Proteinase Produced by Chlamydia psittaci in L Cells

L cells (mouse fibroblasts) infected with Chlamydia psittaci (strain meningopneumonitis) produced a proteinase differing in solubility in ammonium sulfate from the proteinase of uninfected L cells. Synthesis of the enzyme was inhibited by chloramphenicol but not by cycloheximide, indicating that the new proteinase in infected L cells was synthesized by Chlamydia psittaci. The chlamydial proteinase had no demonstrable ion requirements and was not inhibited by a variety of inhibitors of proteinase activity. Gel filtration experiments suggested a molecular weight of approximately 250,000. The proteinase appeared in infected L cells at the time host cells began to die and the large chlamydial cells began to reorganize into small ones. Some possible functions for the chlamydial proteinase were proposed.     

Hematopoietic stem cell expansion: challenges and opportunities

Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo-expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines have failed to support reliable amplification of immature stem cells in culture, suggesting that additional factors are required. In recent years, several novel factors, including developmental factors and chemical compounds, have been reported to affect HSC self-renewal and improve ex vivo stem cell expansion protocols. Here, we highlight early expansion attempts and review recent development in the extrinsic control of HSPC fate in vitro.     

Effect of Circulating Forms of Soy Isoflavones on the Oxidation of Low Density Lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.     

Biology of the Galapagos shark,Carcharhinus galapagensis, in Hawai'i

Synopsis Catch records from the Hawai'i Cooperative Shark Research and Control Program, which operated in Hawai'i from 1967–1969, were examined and data on the Galapagos shark,Carcharhinus galapagensis were analyzed. A total of 304 Galapagos sharks was caught, predominantly with longlines. More female sharks were caught than males, and the catch was skewed geographically. On the island of O'ahu the highest catch rates occurred along the north and south coasts. High catch rates also occurred near points of land, where longshore currents converge. Average depth of capture was greater for juveniles (45.1 m) and mature males (60.2 m), than for subadults (38.8 m) and mature female sharks (34.2 m). Males appear to reach maturity between 205 and 239 cm total length, and females between 215 and 245 cm. Litter size ranged from 4 to 16 pups, with an average of 8.7. In Hawaiian waters Galapagos sharks are born at just over 80 cm total length. Mating and parturition apparently occur early in the year, and gestation is estimated to be about 12 months. Stomach contents consisted mainly of teleosts and benthic prey, and ontogenetic changes in diet occurred as sharks increased in size. Sharks consumed a smaller proportion of teleosts and more elasmobranchs with increasing size. Dietary diversity also increased with increasing size of shark.     

Chlamydia and apoptosis: life and death decisions of an intracellular pathogen

The chlamydiae are important obligate intracellular prokaryotic pathogens that, each year, are responsible for millions of human infections involving the eye, genital tract, respiratory tract, vasculature and joints. The chlamydiae grow in cytoplasmic vesicles in susceptible host cells, which include the mucosal epithelium, vascular endothelium, smooth muscle cells, circulating monocytes and recruited or tissue-specific macrophages. One important pathogenic strategy that chlamydiae have evolved to promote their survival is the modulation of programmed cell death pathways in infected host cells. The chlamydiae can elicit the induction of host cell death, or apoptosis, under some circumstances and actively inhibit apoptosis under others. This subtle pathogenic mechanism highlights the manner in which these highly successful pathogens take control of infected cells to promote their own survival - even under the most adverse circumstances.     

Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.     

Effect of Shaking Speed on the Secretion of Enterotoxin B by Staphylococcus aureus

The concentration of enterotoxin B secreted by four strains of Staphylococcus aureus was dependent upon the shaking speed. For the conditions established, each strain demonstrated an optimal shaking speed, and speeds in excess of the optimum resulted in decreased secretion of toxin. At the optimal shaking speed, maximum secretion occurred at 37 C. At 45 C, both growth and toxin secretion were absent. By using agar containing antienterotoxin B sera, studies with strain S-6 at optimal and suboptimal shaking speeds demonstrated that individual cells vary in their toxin-synthesizing ability and that the relative numbers of high and low producers change during the growth cycle. Although most of the toxin was secreted during the first 12 hr of growth, a portion was secreted during the subsequent 6 hr, even though growth as measured by colony-forming units per milliliter decreased and Klett units increased.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

The influence of plant hormones on phospholipid monolayer stability

The influence of hormones in water subphase on the stability of monolayers built of phospholipid mixtures extracted from embryogenic (PLE) and nonembryogenic (PLNE) wheat calli was examined. Additionally, experiments on individual lipids, dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA), were performed. DPPC was chosen because it was the main phospholipid present in both calli. Negatively charged DPPA could mimic a negatively charged natural mixture of lipids. As hormones, auxins (IAA and 2,4-D), cytokinins (zeatin and kinetin) and zearalenone were chosen. The time of monolayer stability for PLNE calli was much longer than for PLE calli. Kinetics of monolayer stability of PLNE was similar to DPPA, whereas that of PLE was similar to DPPC. Generally, hormones increased the time after which the monolayer stability was reached and decreased the surface pressure. The greatest effect was observed for auxins (especially IAA), whereas cytokinins affected the monolayer stability to a lesser degree.     

Design,synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives: Potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one—1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 μM). The IC₅₀ values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 μM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure–activity relationship studies, in vitro screening and X-ray crystallography.