Molecular activity of 1,25-dihydroxyvitamin D3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] exerts anti-proliferative, differentiating and apoptotic effects on prostatic cells. These activities, in addition to epidemiologic findings that link Vitamin D to prostate cancer risk, support the use of 1,25(OH)(2)D(3) for prevention or therapy of prostate cancer. The molecular mechanisms by which 1,25(OH)(2)D(3) exerts antitumor effects on prostatic cells are not well-defined. In addition, there is heterogeneity among the responses of various prostate cell lines and primary cultures to 1,25(OH)(2)D(3) with regard to growth inhibition, differentiation and apoptosis. To understand the basis of these differential responses and to develop a better model of Vitamin D action in the prostate, we performed cDNA microarray analyses of primary cultures of normal and malignant human prostatic epithelial cells, treated with 50 nM of 1,25(OH)(2)D(3) for 6 and 24 h. CYP24 (25-hydroxyvitamin D(3)-24-hydroxylase) was the most highly upregulated gene. Significant and early upregulation of dual specificity phosphatase 10 (DUSP10), validated in five additional primary cultures, points to inhibition of members of the mitogen-activated protein kinase (MAPK) superfamily as a key event mediating activity of 1,25(OH)(2)D(3) in prostatic epithelial cells. The functions of other regulated genes suggest protection by 1,25(OH)(2)D(3) from oxidative stress. Overall, these results provide new insights into the molecular basis of antitumor activities of Vitamin D in prostate cells.     

High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification

Identification of anonymous proteins from two-dimensional (2-D) gels by peptide mass fingerprinting is one area of proteomics that can greatly benefit from a simple, automated workflow to minimize sample contamination and facilitate high-throughput sample processing. In this investigation we outline a workflow employing robotic automation at each step subsequent to 2-D gel electrophoresis. As proof-of-concept, 96 protein spots from a 2-D gel were analyzed using this approach. Whole protein (1 mg) from mature, dry soybean (Glycine max [L.] Merr.) cv. Jefferson seed was resolved by high resolution 2-D gel electrophoresis. Approximately 150 proteins were observed after staining with Coomassie Blue. The rather low number of detected proteins was due to the fact that the dynamic range of protein expression was greater than 100-fold. The most abundant proteins were seed storage proteins which in total represented over 60% of soybean seed protein. Using peptide mass fingerprinting 44 protein spots were identified. Identification of soybean proteins was greatly aided by the use of annotated, contiguous Expressed Sequence Tag (EST) databases which are available for public access (UniGene, ftp.ncbi.nih.gov/repository/UniGene/). Searches were orders of magnitude faster when compared to searches of unannotated EST databases and resulted in a higher frequency of valid, high-scoring matches. Some abundant, non seed storage proteins identified in this investigation include an isoelectric series of sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins. This survey of anonymous seed proteins will serve as the basis for future comparative analysis of seed-filling in soybean as well as comparisons with other soybean varieties.     

Synthesis, characterization, EXAFS investigation and antibacterial activities of new ruthenium(III) complexes containing tetradentate Schiff base

A series of new hexa-coordinated ruthenium(III) complexes of the type [Ru(X)(2-atmp-ba)(EPh3)] (where H2-2-atmp-ba=N,N'-bis(2-aminothiophenol)benzoylacetone; X=Cl or Br; E=P or As) have been prepared by reacting [RuX3(EPh3)3] (where X=Cl or Br; E=P or As) with tetradentate Schiff base ligand (H2-2-atmp-ba) in 1:1 molar ratio. The complexes have been characterized by elemental analyses, Infra red, electronic, electron paramagnetic resonance spectroscopy and cyclic voltammetry. In order to confirm the coordination and structure of the complexes extended X-ray absorption fine structure spectroscopy (EXAFS) studies have been carried out. Based on the above data, an octahedral structure has been confirmed for the complexes. The new complexes were also screened for their antibacterial properties.     

Uncovering directional sensing: where are we headed?

We survey aspects of directional sensing, i.e. how a cell interprets differences in the external concentration of a chemoattractant to guide its motion, from the perspective of systems biology. We focus on questions that need to be addressed using a combination of modelling and experimental approaches. After briefly summarising the ideas underlying recent modelling efforts, we discuss a variety of experimental questions which are motivated by these models. Some of these questions focus on basic features of the chemotactic response, without involving much biochemistry, while others focus on filling some of the gaps in the biochemistry, which have been brought to light by the models. The emphasis is on systematic quantitative experiments that will unambiguously resolve many of these issues. Finally, we describe some current challenges for theoretical modelling and survey some of the theoretical tools and approaches employed to model the chemotaxis pathways.     

