In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma 180.

Withania somnifera is a medicinal plant used in the treatment of a variety of ailments in the Ayurvedic system. Alcoholic extract of the root of the plant was injected(ip) at daily doses of 200 to 1000 mg/kg body wt for 15 days starting from 24 hr after intradermal inoculation of 5 x 10(5) cells of S-180 in BALB/c mice. Solid tumor growth was monitored for 100 days. Doses of 400 mg/kg and above produced complete regression of tumor after an initial growth, the percentage of complete response (CR) increasing with increasing drug dose. A 55% CR was obtained at 1000 mg/kg drug administration, but this dose also produced some mortality among the animals. A significant increase in the volume doubling time and growth delay was seen when the drug dose was increased from 500 to 750 mg/kg body wt, but further increase in drug dose to 1000 mg/kg did not produce any significant increase in these responses. Cumulative doses of 7.5 to 10 g at daily doses of 500 or 750 mg/kg seems to produce a good response in this tumor.     

Protein synthesis in yeast. Purification of elongation factor 3 from temperature-sensitive mutant 13-06 of the yeast Saccharomyces cerevisiae

An altered form of the elongation factor 3 (EF-3) has been purified to near homogeneity from a thermolabile yeast mutant ts 13-06. The isolation procedure involved chromatography on DEAE-Sephadex, CM-Sepharose, and hydroxylapatite columns. The final purification of this protein was obtained by affinity chromatography on an ATP-Sepharose column. Because of the extreme lability of the mutant protein, the yield was very poor. Silver stain analysis of the sodium dodecyl sulfate electrophoretograms indicated that the affinity-purified protein was better than 90% pure. From the studies of the physical and biochemical properties, the following characteristics of the purified wild type and the mutant protein have been established. The two proteins were indistinguishable by their molecular weight, amino acid composition, and isoelectric point. Purified mutant EF-3 was rapidly inactivated between 37 and 39 degrees C. Under this condition, wild type EF-3 was completely stable. Ribosome-dependent GTPase and ATPase activities of the mutant EF-3 were heat sensitive; GTPase activity was more labile than the ATPase activity. Mutant EF-3, after exposure to a nonpermissive temperature, failed to stimulate binding of the ternary complex of EF-1 X GTP X aminoacyl-tRNA to ribosome. The wild type protein was fully active under this condition. Other biochemical and physical properties of these two proteins are under current investigation.     

Plasma orosomucoid metabolism and susceptibility to malarial infection in rodents

The effects of Plasmodium berghei infection on liver function and plasma orosomucoid metabolism were investigated in Wistar rats. Infected rats with 20-25% parasitaemia manifested increased serum transaminase levels, hypoalbuminaemia and hypoproteinaemia. In spite of such indications of deranged liver function, the hepatic synthesis rate (as measured by 14C-amino acid incorporation) of seromucoids predominantly orosomucoid or alpha 1-acid glycoprotein) was increased by 73%. The circulating levels of this glycoprotein were also doubled in infected animals. The albumin synthesis rate was not increased. This preferential synthesis and increase in circulating levels of orosomucoid may have in vivo significance in malarial infection, in view of reports that orosomuocid has influence on in vitro invasion of red cells by malarial parasites.     

Sperm competition and the evolution of sperm design in mammals

Background  

The influence of sperm competition upon sperm size has been a controversial issue during the last 20 years which remains unresolved for mammals. The hypothesis that, when ejaculates compete with rival males, an increase in sperm size would make sperm more competitive because it would increase sperm swimming speed, has generated contradictory results from both theoretical and empirical studies. In addition, the debate has extended to which sperm components should increase in size: the midpiece to accommodate more mitochondria and produce more energy to fuel motility, or the principal piece to generate greater propulsion forces.     

Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry

Snow crab (Chionoecetes opilio) proteins have been recognized as an important source of both food and occupational allergens. While snow crab causes a significant occupational allergy, only one novel allergen has recently been fully characterized. The muscle proteins from snow crab legs were profiled by SDS-PAGE. Several of these proteins were characterized using tandem mass spectrometry. Five proteins were identified; sarcoplasmic Ca-binding (20kDa), arginine kinase (40), troponin (23kDa) and α-actine (42kDa) and smooth endoplasmic reticulum Ca(2+)ATPase (113kDa). Immunoblotting using serum of sixteen allergic patients resulted in strong reactivity with the 40-kDa protein in seven patients (43%). This protein was purified by chromatography and subsequently de novo sequenced using matrix assisted laser desorption ionization and electrospray tandem mass spectrometry. We identified a second important allergen, arginine kinase, in snow crab, designated Chi o 3. Based on identity and homology analysis, using bioinformatics tools, a signature peptide was identified as a chemical surrogate for arginine kinase. The suitability of this signature peptide was tested for analytically representing the arginine kinase, by performing a multi-reaction monitoring tandem mass spectrometry approach on actual air filter samples collected from a simulated crab processing plant.     

