http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:12929 Human myometrium develops phasic contractions during labor. Phosphorylation of caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) has been implicated in development of these contractions, however the phospho-regulation of these proteins is yet to be examined during periods of both contraction and relaxation. We hypothesized that protein phosphorylation events are implicated in the phasic nature of myometrial contractions, and aimed to examine h-CaD and ERK 1/2 phosphorylation in myometrium snap frozen at specific stages, including; (1) prior to onset of contractions, (2) at peak contraction and (3) during relaxation. We aimed to compare h-CaD and ERK 1/2 phosphorylation in vitro against results from in vivo studies that compared not-in-labor (NIL) and laboring (L) myometrium. Comparison of NIL (n = 8) and L (n = 8) myometrium revealed a 2-fold increase in h-CaD phosphorylation (ser-789; P = 0.012) during onset of labor in vivo, and was associated with significantly up-regulated ERK2 expression (P = 0.022), however no change in ERK2 phosphorylation was observed (P = 0.475). During in vitro studies (n = 5), transition from non-contracting tissue to tissue at peak contraction was associated with increased phosphorylation of both h-CaD and ERK 1/2. Furthermore, tissue preserved at relaxation phase exhibited diminished levels of h-CaD and ERK 1/2 phosphorylation compared to tissue preserved at peak contraction, thereby producing a phasic phosphorylation profile for h-CaD and ERK 1/2. h-CaD and ERK 1/2 are phosphorylated during myometrial contractions, however their phospho-regulation is dynamic, in that h-CaD and ERK 1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively. Comparisons of NIL and L tissue are at risk of failing to detect these changes, as L samples are not necessarily preserved in the midst of an active contraction. 2013-05-21T23:24:20.350Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10953 Objectives: To determine which laboratory tests are first associated with severe envenoming after a snakebite, when (ie, how long after the bite) the test results become abnormal, and whether this can determine a safe observation period after suspected snakebite. Design, patients and setting: Prospective cohort study of 478 patients with suspected or confirmed snakebite recruited to the Australian Snakebite Project from January 2002 to April 2009, who had at least three sets of laboratory test results and at least 12 hours of observation in hospital after the bite. Severe envenoming was defined as venom-induced consumption coagulopathy (VICC), myotoxicity, neurotoxicity or thrombotic microangiopathy. Main outcome measures: International normalised ratio (INR), activated partial thromboplastin time (aPTT), creatine kinase (CK) level, and neurological examination. Results: There were 240 patients with severe envenoming, 75 with minor envenoming and 163 non-envenomed patients. Of 206 patients with VICC, 178 had an INR > 1.2 (abnormal) on admission, and the remaining 28 had an INR > 1.2 within 12 hours of the bite. Of 33 patients with myotoxicity, a combination of CK > 250 U/L and an abnormal aPTT identified all but two cases by 12 hours; one of these two was identified within 12 hours by leukocytosis. Nine cases of isolated neurotoxicity had a median time of onset after the bite of 4 hours (range, 35 min - 12 h). The combination of serial INR, aPTT and CK tests and repeated neurological examination identified 213 of 222 severe envenoming cases (96%) by 6 hours and 238 of 240 (99%) by 12 hours. Conclusion: Laboratory parameters (INR, aPTT and CK) and neurological reassessments identified nearly all severe envenoming cases within 12 hours of the bite, even in this conservative analysis that assumed normal test results if the test was not done. 2012-06-22T06:01:40.313Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10405 Imatinib, dasatinib, sunitinib, CEP-701, and PKC-412, ATP-competitive small molecule inhibitors of type III receptor tyrosine kinases c-KIT and/or FLT3, were evaluated for binding to the closely related receptor, FMS, by docking into models of inactive and active conformations of the FMS kinase domain. To confirm the docking predictions, the drugs were tested for their activity and selectivity in inhibiting cell proliferation and FMS phosphorylation upon stimulation by the FMS ligand, CSF-1. All five drugs inhibited FMS activity. Imatinib, dasatinib and CEP-701 represent three different types of interactions determining drug potency and selectivity. 