Effects of the Antiandrogen Flutamide on the Expression of Protein Kinase C Isoenzymes in LNCaP and PC3 Human Prostate Cancer Cells

Flutamide is a nonsteroidal antiandrogen that is frequently used for total androgen blockage in the treatment of advanced prostate cancer. We investigated the effect of this antiandrogen on the expression of protein kinase C (PKC) isoenzymes (alpha, beta1, epsilon, zeta) that are involved in cell growth, apoptosis and neoplastic transformation. Androgen-dependent (LNCaP) and independent (PC3) human prostate cancer cells were cultured in a medium that contained fetal bovine serum (FBS) or charcoal-stripped serum (CSS) and treated with 10 microM flutamide. The expression of PKC isoenzymes and the androgen receptor (AR) were analyzed by Western blot and RT-PCR, respectively. Serum steroids differentially regulate the expression of PKC isoenzymes in LNCaP and PC3 cells. Flutamide up-regulated the expression of alpha, beta1 and zeta, but not epsilon, PKC isoenzymes in CSS-LNCaP cells. These results were not homogeneously reproduced in the presence of androgens. We observed an opposite effect of flutamide, compared to CSS, on PKCbeta1 isoform expression in CSS-LNCaP suggesting that this antiandrogen exerts an agonistic effect. In PC3 cells flutamide potentiated the expression of the four PKC isoenzymes in almost all conditions tested (FBS- and CSS-cultured cells). Such effect of flutamide in PC3 cells is independent of AR since no expression of AR was detected. These results provide new evidence on antagonistic/agonistic responses of prostate cancer cells to antiandrogen drugs that are widely used in therapy and show that flutamide can elicit responses in prostate cancer cells that do not express AR.     

Reduction of adhesions with composite AlloDerm/polypropylene mesh implants for abdominal wall reconstruction

Ventral hernia repair often includes the use of structural prosthetic materials, such as polypropylene mesh, that can induce dense abdominal adhesions to peritoneal structures. AlloDerm (LifeCell Corp., Branchburg, N.J.), a commercially available decellularized human dermal analogue with its native basement membrane components intact, is gradually revascularized and replaced with autologous tissue after implantation. The authors hypothesized that AlloDerm integrated with polypropylene mesh would reduce adhesions and provide a biodegradable scaffold to generate an autologous vascularized tissue layer separating the abdominal viscera from the mesh. Ventral hernia defects (3 x 1 cm) in 19 guinea pigs were repaired using an inlay technique with polypropylene mesh alone (n = 6) or with composite implants constructed by integrating polypropylene mesh and AlloDerm with its basement membrane surface oriented toward (polypropylene/AlloIn, n = 7) or away from (polypropylene/ AlloOut, n = 6) the peritoneal cavity. At 4 weeks, the authors determined the amount of mesh implant surface area covered by adhesions, the strength of the adhesions [graded from 0 (none) to 3], and the incidence of bowel adhesions. Histologic analyses were performed on full-thickness tissue sections from the repair sites. The mean surface areas affected by adhesions and mean adhesion strength were significantly lower in the polypropylene/AlloIn (area, 12.4 percent; mean grade, 1.0) and polypropylene/AlloOut (area, 9.5 percent; mean grade, 0.5) groups than in the polypropylene group (area, 79.5 percent; mean grade, 2.9); there were no such differences between the polypropylene/AlloIn and polypropylene/AlloOut groups. The bowel was adherent to 67 percent of polypropylene repairs and 0 percent of the composite mesh repairs. The AlloDerm was remodeled to form a vascularized tissue layer beneath the mesh in composite repairs, unlike the significantly thinner, dense scar layer that formed in the polypropylene repairs. Immunohistochemical labeling for factor VIII showed neovascularization throughout the AlloDerm.The AlloDerm thus functioned as a biodegradable tissue scaffold, guiding the formation of a thick, well-vascularized tissue layer separating the polypropylene mesh from intraperitoneal structures. This significantly reduced both the amount of surface area covered by adhesions and adhesion strength. Basement membrane orientation had no effect. Composite mesh implants composed of structural prosthetic materials integrated with AlloDerm may have useful clinical applications for abdominal wall reconstruction by reducing adhesions and providing a vascularized tissue layer to separate and protect the peritoneal structures from polypropylene mesh fibers.     

Homologue recognition during meiosis is associated with a change in chromatin conformation

During meiosis, homologous chromosomes are sorted into pairs and are then intimately aligned, or synapsed, along their lengths while a proteinaceous structure, the synaptonemal complex, is assembled between them. However, little is known about how chromosomes first recognise each other. Here we show, by comparing the behaviour of wild-type wheat and wheat mutant for Ph1 (a suppressor of homeologous chromosome pairing), that when chromosomes recognise a partner to pair with, a conformational change to the chromatin is triggered in both partners that is followed by their intimate alignment [corrected]. Thus, a conformational change in the chromosomes at the onset of meiosis can be correlated directly with recognition.     