Thermoluminescence dosimetric attributes of Yb3+-doped BaO–ZnO–LiF–B2O3 glass material after Er3+ co-doping

Motivated by our previous study on Sm³⁺ ions as thermoluminescence (TL) sensitizers to the BaO–ZnO–LiF–B₂O₃–Yb₂O₃ glass system, in the current study we examined the effect of Er³⁺ ion co-doping on the TL characteristics of this glass system. The 4f–4f electronic transitions of the Er³⁺ and Yb³⁺ ions were confirmed via the optical absorption spectrum. Notably, the use of Yb³⁺–Er³⁺ ions failed to improve the TL intensity, sensitivity, and trap density. However, they enabled the glass system to function as an activator–quencher system. The linearity range and effective atomic number remained unaffected after co-doping. In addition, the problem of anomalous fading caused a remnant signal of just 58% after a week of storage of the Yb³⁺ monodoped glass. This was resolved by the optimum co-doping of Er³⁺ ions to achieve an 89% signal. The co-doping of Er³⁺ ions to the BaO–ZnO–LiF–B₂O₃–Yb₂O₃ glass system regulated its thermal stability and therefore supplemented its potential for radiation monitoring in food processing and retrospective dosimetry.     

The development, maturation, and turnover rate of mouse spleen dendritic cell populations

Three distinct subtypes of dendritic cells (DC) are present in mouse spleen, separable as CD4(-)8alpha(-), CD4(+)8alpha(-), and CD4(-)8alpha(+) DC. We have tested whether these represent stages of development or activation within one DC lineage, or whether they represent separate DC lineages. All three DC subtypes appear relatively mature by many criteria, but all retain a capacity to phagocytose particulate material in vivo. Although further maturation or activation could be induced by bacterially derived stimuli, phagocytic capacity was retained, and no DC subtype was converted to the other. Continuous elimination of CD4(+)8(-) DC by Ab depletion had no effect on the levels of the other DC subtypes. Bromodeoxyuridine labeling experiments indicated that all three DC subtypes have a rapid turnover (half-life, 1.5-2.9 days) in the spleen, with none being the precursor of another. The three DC subtypes showed different kinetics of development from bone marrow precursors. The CD8alpha(+) spleen DC, apparently the most mature, displayed an extremely rapid turnover based on bromodeoxyuridine uptake and the fastest generation from bone marrow precursors. In conclusion, the three splenic DC subtypes behave as rapidly turning over products of three independent developmental streams.     

Synthesis and biological evaluation of novel 5(H)-phenanthridin-6-ones, 5(H)-phenanthridin-6-one diketo acid, and polycyclic aromatic diketo acid analogs as new HIV-1 integrase inhibitors

A new series of phenanthridinone derivatives, and diketo acid analogs, as well as related phenanthrene and anthracene diketo acids have been synthesized and evaluated as HIV integrase (IN) inhibitors. Several new beta-diketo acid analogs with the phenanthridinone scaffold replaced by phenanthrene, anthracene or pyrene exhibited the highest IN inhibitory potency. There is a general selectivity against the integrase strand transfer step. The most potent IN was 2,4-dioxo-4-phenanthren-9-yl-butyric acid (27f) with an IC(50) of 0.38microM against integrase strand transfer. The phenanthrene diketo acids 27d-f were more potent (IC(50)=2.7-0.38microM) than the corresponding phenanthridinone diketo acid 16 (IC(50)=65microM), suggesting that the polar amide bridge in the phenanthridinone system decreases inhibitory activity relative to the more lipophilic phenanthrene system. This might have to do with the possible binding of the aryl group of the compounds binding to a lipophilic pocket at the integrase active site as suggested by the docking simulations. Molecular modeling also suggested that effectiveness of chelation of the active site Mg(2+) contributes to IN inhibitory potency. Finally, some of the potent compounds inhibited HIV-1 replication in human peripheral blood mononuclear cells (PBMC) with EC(50) down to 8microM for phenanthrene-3-(2,4-dioxo)butyric acid (27d), with a selectivity index of 10 against PBMCs.     

Human V gamma 2V delta 2 T cells produce IFN-gamma and TNF-alpha with an on/off/on cycling pattern in response to live bacterial products.

Whereas cytokine production in alphabeta T cells is rapidly regulated by exposure to peptide Ag, the mechanisms regulating cytokine production by gammadelta T cells are unknown. In this study, we demonstrate that human Vgamma2Vdelta2 T cells produce IFN-gamma and TNF-alpha as early as 2 h after Ag exposure, and that they produce these cytokines in a dose- and time- dependent manner in response to stimulation with a live bacterial product, iso-butylamine (IBA), but not to dead bacteria or LPS. gammadelta T cells began, ceased, and then resumed IFN-gamma and TNF-alpha generation in an on/off/on cycling pattern, both in vitro and in vivo, depending on the presence or absence of IBA. IFN-gamma and TNF-alpha, whose optimum production was dependent on IBA-stimulated gammadelta T cells, were critical for monocyte-mediated killing of Escherichia coli. By limiting cytokine production to periods of direct contact with live bacteria, gammadelta T cells focus their resources at the site of infection, while limiting systemic immunopathology. Thus, human gammadelta T cells may mediate innate resistance to extracellular bacteria via tightly regulated cytokine production without necessarily expanding in number.     

A novel dual-color reporter for identifying insulin-producing beta-cells and classifying heterogeneity of insulinoma cell lines

Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel "indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS(+)) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS(+) phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS(+) and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.