2012-03-14T03:20:04.666Z ]]> 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:7798 Background: During the development of the Drosophila eye, specific cell types differentiate from an initially equipotent group of uncommitted precursor cells. The lozenge (lz) gene, which is a member of the Runt family of transcriptional regulators, plays a pivotal role in mediating this process through regulating the expression of several fate-specifying transcription factors. However, the regulation of lz, and the control of lz expression levels in different cell types is not fully understood. Results: Here, we show a genetic interaction between Tramtrack69 (Ttk69) a key transcriptional repressor and an inhibitor of neuronal fate specification, and lz, the master patterning gene of cells posterior to the morphogenetic furrow in the Drosophila eye disc. Loss of Ttk69 expression causes the development of ectopic R7 cells in the third instar eye disc, with these cells being dependent upon Lz for their development. Using the binary UAS Gal4 system, we show that overexpression of Ttk69 causes the loss of lz-dependent differentiating cells, and a down-regulation of Lz expression in the developing eye. The loss of lz-dependent cells can be rescued by overexpressing lz via a GMR-lz transgene. We provide additional data showing that factors functioning upstream of Ttk69 in eye development regulate lz in a Ttk69-dependent manner. Conclusions: Our results lead us to conclude that Ttk69 can either directly or indirectly repress lz gene expression to prevent the premature development of R7 precursor cells in the developing eye of Drosophila. We therefore define a mechanism for the tight regulatory control of the master pre-patterning gene, lz, in early Drosophila eye development and provide insight into how differential levels of lz expression can be achieved to effect specific cell fate outcomes. 2012-01-30T05:28:11.770Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6906 Research Doctorate - Doctor of Philosophy (PhD) 2012-01-30T05:16:45.366Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6565 Mammalian eggs arrest at metaphase of the second meiotic division (MetII). Sperm break this arrest by inducing a series of Ca²⁺ spikes that last for several hours. During this time cell cycle resumption is induced, sister chromatids undergo anaphase and the second polar body is extruded. This is followed by decondensation of the chromatin and the formation of pronuclei. Ca²⁺ spiking is both the necessary and solely sufficient sperm signal to induce full egg activation. How MetII arrest is established, how the Ca²⁺ spiking is induced and how the signal is transduced into cell cycle resumption are the topics of this review. Although the roles of most components of the signal transduction pathway remain to be fully investigated, here I present a model in which a sperm-specific phospholipase C (PLCζ) generates Ca²⁺ spikes to activate calmodulin-dependent protein kinase II and so switch on the Anaphase-Promoting Complex/Cyclosome (APC/C). APC/C activation leads to securin and cyclin B1 degradation and in so doing allows sister chromatids to be segregated and to decondense. 2011-08-31T00:10:02.209Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6543 Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell-cycle arrest by a long-lasting series of Ca²⁺ spikes that lead to an activation of the anaphase-promoting complex/cyclosome. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell-cycle resumption. We have used a combination of pharmacological inhibitors and constitutively active cRNA constructs of PKCα and CamKIIα microinjected into mouse eggs to show that it is CamKII and not PKC that is the sufficient trigger for cell-cycle resumption from metaphase II arrest. Constitutively active PKC constructs had no effect on the resumption of meiosis but caused an immediate and persistent elevation in intracellular Ca²⁺ when store-operated Ca²⁺ entry was stimulated. With respect to resumption of meiosis, the effects of constitutively active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced resumption of meiosis, but the Ca²⁺ chelator BAPTA did not. Furthermore direct measurement of Ca²⁺ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca²⁺. Therefore, we conclude that PKCs may play an important role in maintaining Ca²⁺ spiking at fertilization by promoting store-operated Ca²⁺ entry, while CamKII transduces cell-cycle resumption, and lies downstream of sperm-induced Ca²⁺ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell-cycle resumption through stimulation of CamKII. 