: To investigate the effects of chlamydial infection at different ages on allergic airways disease in later life. Methods: Neonatal, infant, or adult BALB/c mice were infected and 6 weeks later were sensitized and subsequently challenged with ovalbumin. Hallmark features of allergic airways disease were compared with uninfected allergic and nonallergic controls. Results: Early-life (neonatal and infant) but not adult chlamydial infection enhanced the development of hallmark features of asthma in ovalbumin-induced allergic airways disease. Notably early-life infection increased mucus-secreting cell numbers, IL-13 expression, and airway hyperresponsiveness. Neonatal infection attenuated eosinophil influx and ovalbumin-specific TH2 cytokine release and numbers of activated myeloid dendritic cells (DCs) in lymph nodes. By contrast, infant infection augmented features of allergic inflammation with increased airway eosinophils, TH2 cytokine, and DC responses. Both neonatal and infant infection increased systemic DC-induced IL-13 release from CD4⁺ T cells. The timing of infection had significant effects on lung structure because neonatal but not infant or adult infection induced increases in alveolar diameter. Conclusion: Early-life respiratory chlamydial infections modulate immune responses, alter lung function and structure, and enhance the severity of allergic airways disease in later life. 2012-07-31T03:45:47.203Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11115 The glioma pathogenesis-related 1 (GLIPR1) family consists of three genes [GLIPR1, GLIPR1-like 1(GLIPR1L1), and GLIPR1-like 2 (GLIPR1L2)] and forms a distinct subgroup within the cysteine-rich secretory protein (CRISP), antigen 5, and pathogenesis-related 1 (CAP) superfamily. CAP superfamily proteins are found in phyla ranging from plants to humans and, based largely on expression and limited functional studies, are hypothesized to have roles in carcinogenesis, immunity, cell adhesion, and male fertility. Specifically data from a number of systems suggests that sequences within the C-terminal CAP domain of CAP proteins have the ability to promote cell-cell adhesion. Herein we cloned mouse Glipr1l1 and have shown it has a testis-enriched expression profile. GLIPR1L1 is posttranslationally modified by N-linked glycosylation during spermatogenesis and ultimately becomes localized to the connecting piece of elongated spermatids and sperm. After sperm capacitation, however, GLIPR1L1 is also localized to the anterior regions of the sperm head. Zona pellucida binding assays indicate that GLIPR1L1 has a role in the binding of sperm to the zona pellucida surrounding the oocyte. These data suggest that, along with other members of the CAP superfamily and several other proteins, GLIPR1L1 is involved in the binding of sperm to the oocyte complex. Collectively these data further strengthen the role of CAP domain-containing proteins in cellular adhesion and propose a mechanism whereby CAP proteins show overlapping functional significance during fertilization. 2012-07-20T03:30:05.808Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11009 In this review we provide an overview of key in vivo experiments undertaken in the cat spinal cord in the 1950s and 1960s, and point out their contributions to our present understanding of glycine receptor (GlyR) function. Importantly, some of these discoveries were made well before an inhibitory receptor, or its agonist, was identifi ed. These contributions include the universal acceptance of a chemical mode of synaptic transmission; that GlyRs are chloride channels; are involved in reciprocal and recurrent spinal inhibition; are selectively blocked by strychnine; and can be distinguished from the GABAA receptor by their insensitivity to bicuculline. The early in vivo work on inhibitory mechanisms in spinal neurons also contributed to several enduring principles on synaptic function, such as the time associated with synaptic delay, the extension of Dale’s hypothesis (regarding the chemical unity of nerve cells and their terminals) to neurons within the central nervous system, and the importance of inhibition for synaptic integration in motor and sensory circuits. We hope the work presented here will encourage those interested in GlyR biology and inhibitory mechanisms to seek out and read some of the “classic” articles that document the above discoveries. 2012-06-28T04:02:00.025Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10991 Although the importance of clathrin- and caveolin-independent endocytic pathways has recently emerged, key aspects of these routes remain unknown. Using quantitative ultrastructural approaches, we show that clathrin-independent carriers (CLICs) account for approximately three times the volume internalized by the clathrin-mediated endocytic pathway, forming the major pathway involved in uptake of fluid and bulk membrane in fibroblasts. Electron tomographic analysis of the 3D morphology of the earliest carriers shows that they are multidomain organelles that form a complex sorting station as they mature. Proteomic analysis provides direct links between CLICs, cellular adhesion turnover, and migration. Consistent with this, CLIC-mediated endocytosis of key cargo proteins, CD44 and Thy-1, is polarized at the leading edge of migrating fibroblasts, while transient ablation of CLICs impairs their ability to migrate. These studies provide the first quantitative ultrastructural analysis and molecular characterization of the major endocytic pathway in fibroblasts, a pathway that provides rapid membrane turnover at the leading edge of migrating cells. 2012-06-26T06:11:20.695Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10904 Background: Concerns regarding the safety of transfused blood, have prompted reconsideration of the use of allogeneic (blood from an unrelated donor) red blood cell (RBC) transfusion, and a range of techniques to minimise transfusion requirements. Objectives: To examine the evidence for the efficacy of cell salvage in reducing allogeneic blood transfusion and the evidence for any effect on clinical outcomes. Search strategy: We identified studies by searching CENTRAL (The Cochrane Library 2009, Issue 2), MEDLINE (1950 to June 2009), EMBASE (1980 to June 2009), the Internet (to August 2009) and bibliographies of published articles. Selection criteria: Randomised controlled trials with a concurrent control group in which adult patients, scheduled for non-urgent surgery, were randomised to cell salvage (autotransfusion), or to a control group, who did not receive the intervention. Data collection and analysis: Data were independently extracted and the risk of bias assessed. Relative risks (RR) and weighted mean differences (WMD) with 95% confidence intervals (CIs) were calculated. Data were pooled using a random effects model. The primary outcomes were the number of patients exposed to allogeneic red cell transfusion, and the amount of blood transfused. Other clinical outcomes are detailed in the review. Main results: A total of 75 trials were included. Overall, the use of cell salvage reduced the rate of exposure to allogeneic RBC transfusion by a relative 38% (RR=0.62: 95% CI 0.55 to 0.70). The absolute reduction in risk (ARR) of receiving an allogeneic RBC transfusion was 21% (95% CI 15% to 26%). In orthopaedic procedures the RR of exposure to RBC transfusion was 0.46 (95% CI 0.37 to 0.57) compared to 0.77 (95% CI 0.69 to 0.86) for cardiac procedures. The use of cell salvage resulted in an average saving of 0.68 units of allogeneic RBC per patient (WMD=-0.68; 95% CI -0.88 to -0.49). Cell salvage did not appear to impact adversely on clinical outcomes. Authors' conclusions: The results suggest cell salvage is efficacious in reducing the need for allogeneic red cell transfusion in adult elective cardiac and orthopaedic surgery. The use of cell salvage did not appear to impact adversely on clinical outcomes. However, the methodological quality of trials was poor. As the trials were unblinded and lacked adequate concealment of treatment allocation, transfusion practices may have been influenced by knowledge of the patients' treatment status potentially biasing the results in favour of cell salvage. 2012-06-20T23:08:58.415Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10897 Background: Most clinical practice guidelines recommend restrictive red cell transfusion practices, with the goal of minimising exposure to allogeneic blood (from an unrelated donor). The purpose of this review is to compare clinical outcomes in patients randomised to restrictive versus liberal transfusion thresholds (triggers). Objectives: To examine the evidence for the effect of transfusion thresholds on the use of allogeneic and/or autologous blood, and the evidence for any effect on clinical outcomes. Search strategy: Trials were identified by: computer searches of the Cochrane Central Register of Controlled Trials (the Cochrane Library Issue 3, 2009), OVID MEDLINE (1966 to August 2009), Current Contents (1993 to November 2004), and the Web of Science (2004 to August 2009). References in identified trials and review articles were checked and experts contacted to identify any additional trials. Selection criteria: Controlled trials in which patients were randomised to an intervention group or to a control group. Trials were included where intervention groups were assigned on the basis of a clear transfusion 'trigger', described as a haemoglobin (Hb) or haematocrit (Hct) level below which an RBC transfusion was to be administered. Data collection and analysis: Relative risks of requiring allogeneic blood transfusion, transfused blood volumes and other clinical outcomes were pooled across trials, using a random effects model. The risk of bias was assessed. Main results: Seventeen trials involving a total of 3746 patients were identified. Restrictive transfusion strategies reduced the risk of receiving a red blood cell (RBC) transfusion by a relative 37% (RR=0.63; 95% CI 0.54 to 0.74). This equates to an average absolute risk reduction (ARR) of 33% (95% CI 21% to 45%). The volume of RBCs transfused was reduced on average by 0.75 units (95% CI 0.20 to 1.30 units). However, heterogeneity between trials was statistically significant (P<0.001; I²≥74%) for these outcomes. Restrictive transfusion strategies did not appear to impact on the rate of adverse events compared to liberal transfusion strategies (i.e. mortality, cardiac events, myocardial infarction, stroke, pneumonia and thromboembolism). Restrictive transfusion strategies were associated with a statistically significant reduction in the rates of infection (RR=0.76; 95% CI 0.60 to 0.97). The use of restrictive transfusion strategies did not reduce hospital or intensive care length of stay. Authors' conclusions: The existing evidence supports the use of restrictive transfusion triggers in patients who are free of serious cardiac disease. The effects of conservative transfusion triggers on functional status, morbidity and mortality, particularly in patients with cardiac disease, need to be tested in further large clinical trials. In countries with inadequate screening of donor blood, the data may constitute a stronger basis for avoiding transfusion with allogeneic red cells. 2012-06-13T23:40:12.539Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8687 This paper examines the effect of cell loadings on the performance of a group based multicast algorithm implemented on the HSDPA air interface. The group based multicast scheme could offer higher QoS and capacity in a HSDPA network. However, the performance of the multicast algorithm could be affected by neighboring cell loadings. In order to compensate the variable SINR conditions caused by the variation in neighboring cell loading it is necessary to optimize the group switching algorithm to maintain high QoS in a cell and to minimize the uplink feedback traffic. The paper presents simulation results showing the effects of cell loading on the performance of the group based multicasting scheme. An OPNET based multi-cell simulation model has been used to obtain the results. 2012-06-05T01:31:05.223Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10824 Calcium/calmodulin-stimulated protein kinase II (CaMKII) is an important mediator of synaptic function that is regulated by multi-site phosphorylation and targeting through interactions with proteins. A new phosphorylation site at Thr253 has been identified in vivo, that does not alter CaMKII activity, but does alter CaMKII function through interactions with binding proteins. To identify these proteins, as well as to examine the specific effects following Thr253 or Thr286 phosphorylation on these interactions, we developed an in vitro overlay binding assay. We demonstrated that the interaction between CaMKII and its binding proteins was altered by the phosphorylation state of both the CaMKII and the partner, and identified a CaMKII-specific sequence that was responsible for the interaction between CaMKII and two interacting proteins. By comparing CaMKII binding profiles in tissue and cell extracts, we demonstrated that the CaMKII binding profiles varied with cell type, and also showed that overexpression of a CaMKII Thr253 phospho-mimic mutant in human neuroblastoma and breast cancer cells dramatically altered the morphology and growth rates when compared to overexpression of non-phosphorylated CaMKII. This data highlights the importance of the microenvironment in regulating CaMKII function, and describes a potentially new mechanism by which the functions of CaMKII can be regulated. 