Tumor interactions with soluble factors and the nervous system

Members of Shc (src homology and collagen homology) family, p46shc, p52shc, p66shc have known to be related to cell proliferation and carcinogenesis. Whereas p46shc and p52shc drive the reaction forward, the role of p66shc in cancers remains to be understood clearly. Hence, their expression in cancers needs to be evaluated carefully so that Shc analysis may provide prognostic information in the development of carcinogenesis. In the present study, the expression of p66shc and its associate targets namely Eps8 (epidermal pathway substrate 8), Rac1 (ras-related C3 botulinum toxin substrate1) and Grb2 (growth factor receptor bound protein 2) were examined in fresh tissue specimens from patients with esophageal squamous cell carcinoma and esophageal adenocarcinoma using western blot analysis. A thorough analysis of both esophageal squamous cell carcinoma and adenocarcinoma showed p66shc expression to be significantly higher in both types of carcinomas as compared to the controls. The controls of adenocarcinoma show a higher basal expression level of p66shc as compared to the controls of squamous cell carcinoma. The expression level of downstream targets of p66shc i.e., eps8 and rac1 was also found to be consistently higher in human esophageal carcinomas, and hence correlated positively with p66shc expression. However the expression of grb2 was found to be equal in both esophageal squamous cell carcinoma and adenocarcinoma. The above results suggest that the pathway operated by p66shc in cancers does not involve the participation of Ras and Grb2 as downstream targets instead it operates the pathway involving Eps8 and Rac1 proteins. From the results it is also suggestive that p66shc may have a role in the regulation of esophageal carcinomas and represents a possible mechanism of signaling for the development of squamous cell carcinoma and adenocarcinoma of esophagus.     

JNK Signalling Controls Remodelling of the Segment Boundary through Cell Reprogramming during Drosophila Morphogenesis

Segments are fundamental units in animal development which are made of distinct cell lineages separated by boundaries. Although boundaries show limited plasticity during their formation for sharpening, cell lineages make compartments that become tightly restricted as development goes on. Here, we characterize a unique case of breaking of the segment boundary in late drosophila embryos. During dorsal closure, specific cells from anterior compartments cross the segment boundary and enter the adjacent posterior compartments. This cell mixing behaviour is driven by an anterior-to-posterior reprogramming mechanism involving de novo expression of the homeodomain protein Engrailed. Mixing is accompanied by stereotyped local cell intercalation, converting the segment boundary into a relaxation compartment important for tension-release during morphogenesis. This process of lineage switching and cell remodelling is controlled by JNK signalling. Our results reveal plasticity of segment boundaries during late morphogenesis and a role for JNK-dependent developmental reprogramming in this process.     

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.     

Ligand Depletion in vivo Modulates the Dynamic Range and Cooperativity of Signal Transduction

Biological signal transduction commonly involves cooperative interactions in the binding of ligands to their receptors. In many cases, ligand concentrations in vivo are close to the value of the dissociation constant of their receptors, resulting in the phenomenon of ligand depletion. Using examples based on rotational bias of bacterial flagellar motors and calcium binding to mammalian calmodulin, we show that ligand depletion diminishes cooperativity and broadens the dynamic range of sensitivity to the signaling ligand. As a result, the same signal transducer responds to different ranges of signal with various degrees of cooperativity according to its effective cellular concentration. Hence, results from in vitro dose-response analyses cannot be applied directly to understand signaling in vivo. Moreover, the receptor concentration is revealed to be a key element in controlling signal transduction and we propose that its modulation constitutes a new way of controlling sensitivity to signals. In addition, through an analysis of the allosteric enzyme aspartate transcarbamylase, we demonstrate that the classical Hill coefficient is not appropriate for characterizing the change in conformational state upon ligand binding to an oligomeric protein (equivalent to a dose-response curve), because it ignores the cooperativity of the conformational change for the corresponding equivalent monomers, which are generally characterized by a Hill coefficient . Therefore, we propose a new index of cooperativity based on the comparison of the properties of oligomers and their equivalent monomers.     

An integrated physical, genetic and cytogenetic map of Brachypodium distachyon, a model system for grass research

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent validation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation.     

β2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.     

Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger

A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.     

Improved assay for R(-)-apomorphine with application to clinical pharmacokinetic studies in Parkinson's disease

A high performance liquid chromatographic assay for the quantitative determination of apomorphine in human plasma is described. Sample clean-up and concentration was optimised using solid-phase extraction on C18 cartridges, enabling rapid and sensitive determination of apomorphine and potential metabolites. The limit of apomorphine quantification, using fluorescence detection, was 0.5 ng/mL. The assay was stability-indicating, and allowed the detection of analytes in the presence of commonly co-administered anti-Parkinsonian drugs. Apomorphine was stable in frozen plasma containing 0.14% (w/v) ascorbic acid for 98 days, and through four freeze-thaw cycles. The assay has been used in clinical pharmacokinetic studies of apomorphine in patients with Parkinson's disease, and in preliminary studies of novel apomorphine delivery devices in volunteers.     

The Effects of Removing the GAT Domain from E. coli GMP Synthetase

E. coli GMP synthetase (GMPS) catalyzes the conversion of XMP to GMP. Ammonia, generated in the amino-terminal glutamine amidotransferase (GAT) domain, is transferred by an unknown mechanism to the ATP-pyrophosphatase (ATPP) domain, where it attacks a highly reactive adenyl-XMP intermediate, leading to GMP formation. To study the structural requirements for the activity of E. coli GMPS, we used PCR to generate a protein expression construct that contains the ATPP domain as well as the predicted dimerization domain (DD). The ATPP/DD protein is active in solution, utilizing NH₄⁺ as an NH₃ donor. Size-exclusion chromatography demonstrates a dimeric mass for the ATPP/ DD protein, providing the first evidence in solution for the structural organization of the intact GMPS. Kinetic characterization of the ATPP/DD domain protein provides evidence that the presence of the GAT domain can regulate the activity of the ATPP domain.