2011-08-30T05:40:06.685Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8338 Screening identified two bisindolylmaleimides as 100 μM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34−2), a 1.3 ± 0.3 μM dynamin I inhibitor. Dynole 34−2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34−2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC50 15 μM; RME IC50 80 μM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells. 2011-07-19T06:00:03.759Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:3455 In both mice and humans alternate splicing results in isoforms of c-Kit characterized by the presence or the absence of a tetrapeptide sequence, GNNK, in the juxtamembrane region of the extracellular domain. Dramatic differences in the kinetics and magnitude of activation of the intrinsic tyrosine kinase activity of c-Kit between the GNNK- and GNNK+ isoforms has previously been shown. Here we report the analysis of downstream targets of receptor signaling, which revealed that the signaling was differentially regulated in the two splice forms. The kinetics of phosphorylation of Shc, previously demonstrated to be phosphorylated by Src downstream of c-Kit, was stronger and more rapid in the GNNK- form, whereas it showed slower kinetics in the GNNK+ form. Inhibition of Src family kinases with the specific Src family kinase inhibitor SU6656 altered the kinetics of activation of the GNNK- form of c-Kit so that it resembled that of the GNNK+ form. In cells expressing the GNNK- form, SCF was rapidly degraded, whereas in cells expressing the GNNK+ form only showed a very slow rate of degradation of SCF. In the GNNK+ form the Src inhibitor SU6656 only had a weak effect on degradation, whereas in the GNNK- form it dramatically inhibited degradation. In summary, the two splice forms show, despite only a four-amino acid sequence difference, remarkable differences in their signaling capabilities. 2011-05-03T00:10:03.531Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6595 At fertilization, the spermatozoon is generally held to generate two important second messengers, inositol trisphosphate and diacylglycerol. A similar situation arises when these signalling molecules are generated after a hormone binds to its plasma membrane receptor. This signalling mechanism releases intracellular Ca²⁺ which causes cortical granule release and initiates meiotic resumption. This review will examine the role played at fertilization by protein kinase C which is a primary target of diacylglycerol. The pharmacological agents phorbol esters, which mimic the action of diacylglycerol, when added to mammalian oocytes induce cortical granule release and may cause meiotic resumption. However, the originally accepted mechanism of fertilization is now questioned with the recent finding of a soluble sperm Ca²⁺-releasing factor expelled directly into the oocyte cytoplasm, bypassing any membrane receptor. Therefore, it is timely to re-evaluate the role played by protein kinase C at fertilization in light of a mechanism that may produce Ca²⁺ without producing diacylglycerol concomitantly. This article will examine the evidence implicating activation of protein kinase C in Ca²⁺ oscillations, cortical granule release and meiotic resumption. It will contend that pharmacological studies relying on the specificity of phorbol esters and other agonists, as well as inhibitors of protein kinase C, have produced conflicting interpretations of the role of this kinase at fertilization. 2010-07-29T22:20:12.296Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:6586 rtPCR and Western blotting were used to determine which members of the PKC family are present in both immature and mature mouse oocytes. Using isoform-specific PCR primers and antibodies PKC-δ and -λ were detected while such techniques failed to observe the conventional isoforms of PKC-α, -β, -γ. This isoform profile was confirmed using an alternative PCR strategy, which allowed discrimination of PCR products derived from conventional and novel PKC isoforms. In addition PKC-ε, -η, -θ and -ζ were not detected by rtPCR. These results suggest that the predominant isoforms in oocytes are PKC-δ and -λ. 2010-07-29T02:10:05.054Z ]]> 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:1483 The receptor tyrosine kinase, KIT, displays activating mutations in the kinase domain, which are associated with various cancers. We have used homology modelling based on the crystal structures of the insulin receptor kinase in active and inactive conformations to predict the corresponding structures of the KIT kinase domain. We have prepared four KIT models, one each for the active and inactive conformations of the wild-type and of the Asp816Val mutant proteins. We have also placed ATP into the active conformations and the inhibitor, STI571, into the inactive conformations. All models have been fully energy minimised. The molecular modelling studies described here explain (i) why Asp816Val KIT is constitutively active, (ii) why the nature of the substituting amino acid at residue 816 is relatively unimportant, and (iii) why the Asp816Val substitution confers resistance to the KIT-inhibitory drug STI571. The models will be valuable for predicting other kinase inhibitory drugs that may be active on wild-type and mutant forms of KIT. During the course of this work, a crystal structure of the active conformation of the KIT kinase domain has been published. Our model of the active conformation of the Asp816Val mutant is strikingly similar to this crystal structure, whereas our model of the active conformation of the wild-type kinase domain of KIT differs from the crystal structure in some respects. The reasons for this apparent discrepancy are discussed. 