2012-05-21T06:37:30.612Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10715 Transfer cells are specialised transport cells containing invaginated wall ingrowths that generate an amplified plasma membrane surface area with high densities of transporter proteins. They trans-differentiate from differentiated cells at sites at which enhanced rates of nutrient transport occur across apo/symplasmic boundaries. Despite their physiological importance, little is known of the molecular mechanisms regulating construction of their intricate wall ingrowths. We investigated the genetic control of wall ingrowth formation in phloem parenchyma transfer cells of leaf minor veins in Arabidopsis thaliana. Wall ingrowth development in these cells is substantially enhanced upon exposing plants to high-light or cold treatments. A hierarchical bioinformatic analysis of public microarray datasets derived from the leaves of plants subjected to these treatments identified GIGANTEA (GI) as one of 46 genes that are commonly up-regulated twofold or more under both high-light and cold conditions. Histological analysis of the GI mutants gi-2 and gi-3 showed that the amount of phloem parenchyma containing wall ingrowths was reduced 15-fold compared with wild-type. Discrete papillate wall ingrowths were formed in gi-2 plants but failed to develop into branched networks. Wall ingrowth development in gi-2 was not rescued by exposing these plants to high-light or cold conditions. In contrast, over-expression of GI in the gi-2 background restored wall ingrowth deposition to wild-type levels. These results indicate that GI regulates the ongoing development of wall ingrowth networks at a point downstream of inputs from environmental signals. 2012-05-01T21:40:06.970Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10662 FtsK is a multifunctional, multidomain protein that acts to co-ordinate chromosome unlinking, segregation and cell division. In this issue of Molecular Microbiology, the report by Dubarry et al. reveals new insight into the surprisingly complex relationship between the different activities of FtsK. The new study makes extensive use of fusion proteins to highlight the role of the FtsK ‘linker’ domain in helping to co-ordinate these processes. This, taken together with previous studies, suggests that FtsK is intimately involved in septum constriction, physically contacting several other divisome proteins. Further, it is attractive to speculate that FtsK can regulate the late stages of septation to act as a checkpoint to ensure DNA is fully cleared from the septum before it is allowed to close, as well as being the driving force to unlink the chromosomes and segregate the DNA away from the septum. 2012-04-17T22:57:38.040Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7911 Lung cancer is one of the most common malignancies in the world. In Australia, lung cancer accounts for 8500 new cases pa, the fifth most common cancer. In addition it is seldom ever cured, with the vast majority of patients succumbing from their disease, accounting for 7000 deaths pa in Australia, the commonest cause of cancer death. In developed countries like Australia, trends in smoking habits are leading to decreasing incidence but it still has a substantial health impact. On the contrary lung cancer has a sharply increasing incidence in developing countries, where it is destined to become a major health problem, including in many Asia Pacific region countries. Most cases of lung cancer are classified as non-small cell lung cancer (NSCLC). 2012-03-22T01:40:04.374Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10470 There is little evidence on roles of growth factors other than cytokines in expansion of cord blood (CB) stem cells. We aimed to explore a novel approach for expansion, using Substance P (SP) and calcitonin gene-related peptide (CGRP) neuropeptides. CB CD34⁺ cells were cultured in different concentrations of SP and/or CGRP in combination with a cytokine cocktail. Phenotypic and functional analysis was performed by flowcytometry and colonogenic assay. Our results show a significant improvement of total expansion of neuropeptide treated cells. There was a selective effect of CGRP on CD34⁺ CD133⁺ cells, SP on CD34⁺ CD45dim cells, and 10⁻⁹ M SP and/or CGRP on expansion of CD34⁺ CD38⁻ cells. There was also a tendency for erythroid and granulocyte–myeloid colony formation in SP and CGRP treated cultures, respectively. Supplementation of cytokines with other growth factors, such as neuropeptides, might enable us to overcome the difficulties of ex vivo expansion of CB cells. 2012-03-20T22:50:07.499Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:219 Previous studies in small groups of patients suggested that immunization of melanoma patients with peptide epitopes recognized by T cells could induce regression of melanoma. This approach was tested in 36 patients with stage IV melanoma. The (MHC class I-restricted) peptides were from gp100, MART-1, tyrosinase, and MAGE-3. The gp100 and MART-1 peptides had been modified to increase their immunogenicity. In half the patients (groups 3 and 4) the peptides were given in the adjuvant Montanide-ISA-720, and half the patients in both groups were given GM-CSF s.c. for 4 days following each injection. Treatment was well tolerated except for two severe erythematous responses to Montanide-ISA-720 and marked inflammatory responses at sites of GM-CSF administration in three patients. There were no objective clinical responses but stabilization of disease for periods from 3 to 12 months were seen in seven patients. Five of these were patients given the peptides in Montanide-ISA-720. Delayed-type hypersensitivity (DTH) skin test responses were also seen mainly in the patients given the peptides in Montanide-ISA-720. GM-CSF did not increase DTH responses in patients in the latter group but may have increased DTH responses in those not given peptides in Montanide-ISA-720. Inflammatory responses around s.c. metastases or regional lymph nodes were observed in two patients. These results suggest that the peptides are more effective when given in the adjuvant Montanide-ISA-720. Nevertheless, results from this study, together with those from a number of comparable studies, indicate that peptide vaccines are currently of minimal benefit to patients and support the need for ongoing development of new strategies in treatment of this disease. © Springer-Verlag 2004. 2012-03-12T07:13:33.437Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1622 Mucosal vaccination using adjuvant protein vaccines may offer a novel approach for tuberculosis. To date, however, development of such a vaccine has been considered unlikely due to the inability to identify a safe adjuvant that stimulates an appropriate immune response. This study was undertaken to determine the potential of a dairy propionibacteria, P. jensenii strain 702 (PJ702), to act as an adjuvant when co-administered orally with soluble tuberculosis protein to mice. The efficacy of the PJ702 to act as an adjuvant was assessed by comparison with cholera toxin. C57 mice were orally immunized with Mycobacterium tuberculosis short-term culture filtrate protein (STCF) (200 μg) with either PJ702 (10⁸ cfu) or cholera toxin (10 μg) in a total volume of 100 $mu$L. A control group was given PJ702 10⁸ cfu alone. Each mouse (eight per group) was vaccinated weekly over a 21-day period. At day 25 the mice were sacrificed, spleens were collected and lymphocyte cultures prepared. After stimulation with STCF (2.5 μg), cell proliferation was measured by ³H thymidine uptake, and the cytokines, IL-4 and IFN-γ, by ELISA. A significantly higher T-cell proliferation was observed for the group given the vaccine containing PJ702, compared to both the control group and the group given the vaccine containing cholera toxin (P < 0.05). The predominant cytokine produced from all groups was IFN-γ. These results indicate potential for future development of an oral tuberculosis vaccine, and also identify PJ702 as a potential living vaccine vector that could be applied to a number of mucosally transmitted diseases. 2012-03-01T01:00:06.062Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:45 Mucosal vaccination using adjuvant protein vaccines may offer a novel approach for tuberculosis. To date, however, development of such a vaccine has been considered unlikely due to the inability to identify a safe adjuvant that stimulates an appropriate immune response. This study was undertaken to determine the potential of a dairy propionibacteria, P. jensenii strain 702 (PJ702), to act as an adjuvant when co-administered orally with soluble tuberculosis protein to mice. The efficacy of the PJ702 to act as an adjuvant was assessed by comparison with cholera toxin. C57 mice were orally immunized with Mycobacterium tuberculosis short-term culture filtrate protein (STCF) (200 mu g) with either PJ702 (10(8) cfu) or cholera toxin (10 mu) in a total volume of 100 mu L. A control group was given PJ702 108 cfu alone. Each mouse (eight per group) was vaccinated weekly over a 21-day period. At day 25 the mice were sacrificed, spleens were collected and lymphocyte cultures prepared. After stimulation with STCF (2.5 mu g), cell proliferation was measured by H-3 thymidine uptake, and the cytokines, IL-4 and IFN-gamma, by ELISA. A significantly higher T-cell proliferation was observed for the group given the vaccine containing PJ702, compared to both the control group and the group given the vaccine containing cholera toxin (P<0.05). The predominant cytokine produced from all groups was IFN-gamma. These results indicate potential for future development of an oral tuberculosis vaccine, and also identify PJ702 as a potential living vaccine vector that could be applied to a number of mucosally transmitted diseases. 2012-03-01T00:59:20.411Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10155 Asthma is characterised by antigen-mediated mast cell degranulation resulting in secretion of inflammatory mediators. Protein phosphatase 2A (PP2A) is a serine/threonine protein phosphatase composed of a catalytic (PP2A-C) subunit together with a core scaffold (PP2A-A) subunit and a variable, regulatory (PP2A-B) subunit. Previous studies utilising pharmacological inhibition of protein phosphatases have suggested a positive regulatory role for PP2A in mast cell degranulation. In support of this we find that a high okadaic acid concentration (1 μM) inhibits mast cell degranulation. Strikingly, we now show that a low concentration of okadaic acid (0.1 μM) has the opposite effect, resulting in enhanced degranulation. Selective downregulation of the PP2A-Cα subunit by short hairpin RNA also enhanced degranulation of RBL-2H3 mast cells, suggesting that the primary role of PP2A is to negatively regulate degranulation. PP2A-B subunits are responsible for substrate specificity, and carboxymethylation of the PP2A-C subunit alters B subunit binding. We show here that carboxymethylation of PP2A-C is dynamically altered during degranulation and inhibition of methylation decreases degranulation. Moreover downregulation of the PP2A-Bα subunit resulted in decreased MK2 phosphorylation and degranulation, whilst downregulation of the PP2A-B′δ subunit enhanced p38 MAPK phosphorylation and degranulation. Taken together these data show that PP2A is both a positive and negative regulator of mast cell degranulation, and this differential role is regulated by carboxymethylation and specific PP2A-B subunit binding. 2012-02-24T02:20:04.795Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9646 Cotton fibres are hair-like single-cells that elongate to several centimetres long after their initiation from the ovule epidermis at anthesis. The accumulation of malate, along with K⁺ and sugars, is thought to play an important role in fibre elongation through osmotic regulation and charge balance. However, there is a lack of evidence for or against such an hypothesis. Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme responsible for the synthesis of malate. The potential role of PEPC in cotton fibre elongation is examined here. Developmentally, PEPC activity was higher at the rapid elongation phase than that at the slow elongation stage. Genotypically, PEPC activity correlated positively with the rate of fibre elongation and the final fibre length attained. Importantly, suppression of PEPC activity by LiCl that reduces its phosphorylation status decreased fibre length. To examine the molecular basis underlying PEPC activity, two cDNAs encoding PEPC, GhPEPC1 and 2, were cloned, which represents the major PEPC genes expressed in cotton fibre. RT-PCR analyses revealed that GhPEPC1 and 2 were highly expressed at the rapid elongation phase but weakly at the slow-to-terminal elongation period. In situ hybridization detected mRNA of GhPEPC1 and 2 in 1 d young fibres but not in the ovule epidermis prior to fibre initiation. Collectively, the data indicate that cotton fibre elongation requires high activity of PEPC, probably through the expression of the GhPEPC1 and 2 genes. 