Sequence requirements for Hid binding and apoptosis regulation in the baculovirus inhibitor of apoptosis Op-IAP. Hid binds Op-IAP in a manner similar to Smac binding of XIAP.

It has been suggested that the Drosophila Hid protein interacts with the baculovirus Op-IAP protein in a manner similar to that of human Smac binding to XIAP, based largely on amino acid sequence homology. However, there is little direct experimental evidence in support of this hypothesis; indeed, evidence exists from previous studies suggesting that the mode of binding is not similar. We have now precisely mapped the interaction between Hid and Op-IAP, and we show clearly for the first time that the biochemical interactions between the amino terminus of Hid and BIR2 of Op-IAP are highly similar to those found between the processed amino terminus of Smac and BIR3 of XIAP. Also similar to Smac, the amino terminus of Hid must be processed to bind Op-IAP. In addition, our data also suggest that a second interaction between Hid and Op-IAP exists that does not involve the amino terminus of Hid, which may explain some of the earlier contradictory results. The evolutionary conservation of this mechanism of binding underscores its importance in apoptotic regulation. Nevertheless, interaction with Hid is not sufficient for Op-IAP to inhibit apoptosis induced by Hid overexpression or by treatment with actinomycin D, indicating that additional sequence elements are required for the anti-apoptotic function of Op-IAP.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Aortic ascorbic acid, trace elements, and superoxide dismutase activity in human aneurysmal and occlusive disease

Altered trace elements and ascorbic acid metabolism have been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, their role in the disease process, or the effect of atherosclerosis on their tissue levels within plaque, is poorly understood. The present study analyzes the concentrations of Fe, Cu, Zn, and Mn, and ascorbic acid and superoxide dismutase (SOD) activity in tissue samples from 29 patients with abdominal aortic aneurysms (AAA) and 14 patients with atherosclerotic occlusive disease (AOD). It was observed that the Fe and Mn concentrations in AAA and AOD tissue were higher than the levels in nondiseased control aorta, whereas Cu and Zn levels in AAA and AOD tissue were similar to the levels in controls. The Zn:Cu ratio was significantly lower in the AAA tissue in comparison to both AOD and control tissue. In addition, AAA and AOD tissue had low ascorbic acid levels and low Cu,Zn-SOD activity with Cu,Zn-SOD:Mn-SOD ratios of 0.27 and 0.19, respectively, compared to a ratio of 3.20 in control aorta. These data indicate that aorta affected by aneurysms and occlusive disease have altered trace element and ascorbic acid concentrations, as well as low Cu,Zn-SOD activity. Although these observations do not directly support the hypothesis that AAA is associated with aortic Cu deficiency they do suggest a role for oxygen radicals or increased lipid peroxidation in occlusive and aneurysmal disease of the aorta.     

Influence of ethanol on the activities of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase and squalene-hopene-cyclase in Zymomonas mobilis

Summary The influence of different primary aliphatic alcohols on the activities of two key enzymes in hopanoid biosynthesis of Zymomonas mobilis was investigated. By use of ¹⁴C- and ³H-labelled substrates the enzymes 3-hydroxy-3-methylglutaryl-CoA-reductase and squalene-hopenecyclase were detected with activities of 1.6 pmol x (min x mg protein)^-1 and 2.3 pmol x- (min x mg protein)^-1, respectively. Cells grown in the presence of 6% (v/v) ethanol did not show higher activities of these enzymes than cells grown in the presence of 1% (v/v) ethanol. Furthermore, 3-hydroxy-3-methylglutaryl-CoA-reductase was not activated by ethanol. However, ethanol activated the squalene-hopene-cyclase when added to the enzyme test system. Besides ethanol, propanol also had a positive effect on the squalene-hopene-cyclase: the enzyme's activity increased 1.7-fold in the presence of either alcohol at a concentration of 6% (v/v). This corresponded with a similar increase of hopanoid content of whole cells when grown in the presence of 6% (v/v) added ethanol or propanol. These results indicated that the squalene-hopene-cyclase has a regulatory function in the alcohol dependent hopanoid biosynthesis of Z. mobilis.Abbreviation HMG-CoA-reductase 3-hydroxy-3-methylglutaryl-coenzyme A-reductase     

Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1.     

The Boer goat. II. Growth, nutrient requirements, carcass and meat quality

Growth rates of Boer goats were generally lower than sheep and, under favorable nutritional conditions, weight gains of more than 200 g per day were obtained, against values of up to 176 g per day under extensive subtropical conditions. Lactation and pregnancy had a marked effect on ME intake, and the latter had an improved feed conversion ratio (6.06 kg/kg) in comparison to that of virgin does (10.96 kg/kg). Below 6% crude protein in the diet, feed intake is reduced and has negative effects on birth weights, growth and milk production. Little information is available on mineral requirements of goats. The carcass of Boer goats is generally leaner, less compact and has different carcass proportions than sheep. The relatively high collagen contents with lower solubility of Boer goat meat, has meant that the eating quality has been regarded as inferior to that of lamb and mutton. Breeding holds the key to improving tenderness of goat meat; different slaughtering techniques can be used as well. Boer goats have high potential as meat animals when yielding three kid crops in 2 years and when fed to gain more than 200 g/day.