2010-04-27T06:50:27.522Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:3007 The objectives of this study were to map the ontogeny of tyrosine phosphorylation signal transduction pathways during germ cell development and to determine their association with the differentiation of a functional gamete. Until testicular germ cells differentiate into spermatozoa, cAMP-induced tyrosine phosphorylation is not detectable. Entry of these cells into the epididymis is accompanied by sudden activation of the tyrosine phosphorylation pathway, initially in the principal piece of the cell and subsequently in the midpiece. In the caput and corpus epididymides, the potential to express this pathway is inhibited by the presence of calcium in the extracellular medium. However, calcium has no effect on the expression of this pathway in caudal epididymal sperm. The competence of these cells to phosphorylate the entire sperm tail, from the neck to the tail-end piece, is accompanied by a capacity to exhibit hyperactivated motility on stimulation with cAMP. A distinctly different pattern of tyrosine phosphorylation, involving the acrosomal domain of the sperm head, is invoked as spermatozoa enter the caput epididymis, and phosphorylation remains high until these cells enter the distal corpus and cauda. The proportion of cells exhibiting this form of tyrosine phosphorylation is not affected by extracellular calcium or cAMP but is negatively correlated (R² = 0.99) with their ability to acrosome-react. However, this relationship is not causative. Our findings indicate that the development of functional spermatozoa is accompanied by carefully orchestrated changes in tyrosine phosphorylation, controlled by independent regulatory mechanisms in distinct subcellular compartments of these highly specialized cells. 2010-04-27T06:47:09.439Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:2957 Calcium/calmodulin-dependent protein kinase II (CaMPK-II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn²⁺ has multiple functional effects on CaMPK-II. Zn²⁺ generated a Ca²⁺/CaM-independent activity that correlated with the autophosphorylation of Thr²⁸⁶, inhibited Ca²⁺/CaM binding that correlated with the autophosphorylation of Thr³⁰⁶, and inhibited CaMPK-II activity at high concentrations that correlated with the autophosphorylation of Ser²⁷⁹. The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn²⁺ binding, generated an increased mobility form of CaMPK-II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn²⁺ converts CaMPK-II into a different form than the binding of Ca2+/CaM. In certain nerve terminals, where Zn²⁺ has been shown to play a neuromodulatory role and is present in high concentrations, Zn²⁺ may turn CaMPK-II into a form that would be unable to respond to calcium signals. 2010-04-27T06:46:10.060Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1182 Medicago truncatula, a model for legume genomics, can be regenerated by somatic embryogensis by the use of a suitable genotype and an auxin plus cytokinin. The stress response induced by explant wounding and culture is increasingly recognized as an important component of somatic embryo induction. We have cloned and investigated the stress kinase gene MtSK1 in relation to somatic embryogenesis in M. truncatula, using the highly embryogenic mutant Jemalong 2HA (2HA) and its progenitor Jemalong. The main features of the MtSK1 protein of 351 amino acids are an N-terminal kinase domain and a C-terminal glutamic acid-rich region, which is predicted to be a coiled-coil. MtSK1 is a member of the SnRK2 subgroup of the SnRK group of plant kinases. Members of the SnRK2 kinases play a role in stress responses of plants. MtSKI expression is induced by wounding in the cultured tissue independent of auxin or cytokinin. However, in both 2HA and Jemalong, as the callus develops in response to auxin plus cytokinin, MtSK1 expression continues to increase. MtSK1 responds to salt stress in vivo, consistent with its role as a stress kinase. The likely role of MtSK1 in stress-induced signaling will facilitate the relating of stress–response pathways to auxin and cytokinin-induced signaling in the understanding of the molecular mechanisms involved in the induction of somatic embryogenesis in M. truncatula. 