Induction of chromosome aberrations, micronucleus formation and sperm abnormalities in mouse following carbofuran exposure

Carbofuran was tested to study in vivo cytogenetic effects in mouse bone marrow cells and morphological alterations in sperms. The acute oral and intraperitoneal (i.p.) LD(50) of carbofuran was determined to be 9.5 or 2.0 mg/kg b.w. in mice, respectively. The animals were orally administered 1.9, 3.8 or 5.7 mg/kg b.w. (20, 40 and 60% of LD(50)) of carbofuran for 24 h or 1.9 mg/kg b.w. for 4 consecutive days (cumulative 7.6 mg/kg or 80% of LD(50)) to analyse chromosome aberrations (CAs). For micronucleus test (MT) animals were orally exposed to 5.7 mg/kg b.w. for 24 and 48 h or 1.9 mg/kg b.w. for 4 consecutive days. For reference mice were exposed to peanut oil (negative control) and cyclophosphamide (20 mg/kg) or ethyl methanesulfonate (EMS: 100 mg/kg) positive control for CAs and MT respectively. To analyse the effect on sperm morphology mice were exposed to single i.p. dose of 1 and 2 mg/kg b.w. of carbofuran and repeatedly to 0.5 mg/kg for 5 consecutive days. Cytogenetic analysis revealed that all the test doses induced mitotic inhibition, CAs, micronucleus (MN) formation and sperm abnormalities in a dose dependent manner. Present observations concurrent with earlier reports substantiate the genotoxic potential of carbofuran and possible risk to human beings.     

Protein-protein interaction revealed by NMR T(2) relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

T(2) relaxation experiments in combination with chemical shift and site-directed mutagenesis data were used to identify sites involved in weak but specific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The pyruvate decarboxylase component, a heterotetramer E1(alpha(2)beta(2)), is responsible for the first committed and irreversible catalytic step. The accompanying reductive acetylation of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between E1 and the lipoyl domain of the E2 polypeptide chain. The interactions between the free lipoyl domain (9 kDa) and free E1alpha (41 kDa), E1beta (35 kDa) and intact E1alpha(2)beta(2) (152 kDa) components, all the products of genes or sub-genes over-expressed in Escherichia coli, were investigated using heteronuclear 2D NMR spectroscopy. The experiments were conducted with uniformly (15)N-labeled lipoyl domain and unlabeled E1 components. Major contact points on the lipoyl domain were identified from changes in the backbone (15)N spin-spin relaxation time in the presence and absence of E1(alpha(2)beta(2)) or its individual E1alpha or E1beta components. Although the E1alpha subunit houses the sequence motif associated with the essential cofactor, thiamin diphosphate, recognition of the lipoyl domain was distributed over sites in both E1alpha and E1beta. A single point mutation (N40A) on the lipoyl domain significantly reduces its ability to be reductively acetylated by the cognate E1. None the less, the N40A mutant domain appears to interact with E1 similarly to the wild-type domain. This suggests that the lipoyl group of the N40A lipoyl domain is not being presented to E1 in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain structure, especially in the lipoyl-lysine beta-turn region, as indicated by chemical shift data. Interaction with E1 and subsequent reductive acetylation are not necessarily coupled.     

Antiinflammatory activity of Muktashukti bhasma.

Muktashukti bhasma (MSB), an Ayurvedic compound, consisting of pearl, Aloe vera and vinegar, inhibited acute and subacute inflammation in albino rats as induced by subplanter injection of carrageenan, histamine, 5-HT, nystatin and subcutaneous implant of cotton pellets. In all the test procedures the antiinflammatory response of 1000 mg/kg MSB was comparable to the response observed with 300 mg/kg acetylsalicylic acid (ASA). Oral premedication with MSB delayed castor oil-induced diarrhoea in rats, indicating its prostaglandin inhibitory activity. The antiinflammatory activity of the compound is attributed to its ability to cause inhibition of prostaglandins, histamine and 5-HT and also by stabilization of the lysosomal membranes. The antiinflammatory activity of MSB seems one third to half as potent as ASA.     

