Analysis of the binding of p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime) to mitochondrial cytochrome c reductase.

Cytochrome c reductase is inhibited by p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime). CPMB-oxime induces a red-shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the QP center of this enzyme in a non-competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent Kd in a red-shift assay was 6.9 +/- 0.6 microM. All binding characteristics analysed in this study were very similar to those of the E-beta-methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a 'catalytic switch' proposed recently for the E-beta-methoxyacrylate inhibitors (MOA-inhibitors (Brandt and von Jagow, Eur. J. Biochem.     

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.     

A familial mutation in the testis-determining gene SRY shared by both sexes

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRY^F109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.This paper is dedicated by G. S. to Professor Ulrich Wolf on the occasion of his 60th birthday     

Comparative bacterial clearances of muscle and skin/subcutaneous tissues with and without dead bone: a laboratory study

Seventeen New Zealand White rabbits underwent implantation of three different concentrations of bacteria and a sterile saline control solution with and without dead autologous bone in eight separate muscular and eight separate subcutaneous sites. Following a period of 1 week, each site was surgically explored and samples of tissue were taken for histology and quantitative culture. Results reveal that final bacterial concentrations in the subcutaneous sites were significantly lower than in the muscle sites (p less than or equal to 0.0001) for each concentration of bacteria, with and without dead bone. Dead bone resulted in very significantly greater bacterial concentrations in both subcutaneous and muscle sites. Clinically, these results indicate that a thorough bony wound debridement is more important than the type of tissue used to close the wound. Flap tissue should be selected with regard to the perfusion, contour, and appearance of the recipient site.     

Genetic diversity and restoration of a recalcitrant clonal sedge (Tetraria capillaris Cyperaceae)

The genetic diversity of a clonal sedge (Tertraria capillaris) was assessed using isozyme analysis of 11 loci. Of 29 enzyme systems tested, eight were selected which gave interpretable bands with consistently good resolution. Though seedlings of the species are rarely observed in nature, isozyme analysis showed that for the study transects containing 100 sample plants, the majority of plants at the site were sexually rather than clonally derived. Young plants generated from embryos grown in vitro from excised seeds showed a high level of genetic diversity which could account for the genetic diversity measured in the parent population. In terms of restoration of the species, 85% of the assessed genetic diversity at the study site could be retained if 25 T. capillaris plants were taken at random. The study illustrates how genetic assessment coupled with tissue culture methods provides a feasible model for the recovery of most of the assessed local genetic diversity of a clonal species.     

Complete nucleotide sequence of a cryptic plasmid, pbaw301, from the ruminai anaerobe Ruminococcus flavefaciens R13e2

Abstract The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminai bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli —10 and —35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.