Analysis of among-site variation in substitution patterns

Substitution patterns among nucleotides are often assumed to be constant in phylogenetic analyses. Although variation in the average rate of substitution among sites is commonly accounted for, variation in the relative rates of specific types of substitution is not. Here, we review details of methodologies used for detecting and analyzing differences in substitution processes among predefined groups of sites. We describe how such analyses can be performed using existing phylogenetic tools, and discuss how new phylogenetic analysis tools we have recently developed can be used to provide more detailed and sensitive analyses, including study of the evolution of mutation and substitution processes. As an example we consider the mitochondrial genome, for which two types of transition deaminations (C⇒T and A⇒G) are strongly affected by single-strandedness during replication, resulting in a strand asymmetric mutation process. Since time spent single-stranded varies along the mitochondrial genome, their differential mutational response results in very different substitution patterns in different regions of the genome. Published: September 2, 2004.     

nodD alleles of Sinorhizobium fredii USDA191 differentially influence soybean nodulation, nodC expression, and production of exopolysaccharides

All Rhizobium strains examined to date have one or multiple alleles of nodD. At least one copy of nodD and the presence of flavonoid exudates are required for nod gene induction and nodulation. Sinorhizobium fredii USDA191 has two copies of nodD. In this study, we demonstrate that inactivation of either copy of nodD caused a reduction in basal levels of expression of nodC. Extra copies of nodD1 had no effect on the expression of nodC when compared with the wild type, but extra copies of nodD2 abolished the inducer requirement, thereby rendering nodC constitutive. A nodD1 mutant was unable to nodulate soybean cultivars 'Peking' and 'McCall'. Inactivation of nodD2 or addition of extra copies of nodD1 or nodD2 caused delayed nodulation on Peking, and reduced the number of nodules on McCall. Both nodD alleles of S. fredii USDA191 appear to be involved in regulation of exopolysaccharide production; however, nodD2 appears to be more important in this respect than nodD1.     

The FcR gamma subunit and Syk kinase replace the CD3 zeta-chain and ZAP-70 kinase in the TCR signaling complex of human effector CD4 T cells

The TCR-mediated signals required to activate resting T cells have been well characterized; however, it is not known how TCR-coupled signals are transduced in differentiated effector T cells that coordinate ongoing immune responses. Here we demonstrate that human effector CD4 T cells up-regulate the expression of the CD3zeta-related FcRgamma signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3epsilon, but not CD3zeta. The TCR/CD3/FcRgamma complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases.     

Cartilage interstitial fluid load support in unconfined compression

Under physiological conditions of loading, articular cartilage is subjected to both compressive strains, normal to the articular surface, and tensile strains, tangential to the articular surface. Previous studies have shown that articular cartilage exhibits a much higher modulus in tension than in compression, and theoretical analyses have suggested that this tension–compression nonlinearity enhances the magnitude of interstitial fluid pressurization during loading in unconfined compression, above a theoretical threshold of 33% of the average applied stress. The first hypothesis of this experimental study is that the peak fluid load support in unconfined compression is significantly greater than the 33% theoretical limit predicted for porous permeable tissues modeled with equal moduli in tension and compression. The second hypothesis is that the peak fluid load support is higher at the articular surface side of the tissue samples than near the deep zone, because the disparity between the tensile and compressive moduli is greater at the surface zone. Ten human cartilage samples from six patellofemoral joints, and 10 bovine cartilage specimens from three calf patellofemoral joints were tested in unconfined compression. The peak fluid load support was measured at 79±11% and 69±15% at the articular surface and deep zone of human cartilage, respectively, and at 94±4% and 71±8% at the articular surface and deep zone of bovine calf cartilage, respectively. Statistical analyses confirmed both hypotheses of this study. These experimental results suggest that the tension–compression nonlinearity of cartilage is an essential functional property of the tissue which makes interstitial fluid pressurization the dominant mechanism of load support in articular cartilage.     

Effect of vinblastine sulfate on gamma-radiation-induced DNA single-strand breaks in murine tissues

The effect of vinblastine sulfate on gamma-radiation-induced DNA strand breaks in different tissues of tumour bearing mice, was studied by single-cell gel electrophoresis. Intraperitonial administration of different doses (0.25-2.0mg/kg body weight) of vinblastine sulfate 30 min prior to 4 Gy gamma-radiation exposure showed a dose-dependent decrease in the yield of DNA strand breaks in murine fibrosarcoma, blood leukocytes and bone marrow cells. The dose-dependent protection of cellular DNA against radiation-induced strand breaks as evidenced from comet tail length, tail moment and percent DNA in the tail, was more pronounced in bone marrow cells than in the cells of the tumor fibrosarcoma. In fibrosarcoma cells, the decrease in comet tail length, tail moment and percent DNA in the tail was detected at lower doses of vinblastine sulfate administration and these parameters were not significantly altered at higher doses, from that of the control irradiated. From this study, it appears that in addition to anticancer activity, vinblastine sulfate could offer protection to the normal tissues against gamma-radiation-induced DNA strand breaks.