2010-04-27T06:38:09.151Z ]]> 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:1675 We have cloned a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) gene from Medicago truncatula (MtSERK1) and examined its expression in culture using real time PCR. In the presence of the auxin 1-naphthaleneacetic acid (NAA) alone, root differentiation occurs from the proliferating calli in both the cultured highly embryogenic seed line (2HA) and a low to nonembryogenic seed line (M. truncatula cv Jemalong). Auxin stimulated MtSERK1 expression in both 2HA and M. truncatula cv Jemalong. Embryo induction in proliferating calli requires a cytokinin in M. truncatula and unlike root formation is substantively induced in 2HA, not M. truncatula cv Jemalong. On embryo induction medium containing NAA and the cytokinin 6-benzylaminopurine (BAP), expression of MtSERK1 is elevated within 2 d of initiation of culture in both M. truncatula cv Jemalong and 2HA. However, MtSERK1 expression is much higher when both NAA and BAP are in the medium. BAP potentiates the NAA induction because MtSERK1 expression is not up-regulated by BAP alone. The 2HA genotype is able to increase its embryo formation because of the way it responds to cytokinin, but not because of the cytokinin effect on MtSERK1. Although the studies with M. truncatula indicate that somatic embryogenesis is associated with high SERK expression, auxin alone does not induce somatic embryogenesis as in carrot (Daucus carota) and Arabidopsis. Auxin in M. truncatula induces roots, and there is a clear up-regulation of MtSERK1. Although our analyses suggest that MtSERK1 is orthologous to AtSERK1, which in Arabidopsis is involved in somatic embryogenesis, in legumes, MtSERK1 may have a broader role in morphogenesis in cultured tissue rather than being specific to somatic embryogenesis. 2010-04-27T06:26:06.767Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1051 CD36 is a transmembrane glycoprotein receptor that engages in signal transduction implicated in important physiological and pathophysiological events. CD36 in platelets has been shown physically and functionally to associate with members of the Src family of protein tyrosine kinases, Fyn, Lyn, and Yes, but the nature of this important association has never been rigorously examined. Here, we show that CD36 does not associate with Lyn through a protein-mediated interaction. In COS cells transfected with both CD36 and Lyn these molecules did not co-precipitate, suggesting a requirement for an intermediary molecule absent from the COS cells. Yeast two-hybrid analysis confirmed that the carboxylterminal cytoplasmic tail of CD36 did not bind Lyn directly, and no Lyn binding protein bound to CD36 in a cDNA library screen. Conversely, when the CD36–Lyn association seen in platelets was analysed by biophysical parameters, dissociation occurred at 37 °C and also by solubilisation in octylglucoside, indicative of a lipid-mediated association. Since both CD36 and Lyn are enriched in Triton X-100-insoluble rafts at the plasma membrane, these findings point to the importance of raft-associated lipids in CD36-mediated signal transduction. 2010-04-27T06:08:04.762Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1080 Measurement of the proportion of calcium/calmodulin-stimulated protein kinase II (CaMPK-II) that is autonomously active or phosphorylated on Thr286 is thought to provide an index of the degree to which CaMPK-II in a tissue has been activated. We have examined how various ways of handling hippocampal tissue can alter these properties. Both autonomous activity and phospho-Thr²⁸⁶ content was high in freshly dissected hippocampus or freshly cut hippocampal slices. After incubation of hippocampal slices in artificial cerebrospinal fluid for 120 min, both properties of CaMPK-II decreased to a steady state level. Freeze–thaw or cutting the equilibrated slices could rapidly increase both autonomous activity and phospho-Thr²⁸⁶ immunoreactivity of CaMPK-II. These increases were comparable to changes induced by experimental treatment. Therefore, our results suggest that considerable care needs to be taken over the way in which hippocampal slices are handled. 2010-04-27T06:06:37.639Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:1093 C1 2010-04-27T06:06:07.244Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:2784 Small molecule protein kinase inhibitors show great promise as anti-cancer agents, however, de novo and acquired resistance present problems. These are reviewed and illustrated using the receptor tyrosine kinase, KIT, as an example. Emerging solutions are presented, such as targeting active kinase conformations. 