In vivo and in vitro measurement of cell mediated immunity in Marek's disease: role of reticuloendothelial system

Dinitrofluorobenzene (DNFB) contact-sensitivity test and leucocyte adherence inhibition (LAI) test were performed in this study as in vivo and in vitro tests to measure the cell-mediated immunity (CMI) in chickens subjected to stimulation of reticuloendothelial (RE) system, depletion of RE system and other experimental groups after being challenged with Marek's disease (MD) virus. It was found that CMI was lower in the birds with depleted RE system and infected control birds, whereas CMI was higher in the birds with activated RE system and vaccinated birds as revealed by DNFB contact-sensitivity test. In cases of LAI test, the number of LAI-positive birds were highest in the chicks with depleted RE system particularly in 3rd and 4th month of age, when the incidence of MD was also maximum.     

Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules

Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs.     

Analysis of the Role of Interleukin 6 Receptor Haplotypes in the Regulation of Circulating Levels of Inflammatory Biomarkers and Risk of Coronary Heart Disease

Variants at the interleukin 6 receptor (IL6R) gene regulate inflammation and are associated with risk of coronary heart disease (CHD). The aim of the present study was to investigate the effects of IL6R haplotypes on circulating levels of inflammatory biomarkers and risk of CHD. We performed a discovery analysis in SHEEP, a myocardial infarction (MI) case control study (n = 2,774) and replicated our results in two large, independent European populations, PROCARDIS, a CHD case control study (n = 7,998), and IMPROVE (n = 3,711) a prospective cardiovascular cohort study. Two major haplotype blocks (rs12083537A/G and rs4075015A/T—block 1; and rs8192282G/A, rs4553185T/C, rs8192284A/C, rs4240872T/C and rs7514452T/C—block 2) were identified in the IL6R gene. IL6R haplotype associations with C-reactive protein (CRP), fibrinogen, IL6, soluble IL6R (sIL6R), IL6, IL8 and TNF-α in SHEEP, CRP and fibrinogen in PROCARDIS and CRP in IMPROVE as well as association with risk of MI and CHD, were analyzed by THESIAS. Haplotypes in block 1 were associated neither with circulating inflammatory biomarkers nor with the MI/CHD risk. Haplotypes in block 2 were associated with circulating levels of CRP, in all three study populations, with fibrinogen in SHEEP and PROCARDIS, with IL8 and sIL6Rin SHEEP and with a modest, non significant, increase (7%) in MI/CHD risk in the three populations studied. Our results indicate that IL6R haplotypes regulate the circulating levels of inflammatory biomarkers. Lack of association with the risk of CHD may be explained by the combined effect of SNPs with opposite effect on the CHD risk, the sample size as well as by structural changes affecting sIL6R stability in the circulation.     

Influence of Environmental Factors,Cultural Practices,and Herbicide Application on Seed Germination and Emergence Ecology of Ischaemum rugosum Salisb

Ischaemum rugosum Salisb. (Saramolla grass) is a noxious weed of rice that is difficult to control by chemical or mechanical means once established. A study was conducted to determine the effect of light, temperature, salt, drought, flooding, rice residue mulch, burial depth, and pre-emergence herbicides on seed germination and emergence of I. rugosum. Germination was stimulated by light and inhibited under complete darkness. Optimum temperature for germination was 30/20°C (97.5% germination). Germination reduced from 31 to 3.5% when the osmotic potential of the growing medium decreased from -0.1 to -0.6 MPa and no germination occurred at -0.8 MPa. Germination was 18 and 0.5% at 50 and 100 mM NaCl concentrations, respectively, but was completely inhibited at 150 mM or higher. Residue application at 1–6 t ha^-1 reduced weed emergence by 35–88% and shoot biomass by 55–95%. The efficacy of pre-emergence herbicides increased with increasing application rates and decreased with increasing rice residue mulching. The efficacy of herbicides was in the order of oxadiazon> pendimethalin> pretilachlor. At 6 t ha^-1, all herbicides, regardless of rates, did not differ from the control treatment. I. rugosum seeds buried at 2 cm or deeper did not emerge; however, they emerged by 4.5 and 0.5% at 0.5 and 1 cm depths, respectively, compared to the 39% germination for soil surface seeding. Flooding at 4 DAS or earlier reduced seedling emergence and shoot biomass while flooding at 8 DAS reduced only seedling emergence. The depth and timing of flooding independently reduced root biomass. Flooding at 4 and 6 cm depths reduced the root biomass. Relative to flooding on the day of sowing, flooding at 8 DAS increased root biomass by 89%. Similarly, flooding on the day of sowing and at 2 DAS reduced the root–shoot biomass ratio. Under the no-flood treatment, increasing rates of pretilachlor from 0.075 to 0.3 kg ai ha^-1 reduced weed emergence by 61–79%. At the flooding depth of 2–4 cm, pretilachlor reduced weed emergence and shoot and root biomass, but the differences across rates were non-significant. Information generated in this study will be helpful in developing integrated weed management strategies for managing this weed.     

