Yeast silencers create domains of nuclease-resistant chromatin in an SIR4-dependent manner

Previous analysis of the repression of the silent mating type loci in Saccharomyces cerevisiae has linked the mechanism of silencing to the formation of a chromatin domain at the silenced loci. In this study, a TRP1 reporter gene was used to examine changes in chromatin structure in a neutral environment. This enabled the chromatin structure organized by yeast silencers to be compared directly with changes effected by the yeast α2 repressor. It was found that silencers mediate the formation of lengthy nuclease-resistant domains on the DNA, rather than specifically positioning nucleosomes over promoter regions as the α2 repressor does. Silencing at the TRP1 reporter gene closely resembled silencing at the HMR and HML loci. Repression of the test gene was optimal when two silencers flanking the reporter gene were used, mimicking the situation at the silent loci. In addition, both repression of the reporter gene and the formation of nuclease-resistant chromatin domains was SIR4 dependent. Received: 31 October 1996; in revised form: 6 March 1997 / Accepted: 25 March 1997     

Ultrastructure and synthesis of the extracellular matrix of Pisaster ochraceus embryos preserved by freeze substitution

When asteroid embryos cryoprotected with propylene glycol are rapidly frozen in liquid propane and freeze substituted with ethanol, preservation of the cells and extracellular matrix (ECM) is excellent. The basal lamina, although thicker and less well defined than in conventionally fixed embryos, demonstrates a region of decreased density just below the cells that corresponds to the lamina lucida and a lamina densa. The former region is often occupied by fibrous material. In addition, as was previously described in conventionally fixed tissues, the basal lamina of the ectoderm is generally thicker and more substantial than that of the endoderm, reinforcing an earlier suggestion that the structure of the basal lamina is different in different regions of the embryo. The ECM of the blastocoel consists of thin “twig-like” elements that form a loose meshwork evenly distributed throughout the blastocoel. Bundles of 20 nm fibers, located within the meshwork, are oriented parallel to the base of the cells of the stomodeum. In the long axis of the embryo, similar fibers are present in the dorsal aspect of the animal between the stomach and the ectoderm and radiate out from the esophagus crossing the region between it and the ectoderm. Immunocytochemical work with three different monoclonal antibodies shows that glycoprotein molecules, synthesized in the Golgi apparatus, are also secreted here and form part of the matrix structure. The results suggest that the blastocoel is filled with a gel-like material reinforced with bundles of 20-nm fibers. The manner in which the observed arrangement could contribute to the development and maintainence of the shape of the embryo is discussed. J Morphol 232:133–153, 1997. © 1997 Wiley-Liss, Inc.     

Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin

Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of alpha3 chain of type IV collagen) was identified as possessing anti-angiogenic activity. In the present study, the anti-angiogenic activity of tumstatin was localized to the putative 54-132-amino acid Tum-5 domain, and the activity mediated by alpha(v)beta(3) integrin interaction in an RGD-independent manner. The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris specifically inhibited proliferation and caused apoptosis of endothelial cells with no significant effect on nonendothelial cells. Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G1 endothelial cell cycle arrest. Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo using both a Matrigel plug assay in C57BL/6 mice and human prostate cancer (PC-3) xenografts in nude mice. The in vivo results demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of PC-3 tumors in association with a decrease in CD31 positive vasculature. These in vivo studies also show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin. In addition, these studies for the first time suggest that through the action of endogenous inhibitors, alpha(v)beta(3) integrin may also function as a negative regulator of angiogenesis. Taken together, these findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candidate for the treatment of cancer.     

Degradation of biomolecules in bones: effects of the biological forensics as an example of the stability of isotope ratios in collagen

Modern bone samples were experimentally degraded by incubation into water at increased temperature and examined in terms of their collagen content, the stable C and N isotopic ratios, and the molar C/N ratio. The same analyses were carried out with archaeological human bone of varying age (300 up to 8000 years). The experimentally degraded samples exhibited changes of the collagen's integrity, which influence the stable isotope ratios. In the case of the archaeological material, a correlation between stable delta13C- and delta15N-values and collagen content could be demonstrated. The molar C:N ratio was no suitable criterion for the assessment of the state of preservation of extractable collagen.     