2012-01-30T05:26:33.599Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6975 Ljungan virus (LV) was discovered 20 years ago in Swedish bank voles (Myodes glareolus, previously referred to as Clethrionomys glareolus) during the search for an infectious agent causing lethal myocarditis in young athletes. To date, the genomes of four LV isolates, including the prototype 87-012 strain, have been characterized. Three of these LV strains were isolated from bank voles trapped in Sweden. Sequence analysis of an American virus (M1146), isolated from a montane vole (Microtus montanus) in western USA, indicates that this strain represents a genotype that is different from the Swedish strains. Here, we present genomic analyses of a fifth LV strain (64-7855) isolated from a southern red-backed vole (Myodes gapperi) trapped during arbovirus studies in New York state in the north-eastern USA in the 1960s. Sequence analysis of the 64-7855 genome showed an LV-like genome organization and sequence similarity to other LV strains. Genetic and phylogenetic analyses of the evolutionary relationship between the 64-7855 strain and other viruses within the family Picornaviridae, including previously published LV strains, demonstrated that the 64-7855 train constitutes a new genotype within the LV species. Analyses also showed that different regions of the 64-7855 genome have different phylogenetic relationships with other LV strains, indicating that previous recombination events have been involved in the evolution of this virus. 2012-01-30T05:04:50.953Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7086 We report the long-term follow-up of successful treatment of mucopolysaccharidosis type I H (MPS IH, Hurler syndrome) with combined enzyme replacement therapy and haematopoietic progenitor stem cell transplant. 2012-01-30T05:03:23.934Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6551 Sperm-triggered Ca²⁺ oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca²⁺ oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca²⁺ oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca²⁺ oscillations that are all waves in ascidians. The first of these Ca²⁺ waves begins at the point of sperm-egg fusion while a second phase of Ca²⁺ waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca²⁺ waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca²⁺-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca²⁺ waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca²⁺ waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca²⁺ oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for spermtriggered Ca²⁺ oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophasestage oocytes, lacking CDK activity, failed to induce any Ca²⁺ release even though they responded to microinjection of the Ca²⁺-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca²⁺-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca²⁺-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca²⁺-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca²⁺ waves observed at fertilization with a spatial pattern that mimics those initiated by sperm. 2012-01-30T04:07:31.709Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7426 Research Doctorate - Doctor of Philosophy (PhD) 2011-12-13T01:10:44.572Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9702 Primordial germ cells (PGCs) are embryonic progenitors for the gametes. In the gastrulating mouse embryo, a small group of cells begin expressing a unique set of genes and so commit to the germline. Over the next 3–5 days, these PGCs migrate anteriorly and increase rapidly in number via mitotic division before colonizing the newly formed gonads. PGCs then express a different set of unique genes, their inherited epigenetic imprint is erased and an individual methylation imprint is established, and for female PGCs, the silent X chromosome is reactivated. At this point, germ cells (GCs) commit to either a female or male sexual lineage, denoted by meiosis entry and mitotic arrest, respectively. This developmental program is determined by cues emanating from the somatic environment. 2011-12-12T23:10:08.228Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9655 Modulation of adhesion molecules expression on the surface of cord blood (CB) CD34⁺ cells may assist in overcoming the delay in cord blood engraftment. Likewise, utilization of diverse growth factors such as neuropeptides could also be helpful. Therefore, we aimed to assess the role of Substance P (SP) along with a cytokine cocktail on CB CD34⁺ adhesion molecule expression. CB CD34⁺ cells were cultured in a serum-free media containing different concentrations of SP in combination with a cytokine cocktail (SCF, FL, TPO, IL-3, and IL-6). Expression of adhesion molecules CXCR4, CD44, CD49e, and CD62L was analyzed after 7 and/or 11 days of cell cultivation. Additionally, the colonogenic capacity of cells was analyzed by colony formation unit assay. Our results show an enhanced percentage of CD34⁺cells with CXCR4, CD44, and CD62L on day 7, as compared with control. Furthermore, an increase in frequency was observed for CD49e⁺ CD34⁺cells by day 7 in both test and control groups compared with day 0. Colonogenic assays show occurrence of more total colony formation and immature progenitor cells in SP-treated cells. Our study indicates that SP could act as an effective modulator for expression of cell adhesion molecules. 2011-12-08T00:00:03.323Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6281 Research Doctorate - Doctor of Philosophy (PhD) 2011-12-07T23:50:04.355Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7423 Research Doctorate - Doctor of Philosophy (PhD) 2011-12-07T21:50:29.772Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9634 The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P < 0.05). The results of adhesion assay showed that when probiotic strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P < 0.05). Adhesion percentage of Lb. casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P < 0.05). These results show that the survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products. 2011-12-07T02:00:13.319Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9567 It is well established that Multiple Sclerosis (MS) is an immune mediated disease. Little is known about what drives the differential control of the immune system in MS patients compared to unaffected individuals. MicroRNAs (miRNAs) are small non-coding nucleic acids that are involved in the control of gene expression. Their potential role in T cell activation and neurodegenerative disease has recently been recognised and they are therefore excellent candidates for further studies in MS. We investigated the transcriptome of currently known miRNAs using miRNA microarray analysis in peripheral blood samples of 59 treatment naive MS patients and 37 controls. Of these 59, 18 had a primary progressive, 17 a secondary progressive and 24 a relapsing remitting disease course. In all MS subtypes miR-17 and miR-20a were significantly under-expressed in MS, confirmed by RT-PCR. We demonstrate that these miRNAs modulate T cell activation genes in a knock-in and knock-down T cell model. The same T cell activation genes are also up-regulated in MS whole blood mRNA, suggesting these miRNAs or their analogues may provide useful targets for new therapeutic approaches. 2011-12-05T23:10:03.206Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8830 The computer-assisted microscopy systems can increase the accuracy of the analysis. To guarantee correct results in computer-assisted microscopy, accurate nuclei segmentation is crucially important since images segmentation is the first step towards image understanding and image analysis. In this paper, we present clustering techniques to segment homogeneous clusters in RGB color space and then label each cluster as a different region. According to the evaluation process, 97% of nuclei pixels were correctly delineated with our algorithm and on average 90% of nuclei were correctly detected. Our methods could be of value to computer-based systems designed to objectively interpret microscopic images by accurate nuclei segmentation. 2011-09-05T03:20:04.030Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6574 Mammalian eggs naturally arrest at metaphase of the second meiotic division, until sperm triggers a series of Ca²⁺ spikes that result in activation of the anaphase-promoting complex/cyclosome (APC/C). APC/C activation at metaphase targets destruction-box containing substrates, such as cyclin B1 and securin, for degradation, and as such eggs complete the second meiotic division. Cyclin B1 degradation reduces maturation (M-phase)-promoting factor (MPF) activity and securin degradation allows sister chromatid separation. Here we examined the second meiotic division in mouse eggs following expression of a cyclin B1 construct with an N-terminal 90 amino acid deletion (Δ90 cyclin B1) that was visualized by coupling to EGFP. This cyclin construct was not an APC/C substrate, and so following fertilization, sperm were incapable of stimulating Δ90 cyclin B1 degradation. In these eggs, chromatin remained condensed and no pronuclei formed. As a consequence of the lack of pronucleus formation, sperm-triggered Ca²⁺ spiking continued indefinitely, consistent with a current model in which the sperm-activating factor is localized to the nucleus. Because Ca²⁺ spiking was not inhibited by Δ90 cyclin B1, the degradation timing of securin, visualized by coupling it to EGFP, was unaffected. However, despite rapid securin degradation, sister chromatids remained attached. This was a direct consequence of MPF activity because separation was induced following application of the MPF inhibitor roscovitine. Similar observations regarding the ability of MPF to prevent sister chromatid separation have recently been made in Xenopus egg extracts and in HeLa cells. The results presented here show this mechanism can also occur in intact mammalian eggs and further that this mechanism appears conserved among vertebrates. We present a model in which metaphase II arrest is maintained primarily by MPF levels only. 2011-08-31T00:30:01.369Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6578 Maturation (M-phase) promoting factor (MPF) plays a pivotal role in oocytes during their maturation. This review concentrates on its function at three important time-points. First, its activation during meiotic progression from prophase I arrest at germinal vesicle breakdown. Second, its role during the transition from meiosis I to meiosis II, a defining feature of meiosis involving segregation of homologous chromosomes. Third, maintenance of its activity at metaphase II arrest and the necessity for its destruction during oocyte activation. An understanding of how oocytes switch it on and turn it off underpins much of the basic cell biology of oocyte maturation. 2011-08-31T00:30:01.367Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6569 CDK1-cyclin B1 is a universal cell cycle kinase required for mitotic/meiotic cell cycle entry and its activity needs to decline for mitotic/meiotic exit. During their maturation, mouse oocytes proceed through meiosis I and arrest at second meiotic metaphase with high CDK1-cyclin B1 activity. Meiotic arrest is achieved by the action of a cytostatic factor (CSF), which reduces cyclin B1 degradation. Meiotic arrest is broken by a Ca²⁺ signal from the sperm that accelerates it. Here we visualised degradation of cyclin ∷ GFP in oocytes and found that its degradation rate was the same for both meiotic divisions. Ca²⁺ was the necessary and sufficient trigger for cyclin B1 destruction during meiosis II; but it played no role during meiosis I and furthermore could not accelerate cyclin B1 destruction during this time. The ability of Ca²⁺ to trigger cyclin B1 destruction developed in oocytes following a restabilisation of cyclin B1 levels at about 12 h of culture. This was independent of actual first polar body extrusion. Thus, in metaphase I arrested oocytes, Ca²⁺ would induce cyclin B1 destruction and the first polar body would be extruded. In contrast to some reports in lower species, we found no evidence that oocyte activation was associated with an increase in 26S proteasome activity. We therefore conclude that Ca²⁺ mediates cyclin B1 degradation by increasing the activity of an E3 ubiquitin ligase. However, this stimulation occurs only in the presence of the ubiquitin ligase inhibitor CSF. We propose a model in which Ca²⁺ directly stimulates destruction of CSF during mammalian fertilisation. 