New insights into the mechanisms involved in renal proximal tubular damage induced in vitro by ochratoxin A

The mycotoxin ochratoxin A is a contaminant of human and animal food products. It is a potent nephrotoxin known to damage the proximal tubule. The aim of this work was to investigate the effects of ochratoxin A on a porcine renal proximal tubular epithelial cell line (LLC-PK1), and to identify sensitive endpoints revealing damage at the epithelial barrier level and at the molecular level. Cells exposed for 24 h to 5-10 microM ochratoxin indicated a clear damage to the intactness of the epithelial barrier, as shown by measurements of trans-epithelial resistance and zonula occludens-1 protein expression. At the mitochondrial level we observed alterations of the normal functions, such as an increase of the membrane potential, the formation of straight extensions, and the formation of giant mitochondria. At higher ochratoxin concentrations (50 microM), at which cytotoxicity assays revealed a significant toxicity, alterations of the cytoskeleton organization and induction of apoptosis were evident. In addition, we analyzed the expression of genes by using a cDNA macroarray. Our data indicate that ochratoxin-induced nephrotoxicity can be detected at the barrier and at the mitochondrial level at rather low concentrations, at which conventional cytotoxicity assays are unable to reveal toxic effects.     

Mammalian STAG3 is a cohesin specific to sister chromatid arms in meiosis I

Cohesins, which have been characterized in budding yeast and Xenopus, are multisubunit protein complexes involved in sister chromatid cohesion. Regulation of the interactions among different cohesin subunits and the assembly/disassembly of the cohesin complex to chromatin are key steps in chromosome segregation. We previously characterized the mammalian STAG3 protein as a component of the synaptonemal complex that is specifically expressed in germinal cells, although its function in meiosis remains unknown. Here we show that STAG3 has a role in sister chromatid arm cohesion during mammalian meiosis I. Immunofluorescence results in prophase I cells suggest that STAG3 is a component of the axial/lateral element of the synaptonemal complex. In metaphase I, STAG3 is located at the interchromatid domain and is absent from the chiasma region. In late anaphase I and the later stages of meiosis, STAG3 is not detected. STAG3 interacts with the structural maintenance chromosome proteins SMC1 and SMC3, which have been reported to be subunits of the mitotic cohesin complex. We propose that STAG3 is a sister chromatid arm cohesin that is specific to mammalian meiosis I.     

Secondary structure of prothymosin alpha evidenced for conformational transitions induced by changes in temperature and concentration of n-dodecyltrimethylammonium bromide

Conformational changes of prothymosin alpha (ProTalpha) induced by changes in temperature and concentration of the denaturant n-dodecyltrimethylammonium bromide (C12TAB) were studied by difference spectroscopy. The conformational transition of ProTalpha by C12TAB was followed as a function of denaturant concentration by absorbance measurements at 230 nm and the data were analyzed to obtain the Gibbs energy of the transition in water (deltaG0(w)) and in a hydrophobic environment (deltaG0(hc)) for saturated protein-surfactant complexes. The value of deltaG0(w) was 6.38 kJ mol(-1) and that for deltaG0(hc), which is not affected by temperature, was -18.62 kJ mol(-1). Changes of absorbance at 230 nm of ProTalpha with temperature can be assumed to resemble a transition in the secondary structure. The parameters characterizing the thermodynamics of unfolding, melting temperature (Tm), enthalpy (deltaHm), entropy (deltaSm) and heat capacity (deltaCp) were determined. The values obtained for Tm, deltaHm, and deltaSm are smaller that those found for other globular proteins; deltaCp was found to be much smaller. These results suggest that ProTalpha exhibits some type of secondary structure under these conditions (10 mM glycine buffer, pH 2.4).     

Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides

The polysaccharides obtained from the alkali-extractable, water-soluble fraction (F1SS) from the cell wall of Myrothecium verrucaria and Myrothecium atroviride were shown to be composed of beta-(1-->6)-galactofuranose fully substituted at O-2 by terminal residues of alpha-glucopyranose and alpha-glucuronic acid. Glucuronic acid was substituted at O-4 by glucopyranose in the Myrothecium species M. inundatum, M. setiramosum, M. prestonii, M. tongaense and M. roridum. The acidic polysaccharides from Phaeostilbella atra (=Myrothecium atrum) and Myrothecium gramineum lacked the backbone of 2,6 di-O-substituted galactofuranose and contained a high amount of O-5-substituted beta-galactofuranose. The structures of the polysaccharides isolated from Myrothecium cinctum and Myrothecium penicilloides were unrelated to each other and to the polysaccharides from the other species analysed. The usefulness of these polysaccharides as a characteristic for delimitation of the genus Myrothecium is discussed.