Interactive gene expression pattern in prostate cancer cells exposed to phenolic antioxidants

Dietary phenolic compounds are known to elicite vital cellular responses such as cell cycle arrest, apoptosis and differentiation by activating a cascade of molecular events. As there is an increasing interest to improve the efficacy of these compounds for use as potential chemopreventive agents, we wanted to understand the impact of phenolic compounds on target genes in prostate cancer. In this study we used human cDNA microarrays with 2400 clones consisting of 17 prosite motifs to characterize alterations in gene expression pattern in response to the phenolic antioxidants ellagic acid (EA) and resveratrol (RE). Over a 48-hr exposure of androgen - sensitive LNCaP cells to EA and RE, a total of 593 and 555 genes respectively, showed more than a two fold difference in expression. A distinct set of genes in both EA-and RE-treated cells may represent the signature profile of phenolic antioxidant-induced gene expression in LNCaP cells. Although extensive similarity was found between effects of EA - and RE - responsive genes in prostate cancer cells, out of 246 genes with overlapping responses, 25 genes showed an opposite effect. Quantitative RT-PCR was used to verify and validate the differential expression of selected genes identified from cDNA microarrays. In-depth analysis of the data from this study provided insight into the alterations in the p53 - responsive genes, p300, Apaf-1, NF-kBp50 and p65 and PPAR families of genes, suggesting the activation of multiple signaling pathways that leads to growth inhibition of LNCaP cells. This is a first study to look for changes in a large number of human genes in response to dietary compounds.     

Peptides containing cyclin/Cdk-nuclear localization signal motifs derived from viral initiator proteins bind to DNA when unphosphorylated

A single phosphorylation event at T-antigen residue Thr124 regulates initiation of simian virus 40 DNA replication. To explore this regulatory process, a series of peptides were synthesized, centered on Thr124. These peptides contain a nuclear localization signal (NLS) and a recognition site for cyclin/Cdk kinases. When unphosphorylated, the "CDK/NLS" peptides inhibit T-antigen assembly and bind non-sequence specifically to DNA. However, these activities are greatly reduced upon phosphorylation of Thr124. Similar results were obtained by using peptides derived from the CDK/NLS region of bovine papillomavirus E1. Related studies indicate that residues in the NLS bind to DNA, whereas those in the CDK motif regulate binding. These findings are discussed in terms of the control of T-antigen double hexamer assembly and initiation of viral replication.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

Phosphorylation of simian virus 40 T antigen on Thr 124 selectively promotes double-hexamer formation on subfragments of the viral core origin

Cell cycle-dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DNA replication. A T-ag molecule containing a Thr-->Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but to be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of the T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T124A molecules on the SV40 origin. Herein it is demonstrated that hexamer formation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124A molecules are inhibitors of T-ag double hexamer formation. These and related studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV40 DNA replication are discussed.     

Rates and patterns of mitochondrial DNA sequence evolution in fringilline finches (fringilla spp.) and the greenfinch (Carduelis chloris)

Rates and patterns of evolution in partial sequences of five mitochondrial genes (cytochrome b, ATPase 6, NADH dehydrogenase subunit 5, tRNA(Glu), and the control region) were compared among taxa in the passerine bird genera Fringilla and Carduelis. Rates of divergence do not vary significantly among genes, even in comparisons with the control region. Rate variation among lineages is significant only for the control region and NADH dehydrogenase subunit 5, and patterns of variation are consistent with the expectations of neutral theory. Base composition is biased in all genes but is stationary among lineages, and there is evidence for directional mutation pressure only in the control region. Despite these similarities, patterns of substitution differ among genes, consistent with alternative regimes of selective constraint. Rates of nonsynonymous substitution are higher in NADH dehydrogenase subunit 5 than in other protein-coding genes, and transitions exist in elevated proportions relative to transversions. Transitions appear to accumulate linearly with time in tRNA(Glu), and despite exhibiting the highest overall rate of divergence among species, there are no transversional changes in this gene. Finally, for resolving phylogenetic relationships among Fringilla taxa, the combined protein-coding data are broadly similar to those of the control region in terms of phylogenetic informativeness and statistical support.      

BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed.     

Are Ants Useful Indicators of Restoration Success in Temperate Grasslands?

Assessments of restoration are usually made through vegetation community surveys, leaving much of the ecosystem underexamined. Invertebrates, and ants in particular, are good candidates for restoration evaluation because they are sensitive to environmental change and are particularly important in ecosystem functioning. The considerable resources currently employed in restoring calcareous grassland on ex‐arable land mean that it is important to gather as much information as possible on how ecosystems change through restoration. We compared ant communities from 40 ex‐arable sites where some form of restoration work had been implemented between 2 and 60 years previously, with 40 paired reference sites of good quality calcareous grassland with no history of improvement or cultivation. A total of 11 ant species were found, but only two of these were found to be significantly different in abundance between restoration and reference sites: Myrmica sabuleti was more likely to be present in reference sites, whereas Lasius niger was more likely to be found in restoration sites. Myrmica sabuleti abundance was significantly positively correlated with age of restoration sites. The potential number of ant species found in temperate grasslands is small, limiting the information their assemblages can provide about ecosystem change. However, M. sabuleti is a good indicator species for calcareous grassland restoration success and, alongside information from the plant community, could increase the confidence with which restoration success is judged. We found the survey to be quick and simple to carry out and recommend its use.     

Increasing cell biomass in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

Background  

Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.