2011-08-31T00:10:02.078Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6556 Cdc20 and cdh1 are coactivators of the anaphase-promoting complex (APC). APCcdc²⁰ is necessary for the metaphase–anaphase transition and, at the end of mitosis, vertebrate cdc20 itself becomes a target for degradation through KEN-box-dependent APCcdh1 activity. By studying the degradation of fluorescent protein chimaeras in mammalian oocytes and early embryos, we found that cdc20 was degraded through two independent degradation signals (degrons), the KEN box and a newly described CRY box. In both oocytes and G1-stage embryos, the rate of degradation through the CRY box was greater than through the KEN box, although both were mediated by APCcdh¹. Thus, mammalian oocytes and embryos have the capacity to recognize two degrons in cdc20. 2011-08-30T06:10:01.759Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6544 Metaphase II-arrested mouse eggs are stimulated to complete meiosis by sperm-induced Ca²⁺ spiking. The Ca²⁺ signal causes activation of the E3 ligase anaphasepromoting complex/cyclosome (APC), leading to the destruction of key proteins necessary for meiotic exit. We show, using western blots of mouse eggs, the presence of both APC activators cdc20 and cdh1, which target Dbox and D-box/KEN-box substrates, respectively, for proteolysis. We decided to examine the temporal activation of APCcdc²⁰ and APCcdh¹ by coupling APC substrates to GFP and examining their destruction in real-time following release from second meiotic division arrest. D-box substrates were degraded quickly after the initiation of sperm-induced Ca²⁺ spiking, such that their degradation was complete by the time of second polar body extrusion. By contrast, KEN-box-containing substrates were degraded when CDK1 activity was low, during the period between polar body extrusion and pronucleus formation. This observation of apparent APCcdh¹ activity in meiosis II based on destruction of exogenous GFP-coupled substrates was then confirmed by observing destruction of endogenous APCcdh¹ substrates. These data are consistent with a model of initial APCcdc²⁰ activation on sperminduced activation, followed by APCcdh¹ activation after second polar body extrusion.Interestingly,therefore, we propose that mammalian eggs undergo meiosis II with both APCcdc²⁰ and APCcdh¹, whereas eggs of other species so far described have APCcdc²⁰ activity only. 2011-08-30T05:50:09.796Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8417 Metabolic Cell Support (MC-S), now manufactured by Clinical Health Pty Ltd, is an over the counter immune supplement developed in Australia. Rather than using individual bioactive chemicals, a combined highly standardised multipolysaccharide enriched mix of medicinal mushrooms, the herb Astragalus mebranasis and ascorbic acid was formulated so as not to exclude important components of the original herb that may play a role in clinical outcomes. To examine the potential effect of MC-S on cancer, an in vitro proliferation study using diverse types of cancer cell lines was undertaken. Breast (MCF-7), prostate (DU145), melanoma (MM200) and colon (HT29) cancer cells were cultured under controlled standard conditions in the presence of MC-S at varying doses. After 48 and 72 hours of incubation, proliferation was measured using CellTiter 96 AQueous Non-Radioactive Cell proliferation assay kit. Results MC-S significantly inhibited the growth of breast, prostate, melanoma and bowl cancer cells, over a broad range of doses. Conclusions MC-S in this study was shown to inhibit cancer cell growth. This result, along with previous studies identifying immune stimulation properties, suggests that MC-S may offer additional benefit to standard cancer therapies. 2011-07-21T02:40:09.325Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8411 Somatic embryogenesis (SE) is induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but rarely in wild type Jemalong. The putative WUSCHEL (MtWUS), CLAVATA3 (MtCLV3) and the WUSCHEL-related homeobox gene WOX5 (MtWOX5) were investigated in M. truncatula (Mt) and identified by the similarity to Arabidopsis WUS, CLV3 and WOX5 in amino acid sequence, phylogeny and in planta and in vitro expression patterns. MtWUS was induced throughout embryogenic cultures by cytokinin after 24-48 h and maximum expression occurred after 1 week, which coincides with the induction of totipotent stem cells. During this period there was no MtCLV3 expression to suppress MtWUS. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localised to the embryo, and there was then the onset of MtCLV3 expression. This suggests that the expression of the putative MtCLV3 coincides with the WUS-CLAVATA feedback loop becoming operational. RNAi studies showed that MtWUS expression is essential for callus and somatic embryo production. Based on the presence of MtWUS promoter binding sites, MtWUS may be required for the induction of MtSERF1, postulated to have a key role in the signalling required for SE induced in 2HA. MtWOX5 expressed in auxin-induced root primordia and root meristems and appears to be involved in pluripotent stem cell induction. The evidence is discussed that the homeobox genes MtWUS and MtWOX5 are "hijacked" for stem cell induction, which is key to somatic embryo and de novo root induction. In relation to SE, a role for WUS in the signalling involved in induction is discussed. 2011-07-21T01:30:03.917Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8234 Since 1995 patients with relapsed aggressive non-Hodgkin lymphoma have been treated with high-dose chemotherapy (HDC) instead of standard dose chemotherapy (SC) because of superior survival shown in the ‘Parma study’. As HDC involves hospital admission and intensive supportive care, the cost of HDC would be predicted to be higher than for SC. The aim of this study was to calculate the incremental cost-effectiveness ratio for HDC compared with SC using Australian costs. Cost of treatment was determined on 21 patients receiving HDC with characteristics similar to the Parma study from the HDC database of the Calvary Mater Newcastle Hospital. Drug, transfusion, inpatient and outpatient attendance and additional relevant data from start of treatment for relapse and up to 100 days following HDC were obtained and costed. SC costs required modelling as all suitable patients are planned to receive HDC if possible; therefore there are no concurrent SC arms. A lifetime estimate of patient years gained by HDC versus SC was calculated from the area under survival curves (AUC) of HDC and SC. The incremental cost-effectiveness ratio was calculated according to the following formula: Incremental Cost/Incremental Benefit = (CostsHᴅᴄ-CostsSᴅᴄ)/(AUCHᴅᴄ-AUCSᴅᴄ). Costs for HDC and SC were A$37 490 and A$33 360, respectively, and the AUC0-infinity were 4.09 and 3.5 patient life years, respectively, giving an incremental cost-effectiveness ratio of A$7070 per discounted life year gained. Compared with published studies in multiple myeloma and solid organ transplant, these results support HDC as a cost-effective treatment in relapsed aggressive non-Hodgkin lymphoma. 2011-07-14T01:40:03.977Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8182 Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects the genital and ocular mucosa of humans, causing infections that can lead to pelvic inflammatory disease, infertility, and blinding trachoma. C. pneumoniae is a respiratory pathogen that is the cause of 12−15% of community-acquired pneumonia. Both chlamydial species were believed to be restricted to the epithelia of the genital, ocular, and respiratory mucosa; however, increasing evidence suggests that both these pathogens can be isolated from peripheral blood of both healthy individuals and patients with inflammatory conditions such as coronary artery disease and asthma. Chlamydia can also be isolated from brain tissues of patients with degenerative neurological disorders such as Alzheimer's disease and multiple sclerosis, and also from certain lymphomas. An increasing number of in vitro studies suggest that some chlamydial species can infect immune cells, at least at low levels. These infections may alter immune cell function in a way that promotes chlamydial persistence in the host and contributes to the progression of several chronic inflammatory diseases. In this paper, we review the evidence for the growth of Chlamydia in immune cells, particularly monocytes/macrophages and dendritic cells, and describe how infection may affect the function of these cells. 2011-07-10T23:20:20.215Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8022 The major human tyrosine hydroxylase isoforms (hTH1 and 2) differ in their ability to be phosphorylated in vitro. hTH1 is phosphorylated at Ser31 by extracellular signal-regulated kinase (ERK). This kinase is not capable of phosphorylating hTH2 at Ser35 (the residue that corresponds to Ser31 in hTH1). We have stably transfected SH-SY5Y cells with hTH1 or hTH2 to determine if hTH2 can be phosphorylated at Ser35 in situ. Forskolin increased the phosphorylation of Ser40 in hTH1 and Ser44 in hTH2. Muscarine increased the phosphorylation of both Ser19 and Ser40/44 in both hTH1 and hTH2. EGF increased the phosphorylation of Ser31 in hTH1. Phosphorylation of Ser35 in hTH2 was not detected under any of the conditions tested. Inhibition of ERK by UO126 decreased the phosphorylation of Ser31 and this lead to a 50% decrease in the basal level of phosphorylation of Ser40 in hTH1. The basal level of Ser44 phosphorylation in hTH2 was not altered by treatment with UO126. Therefore, phosphorylation of Ser31 contributes to the phosphorylation of Ser40 in hTH1 in situ; however, this effect is absent in hTH2. This represents a major difference between the two human TH isoforms, and has implications for the regulation of catecholamine synthesis in vivo. 2011-07-01T03:10:10.354Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7995 CaMKII (calcium/calmodulin-stimulated protein kinase II) is a multifunctional protein kinase that regulates normal neuronal function. CaMKII is regulated by multi-site phosphorylation, which can alter enzyme activity, and targeting to cellular microdomains through interactions with binding proteins. These proteins integrate CaMKII into multiple signalling pathways, which lead to varied functional outcomes following CaMKII phosphorylation, depending on the identity and location of the binding partner. A new phosphorylation site on CaMKII (Thr253) has been identified in vivo. Thr253 phosphorylation controls CaMKII purely by targeting, does not effect enzyme activity, and occurs in response to physiological and pathological stimuli in vivo, but only in CaMKII molecules present in specific cellular locations. This new phosphorylation site offers a potentially novel regulatory mechanism for controlling functional responses elicited by CaMKII that are restricted to specific subcellular locations and/or certain cell types, by controlling interactions with proteins that are expressed in the cell at that location. 2011-06-30T04:00:09.310Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7929 Ovarian clear cell carcinoma arising in a mucinous cystadenoma is very rare and its pathogenesis is disputed. We describe a case and comment on its significance in terms of the histogenesis of clear cell carcinoma. 2011-06-23T23:31:25.933Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7747 The mammalian ovary contains numerous immature preantral follicles that are not dependent on endocrine support, unlike the more mature hormone-dependant antral follicles. Preantral follicles can be enzymatically dissociated to yield immature oocytes that survive sub-zero preservation better as they lack a temperature-sensitive meiotic spindle. These techniques are highly applicable to gamete banking, which is an urgent requirement for Australian carnivorous marsupials as several species have rapidly declining populations and risk extinction. The present study developed protocols for the transport, dissociation, preservation and culture of granulosa cell-oocyte complexes (GOC) from the ovaries of dasyurid marsupials. High viability of GOC following enzymatic dissociation is reported and it was demonstrated that GOC are of significantly better quality following refrigerated storage for 24 h compared with storage at room temperature. Oocytes from primary follicles were not damaged by cold shock or the toxicity of vitrification media and following vitrification in liquid nitrogen 69.42 ± 2.44% of oocytes were viable. However, the surrounding granulosa cells demonstrated significant damage post-thaw. These granulosa cells proliferated during a 48-h culture period resulting in significant improvements in GOC quality. The present study is a valuable step towards cryostorage of dasyurid gametes and represents fundamentally important methods by which we can contribute to the conservation of Australia’s native predators. 