Molecular and morphological evidence for conspecificity of two common Indo-Pacific species of Palythoa (Cnidaria: Anthozoa)

Most studies characterize metacommunities based on a single snapshot of the spatial structure, which may be inadequate for taxa with high migratory behavior (e.g., fish). Here, we applied elements of metacommunity structure to examine variations in the spatial distributions of stream fishes over time and to explore possible structuring mechanisms. Although the major environmental gradients influencing species distributions remained largely the same in time, the best-fit pattern of metacommunity structure varied according to sampling occasion and whether or not we included non-native species in the analyses. Quasi-Clementsian and Clementsian structures were the predominant best-fit structures, indicating the importance of species turnover among sites and the existence of more or less discrete community boundaries. The environmental gradient most correlated with metacommunity structure was defined by altitude, area of artificial ponds in the catchment, and dissolved oxygen content. Our results suggest that the best-fit metacommunity structure of the native species can change in time in this catchment due to seasonal changes in distribution patterns. However, the distribution of non-native species throughout the landscape homogenizes the temporal variability in metacommunity structure of native species. Further studies are necessary from other regions to examine best-fit metacommunity structures of stream fishes within relatively short environmental gradients.     

Insolubility of Cr2O3 in Bioaccessibility Tests Points to Requirement for a New Human Oral Reference Dose for Trivalent Chromium

Bioaccessibility measurements have the potential to improve the accuracy of risk assessments and reduce the potential costs of remediation when they reveal that the solubility of chemicals in a matrix (e.g., soil) differs markedly from that in the critical toxicity study (i.e., the key study from which a toxicological or toxicity reference value is derived). We aimed to apply this approach to a brownfield site contaminated with chromium, and found that the speciation was Cr^III, using a combination of alkaline digestion/diphenylcarbazide complexation and X-ray absorption near edge structure analysis. The bioaccessibility of Cr₂O₃, the compound on which a reference dose for Cr^III is based, was substantially lower (<0.1%) than that of the Cr^III in the soils, which was a maximum of 9%, giving relative bioaccessibility values of 13,000% in soil. This shows that the reference dose is based on essentially an insoluble compound, and thus we suggest that other compounds be considered for toxicity testing and derivation of reference dose. Two possibilities are CrCl₃·6H₂O and KCr(SO₄)₂·12H₂O, which have been used for derivation of ecological toxicity reference values and are soluble at a range of dosing levels in our bioaccessibility tests.     

Modeling optimal responses and fitness consequences in a changing Arctic

Animals must balance a series of costs and benefits while trying to maximize their fitness. For example, an individual may need to choose how much energy to allocate to reproduction versus growth, or how much time to spend on vigilance versus foraging. Their decisions depend on complex interactions between environmental conditions, behavioral plasticity, reproductive biology, and energetic demands. As animals respond to novel environmental conditions caused by climate change, the optimal decisions may shift. Stochastic dynamic programming provides a flexible modeling framework with which to explore these trade‐offs, but this method has not yet been used to study possible changes in optimal trade‐offs caused by climate change. We created a stochastic dynamic programming model capturing trade‐off decisions required by an individual adult female polar bear (Ursus maritimus) as well as the fitness consequences of her decisions. We predicted optimal foraging decisions throughout her lifetime as well as the energetic thresholds below which it is optimal for her to abandon a reproductive attempt. To explore the effects of climate change, we shortened the spring feeding period by up to 3 weeks, which led to predictions of riskier foraging behavior and higher reproductive thresholds. The resulting changes in fitness may be interpreted as a best‐case scenario, where bears adapt instantaneously and optimally to new environmental conditions. If the spring feeding period was reduced by 1 week, her expected fitness declined by 15%, and if reduced by 3 weeks, expected fitness declined by 68%. This demonstrates an effective way to explore a species' optimal response to a changing landscape of costs and benefits and highlights the fact that small annual effects can result in large cumulative changes in expected lifetime fitness.     

Analysis of mononuclear cell surfaces with fluoresceinated staphylococcal protein A complexed with IgG antibody of heat-aggregated gamma-globulin.

Fluorescein-conjugated staphylococcal protein A (SPA) was complexed with either: 1) heat-aggregated IgG, 2) B cell specific antibody, or 3) T cell specific antibody and then used for an immunofluorescent analysis of mononuclear cell surfaces. Cellular Fc receptors failed to recognize the Fc region of aggregated IgG that had been blocked by SPA. Moreover, fluoresceinated SPA that had been complexed either with anti-Fab (B-cell specific) or T cell-specific antisera prevented the nonspecific binding of these reagents to the IgG-Fc receptors on mononuclear cells, thereby permitting the latter to be properly identified as B or T lymphocytes. In addition, when unconjugated SPA was added to presensitized target cells in a test for antibody-dependent cell-mediated cytotoxicity, cytolysis was abrogated.