Female sex hormone and nephropathy in Cohen diabetic rat (genetically selected sucrose-fed)

In the Cohen diabetic rat (genetically-selected sucrose-fed) ovariectomy resulted in a significantly decreased incidence of nephropathy, while estradiol treatment to the ovariectomized animal increased the rate of nephropathy to the levels occurring in the uncastrated untreated diabetic female. In the male diabetic animal orchidectomy caused a slight decrease in the rate of nephropathy whereas estradiol treatment to the orchidectomized animal resulted in a slight increase in the rate. No difference was found between the ovariectomized and the uncastrated diabetic female as to the spontaneous blood glucose, plasma insulin, serum cholesterol or TG levels, but a significant difference was found in their plasma estradiol levels.     

Homologs of the yeast neck filament associated genes: isolation and sequence analysis of Candida albicans CDC3 and CDC10

Morphogenesis in the yeast Saccharomyes cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.     

Recent advances in the structure and function of isopenicillin N synthase

Isopenicillin N synthase is a key enzyme in the biosynthesis of penicillin and cephalosporin antibiotics, catalyzing the oxidative ring closure of -(L--aminoadipoyl)-L-cysteinyl-D-valine to form isopenicillin N. Recent advances in our understanding of the unique chemistry of this enzyme have come through the combined application of spectroscopic, molecular genetic and crystallographic approaches and led to important new insights into the structure and function of this enzyme. Here we review new information on the nature of the endogenous ligands that constitute the ferrous iron active site, sequence evidence for a novel structural motif involved in iron binding in this and related non-heme iron dependent dioxygenases, crystal structure studies on the enzyme and its substrate complex and the impact of these and site-directed mutagenesis studies for unraveling the mechanism of the isopenicillin N synthase reaction.     

Octreotide,a Somatostatin Analogue,Fails to Inhibit Hypoxia-induced Retinal Neovascularization in the Neonatal Rat

Objective: Octreotide, a somatostatin analogue, has been shown to prevent angiogenesis in diverse in vitro models. We evaluated its effect on retinal neovascularization in vivo, using a neonatal rat retinopathy model. Methods: We used, on alternating days, hypoxia (10% O₂) and hyperoxia (50% O₂) during the first 14 days of neonatal rats, to induce retinal neovascularization. Half of the rats were injected subcutaneously with octreotide 0.7 μg/g BW twice daily. At day 18 the eyes were evaluated for the presence of epiretinal and vitreal hemorrhage, neovascularization and epiretinal proliferation. Octreotide pharmacokinetics and its effect on serum growth hormone (GH) and insulin-like growth factor I (IGF-I) were examined in 28 rats. Results: Serum octreotide levels were 667 μg/1 two hours after injection, 26.4 μg/1 after nine hours and 3.2 μg/1 after 14 hours. GH levels were decreased by 40% (p = 0.002) two hours after injection but thereafter returned to baseline. IGF-I levels were unchanged two hours after injection and were elevated by 26% 14 hours after injection (p = 0.02). Epiretinal membranes were highly associated with epiretinal hemorrhages (p < 0.001), while retinal neovascularization was notably associated with vitreal hemorrhages (p < 0.001). Conclusions: Twice-daily injections of octreotide failed to produce sustained decrease in serum GH, but produced rebound elevation of serum IGF-I. Accordingly, no statistically significant effect of injections on retinal pathology was noted. This finding, however, does not contradict our assumption that GH suppression may decrease the severity of retinopathy.     

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.     

Factors affecting salmonellae repopulation in composted sludges.

The repopulation potential and recovery of Salmonella sp. and their close relatives Arizona spp. and Citrobacter spp. in sewage sludge which had been composted was examined. Salmonellae growth in previously composted sludge was found to occur in the mesophilic temperature range (20 to 40 degrees c), require a moisture content of greater than or equal to 20%, and require a carbon/nitrogen ratio in excess of 15:1.     