2011-05-20T05:50:06.271Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7699 Incorporation of the anti-CD20 monoclonal antibody rituximab into front-line regimens to treat diffuse large B cell lymphoma (DLBCL) has resulted in improved survival. Despite this progress, however, many patients develop refractory or recurrent DLBCL and then undergo autologous hematopoietic stem cell transplantation (AuHCT). It is unclear to what extent pre-transplant exposure to rituximab affects outcomes after AuHCT Outcomes of 994 patients receiving AuHCT for DLBCL between 1996 and 2003 were analyzed according to whether rituximab was (n = 176; +R cohort) or was not (n = 818; -R cohort) administered with front-line or salvage therapy before AuHCT The +R cohort had superior progression-free survival (PFS; 50% vs 38%; P = .008) and overall survival (OS; 57% vs 45%; P = .006) at 3 years. Platelet and neutrophil engraftment were not affected by exposure to rituximab. Nonrelapse mortality (NRM) did not differ significantly between the 2 cohorts. In multivariate analysis, the +R cohort had improved PFS (relative risk of relapse/progression or death, 0.64; P <.001) and improved OS (relative risk of death, 0.74; P = .039). We conclude that pre-transplant rituximab is associated with a lower rate of progression and improved survival after AuHCT for DLBCL, with no evidence of impaired engraftment or increased NRM. 2011-05-10T01:50:22.682Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7623 The relation between intrauterine growth and risk of childhood acute lymphoblastic leukemia was investigated in an Australian population-based case-control study that included 347 cases and 762 controls aged <15 years recruited from 2003 to 2006. Information on proportion of optimal birth weight, a measure of the appropriateness of fetal growth, was collected from mothers by questionnaire. Data were analyzed by using logistic regression. Risk of acute lymphoblastic leukemia was positively associated with proportion of optimal birth weight; the odds ratio for a 1-standard-deviation increase in proportion of optimal birth weight was 1.18 (95% confidence interval: 1.04, 1.35) after adjustment for the matching variables and potential confounders. This association was also present among children who did not have a high birth weight, suggesting that accelerated growth, rather than high birth weight per se, is associated with risk of acute lymphoblastic leukemia. Similar associations between proportion of optimal birth weight and acute lymphoblastic leukemia were observed for both sexes and across age groups and leukemia subtypes. Results of this study confirm earlier findings of a positive association between rapidity of fetal growth and subsequent risk of acute lymphoblastic leukemia in childhood, and they are consistent with a role for insulin-like growth factors in the causal pathway. 2011-05-02T06:00:06.160Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7617 Past sun exposure is linked to a wide range of disease outcomes but is difficult to measure accurately. Silicone skin casts measure skin damage, but some studies show that age rather than sun exposure is the most important determinant of cast score. We examined skin damage scores from silicone casts of the back of the hand in a large adult sample (n = 534) with a broad range of past cumulative UV radiation (UVR) doses. Participants were ages 18 to 61 years and resided in one of four locations down the eastern Australian seaboard, spanning 27-43 degrees S. Data were collected by questionnaire and during a nurse-led interview and examination. Silicone casts were graded from 1 to 6, where higher score represents greater damage. Higher skin damage score was associated with lighter skin pigmentation [adjusted odds ratio (AOR), 4.51; 95% confidence interval (95% CI), 2.33-8.75], fairer natural hair color, particularly red hair (AOR, 11.31; 95% Cl, 4.08-31.36), and blue/gray eyes (AOR, 1.72; 95% CI, 1.14-2.59). Higher cumulative UVR dose, particularly before age 18 years, was associated with higher skin damage score (AOR, 2.06; 95% CI, 1.15-2.67 per 1,000 KJ/m2) as was number of sunburns, even after adjustment for cumulative UVR dose (AOR, 2.86; 95% CI, 1.50-5.43 for >10 sunburns ever compared with no sunburns ever). Silicone casts of the dorsum of the hand provide a measure of cumulative UVR dose and number of sunburns over the lifetime, which persists after adjustment for chronological age. They can be used as an objective measure of cumulative past sun exposure in epidemiologic studies, but other determinants of skin damage, such as skin pigmentation, should be concurrently evaluated. 2011-05-02T06:00:03.408Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7584 Eosinophils contribute to asthmatic airway inflammation by releasing cysteinyl leukotrienes (cysLT) and other inflammatory mediators, and bradykinin (BK) induces bronchoconstriction in asthmatic patients. The aims of this study were to investigate kinin receptor expression on eosinophils of asthmatic and healthy subjects and to assess the effects of kinin stimulation on eosinophils, which were isolated from peripheral blood of asthmatic (n=27) and healthy subjects (n=14). Kinin B₁ and B₂ receptors (B₁R and B₂R, respectively) and mRNA expression were investigated by quantitative confocal microscopy, flow cytometry, and RT-PCR. Intracellular Ca²⁺ was assessed by live-cell fluorescence confocal microscopy. Production of cysLT and eosinophil migration in response to BK and Lys-des[Arg⁹]-BK were assessed. Eosinophils expressed kinin B₁R and B₂R mRNA and proteins. Quantitative immunofluorescence analysis indicated that expression of B₁R and B₂R proteins was significantly greater in eosinophils of asthmatic patients compared with those of nonasthmatic subjects. However, kinin B₁R and B₂R mRNA expression did not differ significantly between these groups. Expression of kinin B₁R and mRNA was decreased in patients using high doses of inhaled corticosteroids and in eosinophils treated with a corticosteroid in vitro. Kinin B₁ and B₂ agonists up-regulated expression of their respective receptors but did not increase intracellular Ca²⁺ or the production of cysLT or enhance eosinophil migration significantly. Up-regulation of kinin receptor expression in eosinophils of asthmatic patients may be a consequence of inflammation, whereby enhanced release of kinin peptides has a positive-feedback effect on kinin receptor expression. Importantly, anti-inflammatory corticosteroids down-regulated the expression of the kinin B₁R. 2011-04-14T00:20:05.959Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7531 Background: Asthma is a common global health problem. Environmental exposures such as bacteria may protect against asthma development. Objective: This review aims to examine the possible protective role of pneumococcal infection and vaccination in asthma. Methods: A review of known experimental biology and human epidemiology relating to asthma and pneumococcal infection was performed. Results: Pneumococcal infection can modulate components of allergic airways disease such as airways hyperresponsiveness and airway eosinophilia. Exposure to killed pneumococcus can reproduce these effects and the mechanism may involve control by T regulatory cells. Conclusions: Pneumococcal immunoregulatory therapy is a potentially important approach to asthma management that requires further evaluation in well-designed research studies. 2011-04-07T03:00:06.652Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7354 Introduction: Chironex fleckeri is a large box jellyfish that has been labelled the ‘most venomous animal’ in the world. We have recently shown that the primary effect of C. fleckeri venom in vivo is cardiovascular collapse. This study utilised a cell-based assay to examine the effects of C. fleckeri venom on the proliferation of a rat aortic smooth muscle cell line. In addition, the ability of CSL box jellyfish antivenom and/or various potential treatment strategies to neutralise the effects of the venom was examined. Methods: A7r5 cells were cultured in media containing venom. The effect of CSL box jellyfish antivenom (5 U/mL), CSL polyvalent snake antivenom (5 U/mL), lanthanum (5 μM), MgSO4 (50 mM), verapamil (5 μM) or felodipine (5 μM) was examined. Cell viability was determined using a Cell titer 96 AQueous One Solution cell proliferation assay. Results: Incubation of A7r5 cells with serially diluted venom (2–0.004 µg/mL) caused a concentration-dependent inhibition of cell proliferation with an IC50 value of 0.056 μg/mL. This response was not affected by the absence of calcium or the presence of lanthanum in the media. Box jellyfish antivenom (5 U/mL) prevented the inhibition of cell proliferation caused by the venom. Verapamil (5 μM) had no significant effect on the inhibition. In contrast, felodipine (5 µM) or MgSO4 (50 mM) potentiated the effects of the venom and partially negated the protective effect of the antivenom. Discussion: This study displayed the ability to utilise a cell-based assay to determine the effects of C. fleckeri venom on vascular cell viability. It showed that CSL box jellyfish can neutralise the effects of the venom but only if added prior to the venom. In addition, potential adjunct therapies verapamil, felodipine and MgSO4 were found to be ineffective, with felodipine and MgSO4 potentiating the detrimental effects of the venom. 2011-03-10T21:40:23.142Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7337 Mature mammalian eggs are ovulated arrested at meiotic metaphase II. Sperm break this arrest by an oscillatory Ca²⁺ signal that is necessary and sufficient for the two immediate events of egg activation: cell cycle resumption and cortical granule release. Previous work has suggested that cell cycle resumption, but not cortical granule release, is mediated by calmodulin-dependent protein kinase II (CamKII). Here we find that mouse eggs contain detectable levels of only one CamKII isoform, gamma 3. Antisense morpholino knockdown of CamKIIγ3 during oocyte maturation produces metaphase II eggs that are insensitive to parthenogenetic activation by Ca²⁺ stimulation and insemination. The effect is specific to this morpholino, as a 5-base-mismatch morpholino is without effect, and is rescued by CamKIIγ3 or constitutively active CamKII cRNAs. Although CamKII-morpholino-treated eggs fail to exit metaphase II arrest, cortical granule exocytosis is not blocked. Therefore, CamKIIγ3 plays a necessary and sufficient role in transducing the oscillatory Ca²⁺ signal into cell cycle resumption, but not into cortical granule release. 2011-03-02T04:00:06.226Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7164 Background: Anaphylaxis is generally unanticipated and requires emergency management. Therefore, the biological mediators in human beings have been difficult to define. Objective: Our aim was to identify cytokines and chemokines whose concentrations increase during anaphylaxis in human beings and to determine how each correlates with severity. Methods: We measured the concentrations of potential mediators, including cytokines, chemokines, mast cell tryptase (MCT), and histamine, over 3 time points in 76 patients presenting to emergency departments with anaphylaxis and correlated these with a global severity scale, hypotension, and hypoxia. Results: IL-2, IL-6, IL-10, TNF receptor 1, MCT, and histamine were significantly elevated in patients with severe reactions (n = 36) compared with moderate reactions (n = 40) and healthy controls. Histamine levels peaked at emergency department arrival, whereas other mediators peaked later. IL-4, IL-5, IL-13, IFN-γ, and TNF-α were marginally elevated in severe reactions compared with healthy controls but did not correlate with reaction severity. Severe reactions tended to be either hypotensive (n = 19) or hypoxemic (n = 12). Levels of IL-6, IL-10, TNF receptor 1, MCT, and histamine correlated with hypotension. No mediator correlated with hypoxemia or other respiratory features. Conclusion: This study confirms that the concentrations of a number of cytokines are elevated in blood during anaphylaxis in human beings and that some correlate with the presence of hypotension. Others were only marginally elevated within a concentration range that available assays do not reliably detect. During respiratory reactions, mediators may be largely confined to the airways so that blood concentrations do not reflect activity. 2011-02-03T00:10:01.909Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7172 The gas pressure built up within closed cells of porous or cellular metals influences the macroscopic behaviour during deformation. Another important process affected by internal pore pressure is the roll forming of structurally porous metals. During pressing and thermo-mechanical forming, the internal pressure of the pores and the temperature can rise to high levels. In this paper, the influence of the internal gas pressure on the mechanical behaviour of porous metals under different loading conditions is numerically investigated by the finite element method. It has been found that the internal gas pressure significantly influences the initial yielding as well as the macroscopic behaviour if the pressure is clearly less than a half of the yield strength of the base material. This can be found in scientific literature as a limit for a significant influence. 