2010-04-27T06:04:22.036Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:121 We studied pathways involved in the proliferation of rat C6 glioma cells induced by lysophosphatidic acid (LPA), a phospholipid with diverse biological functions. LPA induced a dose-responsive proliferation of C6 cells after 48 h. Proliferation was blocked by inhibitors of the sodium/proton exchanger type 1 (NHE1), Rho-associated kinase, the phosphatidylinositol 3-kinase/Akt pathway (PI3K/Akt), protein kinase C (PKC) and extracellular signal regulated kinase kinase (MEK). Phospho-specific antibodies were used to investigate the pathways involved. LPA induced transient (10 min) phosphorylations of ERK 1/2, Akt and the transcription factor CREB. The LPA-induced phosphorylation of ERK 1/2 and CREB was blocked by inhibition of PI3K, PKC and MEK, but that of Akt was only inhibited by wortmannin, the PI3K inhibitor. Inhibition of Rho kinase or NHE1 did not reduce the LPA-induced phosphorylation of ERK, Akt or CREB. The results were compared with the effects of LPA on transduction pathways in other cell types. 2010-04-27T05:58:22.855Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:233 The Medicago truncatula line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than wild-type Jemalong. We have compared proteomes of tissue cultures from leaf explants of these two lines. Both 2HA and Jemalong explants were grown on media containing the auxin 1-naphthaleneacetic acid and the cytokinin 6-benzylaminopurine. Proteins were extracted from the cultures at different time points (2, 5, and 8 weeks), separated by two-dimensional gel electrophoresis, and detected by silver staining. More than 2,000 proteins could be reproducibly resolved and detected on each gel. Statistical analysis showed that 54 protein spots were significantly (P < 0.05) changed in expression (accumulation) during the 8 weeks of culture, and most of these spots were extracted from colloidal Coomassie-stained two-dimensional gel electrophoresis gels and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry analysis. Using a publicly available expressed sequence tag database and the Mascot search engine, we were able to identify 16 differentially expressed proteins. More than 60% of the differentially expressed protein spots had very different patterns of gene expression between 2HA and Jernalong during the 8 weeks of culture. 2010-04-27T05:56:21.167Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:234 The combination of semi-automation, an elegant synthesis, and parallel solution-phase synthesis approaches has allowed the development of five targeted, symmetrical tyrphostin compound libraries. These libraries on average are comprised of 12 compounds. Notwithstanding this, low micromolar potent growth inhibitors against HT29 ( colorectal carcinoma) and G401 (renal carcinoma) cell lines were discovered. Additionally, significant SAR data was obtained. We noted that the most potent growth inhibitory activity was consistently observed for those analogues that possessed a 2-chlorophenyl (for 10: GI(50 HT29) 5.5 +/- 0.4 muM, GI(50 G401) 2.6 +/- 0.4 muM; for 23: GI(50 HT29) 2.4 +/- 0.2 muM, GI(50 G401) 1.9 +/- 1 muM; for 34: GI(50 HT29) 8.8 +/- 3.1 muM, GI(50 G401) 6.2 +/- 2.9 muM; for 46: GI(50 HT29) 5.2 +/- 0.9 muM, GI(50 G401) 3.7 +/- 0.6 muM; for 57: GI(50 HT29) 4.6 +/- 0.8 muM, GI(50 G401) 2.1 +/- 0.2 muM), a 3-chlorophenyl (for 11: GI(50 HT29) 3.8 +/- 0.7 muM, GI(50 G401) 1.7 +/- 0.7 muM; for 48: GI(50 HT29) 5.9 +/- 0.1 muM, GI(50 G401) 3.4 +/- 0.6 muM; for 58: GI(50 HT29) 4.8 +/- 0.9 muM, GI(50 G401) 3.4 +/- 0.2 muM), or a 3-methoxyphenyl substituent ( for 13: GI(50 HT29) 7.4 +/- 3.8 muM, GI(50 G401) 2.8 +/- 0.5 muM; for 26: GI(50 HT29) 4.5 +/- 0.5 muM, GI(50 G401) 4.9 +/- 1 muM; for 37: GI(50 HT29) 3.7 +/- 0.2 muM, GI(50 G401) 1.6 +/- 0.2 muM; for 49: GI(50 HT29) 3.7 +/- 0.4 muM, GI(50 G401) 3.4 +/- 0.2 muM; for 60: GI(50 HT29) 4.1 +/- 0.6 muM, GI(50 G401) 1.8 +/- 0.3 muM). Finally, we noted that increasing the distance between the terminal aromatic rings had only a minimal effect on the 2-, 3-chlorophenyl, and 3-methoxyphenyl analogues, but did have a favourable effect on OH, COOH, and multiply substituted analogues. 2010-04-27T05:56:09.327Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7 Difference in two-dimensional (2-D) gel electrophoresis (DIGE) is a novel method for analyzing up to three samples in one 2-D gel and using the information gained to study post-translational modifications of proteins. We describe the use of DIGE to isolate and characterize those proteins that undergo processing in spermatozoa as they transit the epididymal tract. We find up to 60 protein spots are significantly modified as sperm traverse the epididymis. In this article, we report eight unambiguous protein identifications and demonstrate that one protein, the beta-subunit of the mitochondrial F1-ATPase, is serine-phosphorylated as sperm undergo epididymal maturation. We suggest that phosphorylation of this particular protein in a cAMP-dependent manner may contribute to the mechanisms by which motility is conferred upon spermatozoa. 2010-04-27T05:34:19.428Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:4738 A commonly used strategy by microorganisms to survive multiple stresses involves a signal transduction cascade that increases the expression of stress-responsive genes. Stress signals can be integrated by a multiprotein signaling hub that responds to various signals to effect a single outcome. We obtained a medium- resolution cryo- electron microscopy reconstruction of the 1.8- megadalton "stressosome" from Bacillus subtilis. Fitting known crystal structures of components into this reconstruction gave a pseudoatomic structure, which had a virus capsid-like core with sensory extensions. We suggest that the different sensory extensions respond to different signals, whereas the conserved domains in the core integrate the varied signals. The architecture of the stressosome provides the potential for cooperativity, suggesting that the response could be tuned dependent on the magnitude of chemophysical insult. 2010-04-27T05:30:56.370Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:4588 The myometrium undergoes substantial remodeling at the time of labor including rearrangement of the cellular contractile machinery. The regulation of this process in human myometrium at the time of labor is poorly defined, but evidence in other muscle types suggests modulation by small heat shock proteins (sHSP). The aim of this study was to investigate whether similar changes in sHSP occur in the myometrium at labor. Using a quantitative proteomic approach (two-dimensional difference gel electrophoresis), we found a 69% decrease in the sHSP αB-crystallin in the myometrium at labor plus multiple isoforms of HSP27. Immunoblotting using phosphospecific HSP27 antibodies (HSP27-serine15, -78, and -82) detected marked changes in HSP27 phosphorylation at labor. Although total HSP27 levels were unchanged, HSP27-Ser15 was 3-fold higher at labor. Coimmunoprecipitation studies showed that HSP27 coprecipitates with αB-crystallin and also smooth muscle α-actin. Coimmunofluorescence studies demonstrated a relocation of HSP27 from the perinuclear region to the actin cytoskeleton at labor. The functional significance of these changes was demonstrated in vitro where myometrial strips stimulated to contract with oxytocin exhibited increased HSP27-Ser15 phosphorylation. Our findings provide data consistent with a novel pathway regulating human myometrial contraction at labor and identify HSP27 and αB-crystallin as potential targets for future tocolytic design. 2010-04-27T04:58:27.324Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5683 The molecular changes involved in the induction of apoptosis by vincristine in melanoma have not yet been well defined. Two human melanoma cell lines showing moderate (Mel-RM) and high (IgR3) sensitivity to vincristine were selected from a panel of eight melanoma lines for analysis. Induction of apoptosis was caspase dependent, and was associated with increases in mitochondrial membrane permeability. Vincristine upregulated the expression of Bax, Bak, PUMA, Noxa, p53 and p21 proteins, and downregulated and/or phosphorylated the Bcl-2 protein. Inhibitors of the Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase, significantly inhibited vincristine-induced apoptosis in both IgR3 and Mel-RM cells. In addition, vincristine induced phosphorylation and reduction in Bcl-2 was prevented by an inhibitor of JNK. Downregulation of mRNA for p53, PUMA or Bim by RNA interference had little or no influence on vincristine-induced apoptosis in IgR3 cells. In addition, silencing Bim mRNA did not affect vincristine-induced apoptosis in Mel-RM cells. These results suggest that vincristine-induced apoptosis of at least some melanoma cell lines is dependent on the activation of JNK. The results are consistent with the phosphorylation of Bcl-2 protein, resulting in the activation of Bax/Bak, release of cytochrome c from the mitochondria and the resulting activation of caspases. 2010-04-27T04:50:01.406Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5428 Integrin αυβ6 plays a very important role in the progression of colon cancer cells and is now defined as a novel, independent prognostic indicator for aggressive colon cancer in humans. Herein, we use the RNA interfering technology to downregulate the expression of αυβ6 in colon cancer cells. Our data demonstrate that plasmid vector based shRNA can effectively down-regulate αυβ6 expression in protein and mRNA levels. Supression of integrin αυβ6 inhibits the phosphorylation and nonphosphorylation level of ERK1/2, the secretion of uPA, pro-MMP-9 and pro-MMP-2 in tumor conditioned medium, and more important, inhibits MAPKdependent [³H] labeled collagen IV degradation via the plasminogen activation cascade. Our study demonstrates in vitro that supression of integrin αυβ6 inhibits extracellular matrix degradation through the MAPK pathway. 