Therapeutically optimized rates of drug release can be achieved by varying the drug-to-lipid ratio in liposomal vincristine formulations

The anti-tumor efficacy of liposomal formulations of cell cycle dependent anticancer drugs is critically dependent on the rates at which the drugs are released from the liposomes. Previous work on liposomal formulations of vincristine have shown increasing efficacy for formulations with progressively slower release rates. Recent work has also shown that liposomal formulations of vincristine with higher drug-to-lipid (D/L) ratios exhibit reduced release rates. In this work, the effects of very high D/L ratios on vincristine release rates are investigated, and the antitumor efficacy of these formulations characterized in human xenograft tumor models. It is shown that the half-times (T(1/2)) for vincristine release from egg sphingomyelin/cholesterol liposomes in vivo can be adjusted from T(1/2) = 6.1 h for a formulation with a D/L of 0.025 (wt/wt) to T(1/2) = 117 h (extrapolated) for a formulation with a D/L ratio of 0.6 (wt/wt). The increase in drug retention at the higher D/L ratios appears to be related to the presence of drug precipitates in the liposomes. Variations in the D/L ratio did not affect the circulation lifetimes of the liposomal vincristine formulations. The relationship between drug release rates and anti-tumor efficacy was evaluated using a MX-1 human mammary tumor model. It was found that the antitumor activity of the liposomal vincristine formulations increased as D/L ratio increased from 0.025 to 0.1 (wt/wt) (T(1/2) = 6.1-15.6 h respectively) but decreased at higher D/L ratios (D/L = 0.6, wt/wt) (T(1/2) = 117 h). Free vincristine exhibited the lowest activity of all formulations examined. These results demonstrate that varying the D/L ratio provides a powerful method for regulating drug release and allows the generation of liposomal formulations of vincristine with therapeutically optimized drug release rates.     

Ultrasonic Telemetry, Tracking and Automated Monitoring Technology for Sharks

Sharks were among the first marine animals to carry telemetry systems because of their size and the need to understand their interactions with humans. Modern telemetry systems can gather many kinds of data (limited only by imagination, funding and sensor types), indicating which animals are near telemetry receivers and what they are doing. Receivers now range from simple autonomous detector units for deployment in mid-water in large-scale grids, to sophisticated automated benthic recorders, to triangulating radio-linked buoy systems (RAP), to ship-borne transponders. In addition, archival tags can now gather and store data even while the shark is away, to be downloaded later. Older types had to be recovered, but popup tags release from sharks automatically, surface and transfer data to satellites, while CHAT tags download whenever queried by a nearby transponding acoustic receiver. Sophisticated animal-borne tags dramatically increase the information gathered about sharks and their environment. The examples provided show the parallel progression of shark biology and acoustic biotelemetry illustrating that telemetry systems are tools for gathering data, which can often be honed to facilitate biological experiments. Future visions include sensors that directly measure shark swimming power and cardiac output, compressing the data so that it can be delivered to RAP systems tracking multiple animals with meter resolution in near real time. CHAT tags as small as 22mm diameter should be able to return similar data from trips of hundreds of kilometers. Continued communication between biologists and engineers is essential to develop these technologies.     

Structure-function studies of the non-heme iron active site of isopenicillin N synthase: some implications for catalysis

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron-dependent oxygenase that catalyzes the ring closure of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies and the crystal structure of IPNS show that the iron atom in the active species is coordinated to two histidine and one aspartic acid residues, and to ACV, dioxygen and H2O. We previously showed by site-directed mutagenesis that residues His212, Asp214 and His268 in the IPNS of Streptomyces jumonjinensis are essential for activity and correspond to the iron ligands identified by crystallography. To evaluate the importance of the nature of the protein ligands for activity, His214 and His268 were exchanged with asparagine, aspartic acid and glutamine, and Asp214 replaced with glutamic acid, histidine and cysteine, each of which has the potential to bind iron. Only the Asp214Glu mutant retained activity, approximately 1% that of the wild type. To determine the importance of the spatial arrangement of the protein ligands for activity, His212 and His268 were separately exchanged with Asp214; both mutant enzymes were completely defective. These findings establish that IPNS activity depends critically on the presence of two histidine and one carboxylate ligands in a unique spatial arrangement within the active site. Molecular modeling studies of the active site employing the S. jumonjinensis IPNS crystal structure support this view. Measurements of iron binding by the wild type and the Asp214Glu, Asp214His and Asp214Cys-modified proteins suggest that Asp214 may have a role in catalysis as well as in iron coordination.     

Practical Method for Detecting Poliovirus in Anaerobic Digester Sludge

A method has been developed which permits reliable detection of low numbers of poliovirus infective units in anaerobic digester sludge and dewatered composted sludge.