2011-02-02T23:40:13.181Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6826 Automated analysis of molecular images has increasingly become an important research in computational life science. In this paper some new and efficient algorithms for recognizing and analyzing cell phases of high-content screening are presented. The conceptual frameworks are based on the morphological features of cell nuclei. The useful preprocessing includes: smooth following and linearization; extraction of morphological structural points; shape recognition based morphological structure; issue of touching cells for cell separation and reconstruction. Furthermore, the novel detecting and analyzing strategies of feed-forward and feed-back of cell phases are proposed based on gray feature, cell shape, geometrical features and difference information of corresponding neighbor frames. Experiment results tested the efficiency of the new method. 2010-11-25T01:10:07.600Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6825 Automated analysis and recognition of cell-nuclear phases using fluorescence microscopy images play an important role for high-content screening. A major task of automated imaging based high-content screening is to segment and reconstruct each cell from the touching cell images. In this paper we present new useful method for recognizing morphological structural models of touching cells, detecting segmentation points, determining the number of segmented cells in touching cell image, finding the related data of segmented cell arcs and reconstructing segmented cells. The conceptual frameworks are based on the morphological structures where a series of structural points and their morphological relationships are established. Experiment results have shown the efficient application of the new method for analysis and recognition of touching cell images of high-content screening. 2010-11-25T01:10:06.971Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6594 At fertilization the union of the sperm and egg has two important functions: it brings together their genetic components and it triggers embryonic development. It is oNen assumed that only the genetic component has an important part to play in further development. In the following review it is argued, on the basis of recent findings, that in disregarding the initial trigger for development, a rise in cytoplasmic Ca²⁺, an important factor in the control of embryonic development is being ignored. The review will concentrate on mammalian eggs due to the very characteristic Ca²⁺ changes which constitute the trigger for egg activation, and so embryonic development. Mammalian eggs show low.frequency Ca²⁺ oscillations over a period of several hours. It will be argued that a novel mechanism of Ca²⁺ release is employed by the sperm at fertilization and such Ca²⁺ changes have a role in subsequent development of the embryo in addition to the initial events of egg activation. For the purposes of this review the female gamete is defined as an 'egg' when it is at a stage that it is normally fertilized, in contrast to an 'oocyte' when it is at an immature stage of maturation and would not normally be fertilized. For all the species discussed here only in the sea urchin is the female gamete correctly termed an egg, having completed all the stages of meiosis. 2010-07-29T22:10:05.608Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6579 At fertilisation of mammalian and ascidian eggs the sperm induces a series of Ca²⁺ oscillations. These Ca²⁺ oscillations are triggered by a sperm-borne Ca²⁺-releasing factor whose identity is still unresolved. In both mammals and ascidians Ca²⁺ oscillations in eggs are associated with the period leading up to exit from meiosis and entry into the first embryonic cell cycle. Thus, in mammals Ca²⁺ oscillations continue for several hours but are complete by within 30 min in the ascidian. In mammals and ascidians Ca²⁺ oscillations stop at around the time when pronuclei form in the 1-cell embryo. There is evidence to show that cell cycle factors are important in regulating the fertilisation Ca²⁺ signal. If the formation of pronuclei is blocked either in mammals (by spindle disruption) or in ascidians (by clamping maturation promoting factor levels high) then Ca²⁺ oscillations continue indefinitely. Here, we explore the nature of the sperm Ca²⁺-releasing factor and examine the relationship between cell cycle resumption and the control of Ca²⁺ oscillations at fertilisation. 2010-07-28T05:40:05.466Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:2810 Decay-accelerating factor (DAF) mediates cellular attachment for many human picornaviruses. In most cases, viral binding to DAF is itself insufficient to permit cell infectivity, with a second, functional internalization receptor being required to facilitate this process. Previously, we postulated that the role of DAF in enterovirus cell infection is as a sequestration receptor, maintaining a reservoir of bound virus in an infectious state, awaiting interaction with functional internalization receptors. Many of these functional receptors possess the capacity to induce relatively rapid changes in capsid conformations, resulting in the formation of altered particles (A-type particles). In this report, we show that antibody-cross-linked DAF, in contrast to endogenous surface-expressed forms, can act as a functional virus receptor to mediate coxsackie A21 virus (CAV21) lytic cell infection. In contrast to the situation with ICAM-1-mediated CAV21 infection, in which high levels of A-type particles are formed, cross-linked DAF-induced CAV21 replication occurs in the absence of detectable A-particle formation. 2010-04-27T07:00:04.594Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1953 Decay-accelerating factor (DAF) functions as cell attachment receptor for a wide range of human enteroviruses. The Kuykendall prototype strain of coxsackievirus A21 (CVA21) attaches to DAF but requires interactions with intercellular cell adhesion molecule 1 (ICAM-1) to infect cells. We show here that a bioselected variant of CVA21 (CVA21-DAFv) generated by multiple passages in DAF-expressing, ICAM-1-negative rhabdomyosarcoma (RD) cells acquired the capacity to induce rapid and complete lysis of ICAM-1-deficient cells while retaining the capacity to bind ICAM-1. CVA21-DAFv binding to DAF on RD cells mediated lytic infection and was inhibited by either antibody blockade with a specific anti-DAF SCR1 monoclonal antibody (MAb) or soluble human DAF. Despite being bioselected in RD cells, CVA21-DAFv was able to lytically infect an additional ICAM-1-negative cancer cell line via DAF interactions alone. The finding that radiolabeled CVA21-DAFv virions are less readily eluted from surface-expressed DAF than are parental CVA21 virions during a competitive epitope challenge by an anti-DAF SCR1 MAb suggests that interactions between CVA21-DAFv and DAF are of higher affinity than those of the parental strain. Nucleotide sequence analysis of the capsid-coding region of the CVA21-DAFv revealed the presence of two amino acid substitutions in capsid protein VP3 (R96H and E101A), possibly conferring the enhanced DAF-binding phenotype of CVA21-DAFv. These residues are predicted to be embedded at the interface of VP1, VP2, and VP3 and are postulated to enhance the affinity of DAF interaction occurring outside the capsid canyon. Taken together, the data clearly demonstrate an enhanced DAF-using phenotype and expanded receptor utilization of CVA21-DAFv compared to the parental strain, further highlighting that capsid interactions with DAF alone facilitate rapid multicycle lytic cell infection. 2010-04-27T06:59:12.709Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1319 We describe the use of scanning electron microscopy to provide novel views of the three-dimensional morphology of the ingrowth wall in epidermal transfer cells of cotyledons of developing Vicia faba seed. Wall ingrowth deposition in these cells amplifies the surface area of plasma membrane available for transport of solutes during cotyledon development. Despite the physiological importance of such amplification, little is known about wall ingrowth morphology and deposition in transfer cells. A detailed morphological analysis of wall deposition in this study clearly established for the first time that wall ingrowths are deposited at scattered, discrete loci as papillate ingrowth projections. The new views of the ingrowth wall revealed that these projections branch and fuse laterally, and fusion occurs by fine connections to form a fenestrated sheet or layer. This sheet of wall material then provides a base for further deposition of ingrowth projections to progressively build many interconnected, fenestrated layers. Consolidation, or filling-in, of the fenestrae in these layers appears to occur from small fingerlike protrusions of wall material which extend laterally from the most recently deposited surface of the fenestrae. We propose that deposition of fenestrated layers may provide a mechanism for maintaining continuous amplification of plasma membrane surface area in the face of turnover of the plasma membrane and transporter proteins associated with it. The techniques reported in this paper will provide new opportunities to investigate wall ingrowth deposition and its regulation in transfer cells. 2010-04-27T06:55:29.068Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1437 A survey is presented of the architecture of secondary wall ingrowths in transfer cells from various taxa based on scanning electron microscopy. Wall ingrowths are a distinguishing feature of transfer cells and serve to amplify the plasma membrane surface area available for solute transport. Morphologically, two categories of ingrowths are recognized: reticulate and flange. Reticulate-type wall ingrowths are characterized by the deposition of small papillae that emerge from the underlying wall at discrete but apparently random loci, then branch and interconnect to form a complex labyrinth of variable morphology. In comparison, flange-type ingrowths are deposited as curvilinear ribs of wall material that remain in contact with the underlying wall along their length and become variously elaborate in different transfer cell types. This paper discusses the morphology of different types of wall ingrowths in relation to existing models for deposition of other secondary cell walls. 2010-04-27T06:52:37.023Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1418 Imatinib (Glivec; STI571) is an ATP-competitive kinase inhibitor of c-Abl, BCR/ABL, c-Kit, and platelet-derived growth factor receptor. Overexpression or constitutive activation of Kit by mutations have been associated with various malignancies. Mutations in the intracellular juxtamembrane region of Kit (e.g., V560G) are common in gastrointestinal stromal tumors and have been linked to poor prognosis. Mutations in the kinase domain of Kit (e.g., D816V) have been detected in mastocytosis, acute myeloid leukemia, and germ-cell tumors. To determine the sensitivity of Kit mutants to Imatinib in the same cellular background, wild-type Kit (WTKit), V560GKit and D816VKit were expressed in FDC-P1 cells. Growth of FDC(WTKit) was inhibited by Imatinib with GI₅₀ (a concentration of drug at which 50% inhibition of growth occurs) of 0.1–0.2 µM but FDC(V560GKit) were more sensitive to Imatinib with a GI₅₀ of 0.01–0.025 µM and FDC(D816VKit) were resistant to Imatinib with a GI₅₀ greater than 5 µM. The naturally occurring isoforms of c-Kit did not differ in their sensitivity to Imatinib. Immunoprecipitation and Western blot analysis indicated that 1 µM Imatinib reduced phosphorylation of WTKit and completely blocked phosphorylation of V560GKit but did not affect D816VKit phosphorylation. In signaling studies, addition of stem cell factor (SCF) induced phosphorylation of ERK and Akt by WTKit, and ERK, Akt and STAT3 by V560GKit, which were all blocked by Imatinib. Imatinib also blocked the constitutive activation of Akt and STAT3 by V560GKit but had no affect on the constitutive activation of ERK, Akt, and STAT3 by D816VKit. Overall, these findings demonstrate the increased susceptibility of the Kit juxtamembrane mutant, V560G, and the resistance of the kinase domain mutant, D816V, to Imatinib compared with WTKit. 