Dissecting Long-Term Glucose Metabolism Identifies New Susceptibility Period for Metabolic Dysfunction in Aged Mice

Metabolic disorders, like diabetes and obesity, are pathogenic outcomes of imbalance in glucose metabolism. Nutrient excess and mitochondrial imbalance are implicated in dysfunctional glucose metabolism with age. We used conplastic mouse strains with defined mitochondrial DNA (mtDNA) mutations on a common nuclear genomic background, and administered a high-fat diet up to 18 months of age. The conplastic mouse strain B6-mt^FVB, with a mutation in the mt-Atp8 gene, conferred β-cell dysfunction and impaired glucose tolerance after high-fat diet. To our surprise, despite of this functional deficit, blood glucose levels adapted to perturbations with age. Blood glucose levels were particularly sensitive to perturbations at the early age of 3 to 6 months. Overall the dynamics consisted of a peak between 3–6 months followed by adaptation by 12 months of age. With the help of mathematical modeling we delineate how body weight, insulin and leptin regulate this non-linear blood glucose dynamics. The model predicted a second rise in glucose between 15 and 21 months, which could be experimentally confirmed as a secondary peak. We therefore hypothesize that these two peaks correspond to two sensitive periods of life, where perturbations to the basal metabolism can mark the system for vulnerability to pathologies at later age. Further mathematical modeling may perspectively allow the design of targeted periods for therapeutic interventions and could predict effects on weight loss and insulin levels under conditions of pre-diabetic obesity.     

Identification of cultivation condition to produce correctly folded form of a malaria vaccine based on Plasmodium falciparum merozoite surface protein-1 in Escherichia coli

The C-terminal, 19-kDa domain of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1₁₉) is among the leading vaccine candidate for malaria due to its essential role in erythrocyte invasion by the parasite. We designed a synthetic gene for optimal expression of recombinant PfMSP-1₁₉ in Escherichia coli and developed a scalable process to obtain high-quality PfMSP-1₁₉. The synthetic gene construct yielded a fourfold higher expression level of PfMSP-1₁₉ in comparison to the native gene construct. Optimization of cultivation conditions in the bioreactor indicated important role of yeast extract and substrate feeding strategy for obtaining enhanced expression of soluble and correctly folded PfMSP-1₁₉. It was observed that the higher expression level of PfMSP-1₁₉ was essentially associated with the generation of higher level of incorrectly folded PfMSP-1₁₉. A simple purification procedure comprising metal affinity and ion exchange chromatography was developed to purify correctly folded form of PfMSP-1₁₉ from cell lysate. Biochemical and biophysical characterization of purified PfMSP-1₁₉ suggested that it was highly pure, homogeneous, and correctly folded.     

Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization

We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. One chimera consists of a FK506-binding protein (FKBP12) fused to a cellular 'address' (nuclear localization signal or nuclear export sequence). The second chimera consists of a target protein fused to a fluorescent protein and the FKBP12-rapamycin-binding (FRB) domain from FKBP-12-rapamycin associated protein 1 (FRAP1, also known as mTor). Rapamycin induces dimerization of the FKBP12- and FRB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment.