Proteolytic cleavage of Hansenula anomala flavocytochrome b2 into its two functional domains. Isolation of a highly active flavodehydrogenase and a cytochrome b2 core

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b2 into its two functional domains: a cytochrome b2 core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1% that of intact flavocytochrome b2. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome b2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80% that of flavocytochrome b2. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains. Our results are discussed in terms of 'gene fusion'.     

The role of domain redundancy in genetic robustness against null mutations

A key question in molecular genetics is why severe gene mutations often do not result in a detectable abnormal phenotype. Alternative networks are known to be a gene compensation mechanism. Gene redundancy, i.e. the presence of a duplicate gene (or paralog) elsewhere in the genome, also underpins many cases of gene dispensability. Here, we investigated the role of partial duplicate genes on dispensability, where a partial duplicate is defined as a gene that has no paralog but which codes for a protein made of domains, each of which belongs to at least another protein. The rationale behind this investigation is that, as a partial duplicate codes for a domain redundant protein, we hypothesised that its deletion might have a less severe phenotypic effect than the deletion of other genes. This prompted us to (re)address the topic of gene dispensability by focusing on domain redundancy rather than on gene redundancy. Using fitness data of single-gene deletion mutants of Saccharomyces cerevisiae, we will show that domain redundancy is a compensation mechanism, the strength of which is lower than that of gene redundancy. Finally, we shall discuss the molecular basis of this new compensation mechanism.     

Excreted steroids in primate feces over the menstrual cycle and pregnancy

Techniques were established for the extraction and measurement of 17 beta-estradiol (E2) and progestins (P4) from feces of Old World primates. Studies were conducted to show the sensitivity of these measures, means of preserving fecal samples in the field, effects of urinary contamination, and means to eliminate these effects. Our results show that excreted steroid measures can be used to distinguish between mid-follicular and luteal phases in the menstrual cycle, and to identify pregnancy by Day 20 of gestation; the steroid measures can also be used to identify ovulatory levels of E2 and to establish the length of the menstrual cycle. Urine was shown to contaminate the fecal sample and to confound the estimate of steroid levels in feces; prolonged storage (less than 6 h) was shown to change the steroid estimate. Both urinary contamination and storage-dependent changes were eliminated by the addition of ethanol to the sample. Preliminary results also suggest that effects of dietary fiber on steroid hormone levels are minimal when controlled quantitatively by adjusting for water content of the fecal sample. We conclude that these measurements of excreted steroids provide a valid, noninvasive measure of physiological state of the hypothalamic-pituitary-ovarian axis among free-ranging animals in the field.     

Gene fusion/fission is a major contributor to evolution of multi-domain bacterial proteins

Most proteins comprise one or several domains. New domain architectures can be created by combining previously existing domains. The elementary events that create new domain architectures may be categorized into three classes, namely domain(s) insertion or deletion (indel), exchange and repetition. Using 'DomainTeam', a tool dedicated to the search for microsyntenies of domains, we quantified the relative contribution of these events. This tool allowed us to collect homologous bacterial genes encoding proteins that have obviously evolved by modular assembly of domains. We show that indels are the most frequent elementary events and that they occur in most cases at either the N- or C-terminus of the proteins. As revealed by the genomic neighbourhood/context of the corresponding genes, we show that a substantial number of these terminal indels are the consequence of gene fusions/fissions. We provide evidence showing that the contribution of gene fusion/fission to the evolution of multi-domain bacterial proteins is lower-bounded by 27% and upper-bounded by 64%. We conclude that gene fusion/fission is a major contributor to the evolution of multi-domain bacterial proteins.     

The myb-related gene stonewall induces both hyperplasia and cell death in Drosophila: rescue of fly lethality by coexpression of apoptosis inducers

We carried out gain-of-function mutagenesis screening and identified a mutant in which GAL4 induction led to both hyperplasia and apoptosis. The gene involved was identified as stonewall (stwl), a myb-related gene involved in germ cell proliferation and differentiation during oogenesis. As observed with dmyb, the ectopic expression of stwl(UY823) inhibited endoreplication in salivary glands. We also found that stwl(UY823) overexpression, like overexpression of the wild-type gene, activated G1/S transition and apoptosis. The apoptosis triggered by stwl(UY823) expression is correlated to induction of the proapoptotic gene reaper. Finally, the death of flies induced by ectopic stwl(UY823) expression is efficiently prevented in vivo by triggering cell death in stwl(UY823)-expressing cells. Our results suggest that stwl(UY823) kills flies by causing inappropriate cell cycle entry, and that triggering the death of these overproliferating cells or slowing their proliferation restores viability.     

Influence of nebivolol and metoprolol on inflammatory mediators in human coronary endothelial or smooth muscle cells. Effects on neointima formation after balloon denudation in carotid arteries of rats treated with nebivolol.

OBJECTIVE AND BACKGROUND: Inflammation plays a critical role in all stages of atherogenesis. Proliferating vascular smooth muscle cells (SMC) and endothelial cells (EC) enhancing the inflammatory response, both contribute to the progression of atherosclerosis. Anti-proliferative, anti-inflammatory and anti-oxidative therapy seems to be a promising therapeutic strategy. The aim of this study was to assess the anti-proliferative and anti-inflammatory effect of the beta-blocker nebivolol in comparison to metoprolol in vitro and to find out whether nebivolol inhibits neointima formation in vivo. METHODS AND RESULTS: Real-time-RT-PCR revealed a decrease in VCAM-1, ICAM-1, PDGF-B, E-selectin and P-selectin mRNA expression in human coronary artery EC and SMC incubated with nebivolol for 72 hours while metoprolol did not have this effect. Nebivolol reduced MCP-1 and PDGF-BB protein in the culture supernatant of SMC and EC, respectively. Sprague-Dawley rats were treated with nebivolol for 0 or 35 days before and 28 days after carotid balloon injury. Immunohistological analyses showed that pre-treatment with nebivolol was associated with a decreased number of SMC layers and macrophages and an increased lumen area at the site of the arterial injury. The intima area was reduced by 43% after pre-treatment. CONCLUSION: We found that nebivolol reduced the expression of proinflammatory genes in endothelial cells and vascular smooth muscle cells in vitro whereas metoprolol did not. In vivo, nebivolol inhibited neointima formation by reducing SMC proliferation and macrophage accumulation.     

Identification of a novel gene (HSN2) causing hereditary sensory and autonomic neuropathy type II through the Study of Canadian Genetic Isolates

Hereditary sensory and autonomic neuropathy (HSAN) type II is an autosomal recessive disorder characterized by impairment of pain, temperature, and touch sensation owing to reduction or absence of peripheral sensory neurons. We identified two large pedigrees segregating the disorder in an isolated population living in Newfoundland and performed a 5-cM genome scan. Linkage analysis identified a locus mapping to 12p13.33 with a maximum LOD score of 8.4. Haplotype sharing defined a candidate interval of 1.06 Mb containing all or part of seven annotated genes, sequencing of which failed to detect causative mutations. Comparative genomics revealed a conserved ORF corresponding to a novel gene in which we found three different truncating mutations among five families including patients from rural Quebec and Nova Scotia. This gene, termed "HSN2," consists of a single exon located within intron 8 of the PRKWNK1 gene and is transcribed from the same strand. The HSN2 protein may play a role in the development and/or maintenance of peripheral sensory neurons or their supporting Schwann cells.