2010-04-27T06:51:13.036Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:999 Purpose: Heterogeneous sensitivity of melanoma cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis may lead to outgrowth of TRAIL-resistant cells and limit successful treatment by TRAIL. The present study aims to better understand the biological characteristics of melanoma cells resistant to TRAIL-induced apoptosis. Experimental Design: We generated TRAIL-resistant melanoma cells by prolonged exposure to TRAIL and characterized the cells in terms of their sensitivity to killing induced by a panel of cytotoxic agents using biological and biochemical methods. Results: TRAIL-resistant melanoma cells are cross-resistant to apoptosis induced by another death ligand FasL, the DNA-damaging agent cisplatin, the histone deacetylase inhibitor suberic bishydroxamate, and the antimicrotubule Vinca alkaloid, vincristine. The apoptotic signaling seemed to be inhibited upstream of mitochondrial apoptotic events and was associated with decreased expression of multiple apoptotic mediators, including pro-caspase-8, Fas-associated death domain, Bid, Bim, p53, and the products of its proapoptotic target genes. Despite being resistant to apoptosis, TRAIL-resistant melanoma cells were more vulnerable to cisplatin-induced nonapoptotic cell death. This was characterized by lack of DNA fragmentation, delayed externalization of phosphatidylserine, caspase and p53 independence, and severe mitochondrial disruption, and was preceded by poly(ADP)ribose polymerase (PARP) activation and depletion of intracellular ATP, indicative of necrotic cell death. Inhibition of PARP activity partially converted the mode of cell death from necrosis to apoptosis. Conclusions: TRAIL-resistant melanoma cells are cross-resistant to apoptosis induced by various apoptotic stimuli but are more sensitive to nonapoptotic cell death induced by cisplatin. Exploration of chemotherapy-induced nonapoptotic cell death may provide an alternative strategy in overcoming resistance of melanoma cells to TRAIL-induced apoptosis. 2010-04-27T06:43:01.324Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1017 Cancer progression is associated with enhanced directional cell migration, both of the tumour cells invading into the stroma and stromal cells infiltrating the tumour site. In cell-based assays to study directional cell migration, phorbol esters are frequently used as a chemotactic agent. However, the molecular mechanism by which these activators of protein kinase C (PKC) result in the establishment of a polarized migratory phenotype is not known. Here we show that CD44 expression is essential for chemotaxis towards a phorbol ester gradient. In an investigation of CD44 phosphorylation kinetics in resting and stimulated cells, Ser316 was identified as a novel site of phosphorylation following activation of PKC. PKC does not phosphorylate Ser316 directly, but rather mediates the activation of downstream Ser316 kinase(s). In transfection studies, a phosphorylation-deficient Ser316 mutant was shown to act in a dominant-negative fashion to impair chemotaxis mediated by endogenous CD44 in response to a phorbol ester gradient. Importantly, this mutation had no effect on random cell motility or the ability of cells to migrate directionally towards a cocktail of chemoattractants. These studies demonstrate that CD44 functions to provide directional cues to migrating cells without affecting the motility apparatus. 2010-04-27T06:41:25.536Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1212 This study examines the safety and immunogenicity of an oral, whole-cell Pseudomonas aeruginosa vaccine administered to healthy volunteers. Thirty subjects received an oral dose of Pseudostat in two timed, measured doses with serological follow-up to 56 days postvaccination. Following vaccination, several individuals were identified as antibody responders for all three immunoglobulin (Ig) isotypes tested, specifically against whole-cell P. aeruginosa extract and outer membrane proteins F and I. The mean pooled lipopolysaccharide antigen-specific IgA showed the most significant and constant increases in titer postdose, with a similar increase in titer for whole-cell P. aeruginosa extract-specific IgA. The results demonstrated an increased phagocytic ability of the selected macrophage cell line in post vaccination sera. Furthermore a significant increase in intracellular macrophage killing of opsonized P. aeruginosa was also demonstrated (82% on day 14 postdose) in the presence of the postvaccination sera. The safety component of the study did not show any vaccine-attributable adverse effects in any of the subjects, as documented by clinical evidence, hematology, and biochemistry profiles. We conclude that Pseudostat is safe and immunogenic in humans at this dose and that further studies to determine the appropriate dosage and efficacy are needed. In our study, we have shown that the most significant and sustained responses to oral vaccination in human adult volunteers were serum IgA levels and that pooled sera collected postimmunization have an increased capacity to promote opsonophagocytotic killing of P. aeruginosa. 2010-04-27T06:39:47.570Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1219 In this study, we characterize the electrophysiological and morphological properties of spiny principal neurons in the rat lateral amygdala using whole cell recordings in acute brain slices. These neurons exhibited a range of firing properties in response to prolonged current injection. Responses varied from cells that showed full spike frequency adaptation, spiking three to five times, to those that showed no adaptation. The differences in firing patterns were largely explained by the amplitude of the afterhyperpolarization (AHP) that followed spike trains. Cells that showed full spike frequency adaptation had large amplitude slow AHPs, whereas cells that discharged tonically had slow AHPs of much smaller amplitude. During spike trains, all cells showed a similar broadening of their action potentials. Biocytin-filled neurons showed a range of pyramidal-like morphologies, differed in dendritic complexity, had spiny dendrites, and differed in the degree to which they clearly exhibited apical versus basal dendrites. Quantitative analysis revealed no association between cell morphology and firing properties. We conclude that the discharge properties of neurons in the lateral nucleus, in response to somatic current injections, are determined by the differential distribution of ionic conductances rather than through mechanisms that rely on cell morphology. 2010-04-27T06:39:07.259Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1168 RNA polymerase (RNAP) requires the interaction of various transcription elongation factors to efficiently transcribe RNA. During transcription of rRNA operons, RNAP forms highly processive antitermination complexes by interacting with NusA, NusB, NusG, NusE, and possibly several unidentified factors to increase elongation rates to around twice those observed for mRNA. In previous work we used cytological assays with Bacillus subtilis to identify the major sites of rRNA synthesis within the cell, which are called transcription foci. Using this cytological assay, in conjunction with both quantitative native polyacrylamide gel electrophoresis and Western blotting, we investigated the total protein levels and the ratios of NusB and NusG to RNAP in both antitermination and mRNA transcription complexes. We determined that the ratio of RNAP to NusG was 1:1 in both antitermination and mRNA transcription complexes, suggesting that NusG plays important regulatory roles in both complexes. A ratio of NusB to RNAP of 1:1 was calculated for antitermination complexes with just a 0.3:1 ratio in mRNA complexes, suggesting that NusB is restricted to antitermination complexes. We also investigated the cellular abundance and subcellular localization of transcription restart factor GreA. We found no evidence which suggests that GreA is involved in antitermination complex formation and that it has a cellular abundance which is around twice that of RNAP. Surprisingly, we found that the vast majority of GreA is associated with RNAP, suggesting that there is more than one binding site for GreA on RNAP. These results indicate that transcription elongation complexes are highly dynamic and are differentially segregated within the nucleoid according to their functions. 2010-04-27T06:37:50.421Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1881 The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially α3β1, α6β1, α7β1, and α6β4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of α3 and α6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation. 2010-04-27T06:35:44.314Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1880 We have previously shown that the phosphorylation of Ser19 in tyrosine hydroxylase can increase the rate of phosphorylation of Ser40 in tyrosine hydroxylase threefold in vitro. In this report we investigated the role of Ser19 on Ser40 phosphorylation in intact cells. Treatment of bovine chromaffin cells with anisomycin produced a twofold increase in Ser19 phosphorylation with no increase in Ser31 phosphorylation and only a small increase in Ser40 phosphorylation. Treatment of bovine chromaffin cells with forskolin produced a fourfold increase in Ser40 phosphorylation but no significant increase in either Ser19 or Ser31 phosphorylation. When chromaffin cells were first treated with anisomycin, the level of Ser40 phosphorylation after treatment by forskolin was 76% greater than the level of Ser40 phosphorylation in cells treated with forskolin alone. This potentiation of Ser40 phosphorylation by anisomycin could be completely blocked by the p38 MAP (mitogen-activated protein) kinase inhibitor SB 203580. The potentiation of Ser40 phosphorylation by anisomycin was not due to an increase in Ser40 kinase activity. Anisomycin treatment of chromaffin cells potentiated the forskolin-induced increase in tyrosine hydroxylase activity by 50%. This potentiation of activity was also blocked by SB 203580. These data provide the first evidence that the phosphorylation of Ser19 can potentiate the phosphorylation of Ser40 and subsequent activation of tyrosine hydroxylase in intact cells. 2010-04-27T06:35:37.963Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1511 We have previously reported that the αvβ6 integrin upregulates its own expression in a protein kinase C-dependent manner with increasing cell density. The wild-type β6 integrin subunit has also been shown to promote tumour growth in vivo and its growth-enhancing effect is regulated by both a MAP kinase binding motif on β6 and the 11 amino acid C-terminal cytoplasmic extension unique to the β6 subunit. Herein, we show that the 11 amino acid cytoplasmic extension is essential for the cell density-dependent increase in β6 expression and that the 11 amino acid tail exerts a dominant negative effect on cell density- and PKC-mediated β5 expression in αvβ6-expressing colon cancer cells. Cells that express β6 lacking the 11 amino acid tail respond to PKC simulation with increased expression of only the β5 subunit as seen for cells that lack constitutive αvβ6 expression. In contrast, loss of the ERK binding site on β6 markedly impairs cell density- and PKC-dependent expression of either β6 or β5 in the presence or absence of the 11 amino acid tail, respectively. Our findings suggest that in αvβ-expressing cells, a hierarchy of kinase signalling cascades exists and that the β6-ERK2 interaction dominates over PKC-mediated signalling pathways responsible for integrin upregulation with cell confluence. Given the dominance of the β6-ERK2 interaction over PKC-mediated expression of both β5 and β6 integrin subunits, targeting the β6-ERK2 interaction may prove useful as an anticancer strategy in colon cancer. 2010-04-27T06:29:37.742Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1517 A sperm-epididymal cell co-culture was shown to be capable of inducing the in vitro maturation of spermatozoa from a marsupial species, the tammar wallaby (Macropus eugenii). This system was able to maintain wallaby epididymal epithelial cells in vitro for more than 2 months. The system also enabled immature wallaby spermatozoa to differentiate from a T-shaped to a streamlined form, accompanied by the development of progressive motility after co-culture with epididymal cell monolayers that had been cultured for 7 days. The addition of inhibitors of actin polymerization (latrunculin A or B) to the co-culture system showed that wallaby sperm maturation was impaired by the interruption of actin organization within the immature spermatozoa. These results indicate that actin filaments play a significant role in sperm transformation during post-testicular maturation in marsupials. These observations also indicate that the marsupial co-culture system has the potential to greatly increase understanding of sperm-epididymal cell interactions and the mechanism of sperm maturation in these species. 