2010-04-27T04:48:41.233Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5540 Ca²⁺ -stimulated protein kinase II (CaMKII) is critically involved in the regulation of synaptic function and is implicated in the neuropathology associated with ischemia and status epilepticus (SE). The activity and localization of CaMKII is regulated by multi-site phosphorylation. In the present study we investigated the effects of global ischemia followed by reperfusion and of SE on the phosphorylation of CaMKII on T253 in rat forebrains and compared this to the phosphorylation of T286. Both ischemia and SE resulted in marked increases in the phosphorylation of T253, and this was particularly marked in the postsynaptic density (PSD). Phosphorylation of T286 decreased rapidly towards basal levels following ischemia whereas phosphorylation of T253 remained elevated for between 1 and 6 h before decreasing to control values. Following SE, phosphorylation of T253 remained elevated for between 1 and 3 h before decreasing to control levels. In contrast, phosphorylation of T286 remained elevated for at least 24 h following the termination of SE. Total CaMKII associated with PSDs transiently increased 10 min following ischemia, but only several hours following SE. The results demonstrate that phoshorylation of CaMKII on T253 is enhanced following both ischemia/reperfusion and SE and indicate that the phosphorylation of T253 and T286 are differentially regulated. 2010-04-27T04:46:15.626Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5158 Because brain maturation in chickens is protracted and occurs well after the major developmental period of synaptogenesis, chicken forebrain is suitable to investigate whether the molecular mechanisms underlying memory consolidation are different in immature and mature animals. We have used antibodies and western blotting to analyze subcellular fractions from the intermediate medial mesopallium region of 14-day and 8-week chicken forebrain prepared 0, 45, and 120 min after learning a discriminative taste avoidance task. At both ages learning induced changes in the phosphorylation of the glutamate receptor subunit 1 at Ser831, the levels of calcium-calmodulin stimulated/dependent protein kinase II and the phosphorylation of calcium-calmodulin stimulated/dependent protein kinase II at Thr286 were observed only in the fraction enriched in post-synaptic densities. The changes were of the same type at the two ages but occurred faster in mature animals. The changes in extracellular signal regulated kinase and phosphorylated-extracellular signal regulated kinase were more complex with different subcellular fractions showing different patterns of change at the two ages. These results imply that the molecular changes induced by learning a behavioral task are faster in mature than immature brain and may involve a different balance of intracellular signaling pathways. 2010-04-27T04:44:38.899Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5128 Data from several studies have suggested that polymorphisms in A-kinase anchoring proteins (AKAPs), which are key components of signal transduction, contribute to carcinogenesis. To evaluate the impact of AKAP variants on breast cancer risk, we genotyped six nonsynonymous single-nucleotide polymorphisms that were predicted to be deleterious and found two (M463I, 1389G>T and N2792S, 8375A>G) to be associated with an allele dose–dependent increase in risk of familial breast cancer in a German population. We extended the analysis of AKAP9 M463I, which is in strong linkage disequilibrium with AKAP9 N2792S, to 9523 breast cancer patients and 13 770 healthy control subjects from seven independent European and Australian breast cancer studies. All statistical tests were two-sided. The collaborative analysis confirmed the association of M463I with increased breast cancer risk. Among all breast cancer patients, the combined adjusted odds ratio (OR) of breast cancer for individuals homozygous for the rare allele TT (frequency = 0.19) compared with GG homozygotes was 1.17 (95% confidence interval [CI] = 1.08 to 1.27, P = .0003), and the OR for TT homozygotes plus GT heterozygotes compared with GG homozygotes was 1.10 (95% CI = 1.04 to 1.17, P = .001). Among the combined subset of 2795 familial breast cancer patients, the respective ORs were 1.27 (95% CI = 1.12 to 1.45, P = .0003) and 1.16 (95% CI = 1.06 to 1.27, P = .001). 2010-04-27T04:41:59.677Z ]]>