A mutation in the STN1 gene triggers an alternative lengthening of telomere-like runaway recombinational telomere elongation and rapid deletion in yeast

Some human cancer cells achieve immortalization by using a recombinational mechanism termed ALT (alternative lengthening of telomeres). A characteristic feature of ALT cells is the presence of extremely long and heterogeneous telomeres. The molecular mechanism triggering and maintaining this pathway is currently unknown. In Kluyveromyces lactis, we have identified a novel allele of the STN1 gene that produces a runaway ALT-like telomeric phenotype by recombination despite the presence of an active telomerase pathway. Additionally, stn1-M1 cells are synthetically lethal in combination with rad52 and display chronic growth and telomere capping defects including extensive 3' single-stranded telomere DNA and highly elevated subtelomere gene conversion. Strikingly, stn1-M1 cells undergo a very high rate of telomere rapid deletion (TRD) upon reintroduction of STN1. Our results suggest that the protein encoded by STN1, which protects the terminal 3' telomere DNA, can regulate both ALT and TRD.     

PorA protein of Campylobacter jejuni is not a cytotoxin mediating inflammatory diarrhoea

Campylobacter jejuni is a major food-borne pathogen and a leading cause of diarrhoea. A cytotoxin is most likely involved in the pathogenesis of inflammatory diarrhoea due to C. jejuni. A 45-kDa outer membrane protein encoded by the porA gene was reported to exhibit cytotoxic activity for cultured mammalian cells in vitro. We cloned and expressed the porA gene in Escherichia coli BL21 codon plus RIL strain using the fusion vector pGEX-4T-1. The fusion protein solubilised in urea in denatured form or solubilised in Empigen BB in native form or their thrombin-cleaved products did not exhibit cytotoxic activity for Chinese hamster ovary (CHO) cells. The urea-solubilised fusion protein did not induce fluid accumulation in the rabbit ileal loop assay. All 76 clinical isolates of Campylobacter spp. tested were positive for porA by PCR, but only 13 isolates were positive for cytotoxin on CHO cells. Both cytotoxin-positive as well as cytotoxin-negative strains expressed PorA as determined by immunoblot analysis. These findings show that the porA gene product of C. jejuni is not a cytotoxin mediating inflammatory diarrhoea.     

Platelet-derived Growth Factor-mediated Induction of the Synaptic Plasticity Gene Arc/Arg3.1

Platelet-derived growth factor (PDGF) is a pleiotropic protein with critical roles in both developmental as well as pathogenic processes. In the central nervous system specifically, PDGF is critical for neuronal proliferation and differentiation and has also been implicated as a neuroprotective agent. Whether PDGF also plays a role in synaptic plasticity, however, remains poorly understood. In the present study we demonstrated that in the rat hippocampal neurons PDGF regulated the expression of Arc/Arg3.1 gene that has been implicated in both synapse plasticity and long term potentiation. Relevance of these findings was further confirmed in vivo by injecting mice with intracerebral inoculations of PDGF, which resulted in a rapid induction of Arc in the hippocampus of the injected mice. PDGF induced long term potentiation in rat hippocampal slices, which was abolished by PDGF receptor-tyrosine kinase inhibitor STI-571. We also present evidence that PDGF-mediated induction of Arc/Arg3.1 involved activation of the MAPK/ERK (MEK) pathway. Additionally, induction of Arc/Arg3.1 also involved the upstream release of intracellular calcium stores, an effect that could be blocked by thapsigargin but not by EGTA. Pharmacological approach using inhibitors specific for either MAPK/ERK phosphorylation or calcium release demonstrated that the two pathways converged downstream at a common point involving activation of the immediate early gene Egr-1. Chromatin immunoprecipitation assays demonstrated the binding of Egr-1, but not Egr-3, to the Arc promoter. These findings for the first time, thus, suggest an additional role of PDGF, that of induction of Arc.     

Synthesis, regulation and production of urokinase using mammalian cell culture: a comprehensive review

Urokinase, a serine protease, catalyzes the conversion of plasminogen to plasmin, which is responsible for dissolution of clots in blood vessels. It is an important drug for treatment of thromboembolic disease. Production of urokinase by mammalian cell culture has the following important steps: synthesis, regulation and secretion. Production and accumulation of this product in a bioreactor is a real challenge for biochemical engineers. Considerable information at molecular level needs to be understood for production of urokinase in order to correlate different parameters, which in turn can maximize the productivity. This information will be highlighted in this review. Moreover, urokinase production is a product-inhibited process. Therefore, in situ urokinase separation strategy is required to operate a bioreactor at its maximum urokinase formation rate. Integrated urokinase production and isolation processes developed recently will also be discussed briefly in this review.     

Chemokine receptor CXCR3 desensitization by IL-16/CD4 signaling is dependent on CCR5 and intact membrane cholesterol

Previous work has shown that IL-16/CD4 induces desensitization of both CCR5- and CXCR4-induced migration, with no apparent effect on CCR2b or CCR3. To investigate the functional relationship between CD4 and other chemokine receptors, we determined the effects of IL-16 interaction with CD4 on CXCR3-induced migration. In this study we demonstrate that IL-16/CD4 induced receptor desensitization of CXCR3 on primary human T cells. IL-16/CD4 stimulation does not result in surface modulation of CXCR3 or changes in CXCL10 binding affinity. This effect does require p56(lck) enzymatic activity and the presence of CCR5, because desensitization is not transmitted in the absence of CCR5. Treatment of human T cells with methyl-beta-cyclodextrin, a cholesterol chelator, prevented the desensitization of CXCR3 via IL-16/CD4, which was restored after reloading of cholesterol, indicating a requirement for intact cholesterol. These studies demonstrate an intimate functional relationship among CD4, CCR5, and CXCR3, in which CCR5 can act as an adaptor molecule for CD4 signaling. This process of regulating Th1 cell chemoattraction may represent a mechanism for orchestrating cell recruitment in Th1-mediated diseases.