2010-04-27T06:29:02.532Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1661 Whereas it is widely accepted that the adult cortex is capable of a remarkable degree of functional plasticity, demonstrations of accompanying structural changes have been limited. We examined the basal dendritic field morphology of dye-filled neurons in layers III and IV of the mature barrel cortex after vibrissal-deafferentation in adult rats. Eight weeks later, the tendency for these neurons to orient their dendritic arbors toward the center of their home barrel was found to be disrupted by the resultant reduced activity of thalamocortical innervation. Measures of spine density and total dendritic length were normal, indicating that the loss of dendritic bias was accompanied by growth of dendrites directed away from the barrel center. This finding suggests that in the mature cortex, the apparently static structural attributes of the normal adult cortex depend on maintenance of patterns of afferent activity; with the corollary that changes in these patterns can induce structural plasticity. 2010-04-27T06:28:03.033Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1677 Transfer cells are plant cells with secondary wall ingrowths. These cells are ubiquitous, occurring in all plant taxonomic groups and in algae and fungi. Transfer cells form from differentiated cells across developmental windows and in response to stress. They are considered to play a central role in nutrient distribution by facilitating high rates of transport at bottlenecks for apo-/symplasmic solute exchange. These properties are conferred by their unique structural features—an invaginated secondary wall ensheathed by an amplified area of plasma membrane enriched in a suite of solute transporters. Recent development of transfer cell experimental systems, combined with technologies to image the three-dimensional structure of wall ingrowths, is allowing identification of inductive and regulatory signals, discovery of sequential processes involved in their differentiation, and a search for transfer cell identity genes. A model of key events in differentiation of a transfer cell is presented to highlight areas for future investigation. 2010-04-27T06:26:04.628Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1801 Previously we reported the isolation of three sucrose transporter genes, TaSUT1A, 1B and 1D, all expressed at high levels in the developing grains of hexaploid wheat (Triticum aestivum L.), but also in a variety of other tissues [N. Aoki et al. (2002) Plant Mol Biol 50:453–462]. In order to further characterise the expression of the TaSUT1 genes in wheat plants, we have analysed TaSUT1 expression in their vegetative tissues using semi-quantitative reverse transcription–polymerase chain reaction, in situ hybridisation and immunolocalisation. The three TaSUT1 genes, which encode 98% identical SUT proteins, all appeared to be expressed at the same level in leaf blades, leaf sheaths and internodes, as well as developing grains, of hexaploid wheat. In mature leaf blades, TaSUT1 protein localised to the plasma membrane of phloem sieve elements in all classes of veins. In contrast, TaSUT1 mRNA was found to be localised to phloem companion cells. A similar localisation pattern for TaSUT1 protein was observed in veins of leaf sheaths and internodes. These results suggest that the wheat SUT1 has a transport function in enucleate sieve elements, in both veins responsible for loading photoassimilates, and in veins for axial transport. Furthermore, transport of the fluorescent dye carboxyfluorescein was used to investigate symplasmic connectivity between sieve element–companion cell complexes and non-phloem cells. Observations in source leaves indicated that sieve element–companion cell complexes of minor veins were symplasmically restricted, suggesting a role of TaSUT1 in apoplasmic phloem loading. In contrast, the dye was able to move symplasmically out of the phloem in internodes. In these circumstances TaSUT1 may also have a role in retrieving sucrose leaked to the phloem apoplasm. 2010-04-27T06:10:59.723Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1087 Expression of the growth-promoting integrin ανβ6 in colon cancer cells induces gelatinase B secretion and activation, the inhibition of which abolishes ανβ6-mediated tumour cell growth within a collagen matrix. Herein, we show that high cell density selectively enhances ανβ6 expression in a protein kinase C (PKC)-dependent manner in preference to other integrin subunits, resulting in a marked increase in gelatinase B secretion as cells reach confluence. Moreover, PKC activity increases with cell confluence, and the rise in PKC activity is much greater for ανβ6-expressing cells than for colon cancer cells which lack ανβ6. We propose a self-perpetuating system of colon cancer progression in which the integrin ανβ6 provides a means of sustaining tumour cell proliferation. In this model, ανβ6 regulates its own expression via a PKC-mediated signalling pathway as tumour cells become crowded and quiescent. The ανβ6-mediated induction of gelatinase B secretion facilitates peri-cellular matrix degradation, which helps overcome crowding and restores cell proliferation. 2010-04-27T06:06:39.483Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:146 Bacterial RNA polymerases (RNAPs) are capable of producing full-length transcripts in the absence of additional factors using in vitro assays. However, in vivo RNAP can become stalled during the elongation phase of transcription due to the presence of various sequence motifs. Subsequently, a host of elongation factors are required to modulate the activity of RNAP. NusA, the most intensively studied elongation factor, plays a role in increasing RNAP pausing and termination. Conversely, it is also important in transcription of rRNA where it functions as an anti-termination factor, helping to ensure only full-length transcripts are produced. Here we show that NusA is closely associated with RNAP within the bacterial nucleoid and that it is preferentially recruited to sites of rRNA synthesis. In vivo and in vitro analyses indicate this results in a change in stoichiometry of NusA:RNAP from 1:1 to approximately 2:1 at the subcellular sites of rRNA synthesis. A model is presented showing how the ratio of NusA:RNAP could affect the activity of the elongation complex so that it functions as an anti-terminator complex during rRNA synthesis. 2010-04-27T06:00:20.162Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:108 1. The topographic representation of the body surface in the somatosensory cortex provides an important model system for the in vivo study of neuronal plasticity, induced changes in somatotopy providing a direct measure of plasticity not available in most parts of the central nervous system. 2. Over the past two decades, animal experimentation in a number of laboratories has shown a remarkable degree of adaptability of the cortical representation following peripheral lesions and has had a widespread influence by challenging the once-accepted dogma that the brain is a structurally fixed organ. 3. Although some aspects of original stimulation will be missing, it is likely that receptors stimulated through bone conduction and compression by bone-mounted dental prostheses preserve some of the geometric and temporal relationships of original stimulation. By analogy with data obtained from the forearm representations, it would be expected that many features of the original cortical representations will be recreated. 4. There are also examples in the literature of perceptual learning without gross changes to the cortical representation (some being within a class of adaptability known as gain control) and it is likely that perceptual integration of many dental prostheses occurs within the limits of these neural adaptation mechanisms. 2010-04-27T05:58:10.323Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:322 In rats immunized systemically with tetanus toxoid the concentration of specific anti-tetanus-toxoid-specific IgG in fluid from the rete testis and cauda epididymidis were respectively 0.6% and 1.4% the concentration in blood serum. The extratesticular duct system reabsorbed 97% of the IgG and 99% of the fluid leaving the rete, but estradiol administration affected the site of reabsorption. In untreated rats, the ductuli efferentes reabsorbed 94% of the IgG and 96% of the fluid leaving the rete, whereas estradiol-treated rats reabsorbed 83% of the IgG and 86% of the fluid, and the ductus epididymidis fully compensated for these different effects of estradiol on the ductuli efferentes. The concentrations of IgG in secretions of the seminal vesicles and prostate gland were lower (0.1% and 0.3% respectively of the titers in blood serum) than in fluids from the extratesticular ducts, and were not affected by,the administration of estradiol. RT-PCR showed that Fcgrt (neonatal Fc receptor, also known as FcRn) is expressed in the reproductive ducts, where IgG is probably transported across epithelium, being particularly strong in the ductuli efferentes (where most IgG was reabsorbed) and distal caput epididymidis. it is concluded that IgG enters the rete testis and is concentrated only 2.5-fold along the extratesticular duct system, unlike spermatozoa, which are concentrated 95-fold. Further, the ductus epididymidis can recognize and compensate for changes in function of the ductuli efferentes. 2010-04-27T05:49:08.452Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:442 This study aimed to explore compliance with international recommendations on solaria use in a unregulated setting. Simulated customers visited 176 solaria operating in Australia and two face-to-face visits and one telephone contact were made for each establishment. From the survey, establishments compliant with the recommendations ranged from: 1.1 % refusing access to the customer with skin type I; 9.7 % recommending to the customer with skin type I against solaria use and up to 87.5 % assessing skin type and recommending eye protection. Few (15.9 %) were compliant with more than 10 of the 13 recommendations. Establishment type and number of sunbeds were significantly associated with compliance. This study has shown that a much higher level of compliance with recommendations, particularly those excluding higher-risk groups, is required to reduce the harm associated with use of solaria. While new legislation may be useful, other harm minimisation strategies including mandatory staff training and taxation should be considered. 2010-04-27T05:43:06.505Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:522 Whereas it is widely accepted that the adult cortex is capable of a remarkable degree of functional plasticity, demonstrations of accompanying structural changes have been limited. We examined the basal dendritic field morphology of dye-filled neurons in layers III and IV of the mature barrel cortex after vibrissal-deafferentation in adult rats. Eight weeks later, the tendency for these neurons to orient their dendritic arbors toward the center of their home barrel was found to be disrupted by the resultant reduced activity of thalamocortical innervation. Measures of spine density and total dendritic length were normal, indicating that the loss of dendritic bias was accompanied by growth of dendrites directed away from the barrel center. This finding suggests that in the mature cortex, the apparently static structural attributes of the normal adult cortex depend on maintenance of patterns of afferent activity; with the corollary that changes in these patterns can induce structural plasticity. 2010-04-27T05:38:31.832Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:19 2010-04-27T05:35:32.334Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:39 Recently, we have reported that cell wall pectins are internalized into apical meristem root cells. In cells exposed to the fungal metabolite brefeldin A, all secretory pathways were inhibited, while endocytic pathways remained intact, resulting in accumulation of internalized cell wall pectins within brefeldin A-induced compartments. Here we report that, in addition to the already published cell wall epitopes, rhamnogalacturonan I and xyloglucans also undergo large-scale internalization into dividing root cells. Interestingly, multilamellar endosomes were identified as compartments internalizing arabinan cell wall pectins reactive to the 6D7 antibody, while large vacuole-like endosomes internalized homogalacturonans reactive to the 2F4 antibody. As all endosomes belong topographically to the exocellular space, cell wall pectins deposited in these "cell wall islands", enclosed by the plasma-membrane-derived membrane, are ideally suited to act as temporary stores for rapid formation of cell wall and generation of new plasma membrane. In accordance with this notion, we report that all cell wall pectins and xyloglucans that internalize into endosomes are highly enriched within cytokinetic cell plates and accumulate within brefeldin A compartments. On the other hand, only small amounts of the pectins reactive to the JIM7 antibody, which are produced in the Golgi apparatus, localize to cell plates and they do not accumulate within brefeldin A compartments. In conclusion, meristematic root cells have developed pathways for internalization and recycling of cell wall molecules which are relevant for plant-specific cytokinesis. 2010-04-27T05:35:20.902Z ]]>