High cycling cadence reduces carbohydrate oxidation at given low intensity metabolic rate

Cycling cadence (RPM)-related differences in blood lactate concentration (BLC) increase with increasing exercise intensity, whilst corresponding divergences in oxygen uptake (V.O₂) and carbon dioxide production (V.CO₂) decrease. Aim of the present study was to test whether a higher RPM reduces the fraction (%) of the V.O₂ used for carbohydrate oxidation (relCHO) at a given BLC. Eight males (23.9 ± 1.6 yrs; 177 ± 3 cm; 70.3 ± 3.4 kg) performed incremental load tests at 50 and 100 RPM. BLC, V.O₂ and V.CO₂ were measured. At respiratory exchange ratios (RER) < 1, relCHO were calculated and the constant determining 50 % relCHO (k_CHO) was approximated as a function of the BLC. At submaximal workload V.O₂, V.CO₂, and relCHO were lower (all p < 0.002; η² > 0.209) at 50 than at 100 RPM. No differences were observed in V.O₂peak (3.96 ± 0.22 vs. 4.00 ± 0.25 l · min⁻¹) and RER_peak (1.18 ± 0.02 vs. 1.15 ± 0.02). BLC was lower (p < 0.001; η² = 0.680) at 50 than at 100 RPM irrespective of cycling intensity. At 50 RPM, kCHO (4.2 ± 1.4 (mmol · l⁻¹)³) was lower (p = 0.043; η² = 0.466) than at 100 RPM (5.9 ± 1.9 (mmol · l⁻¹)³). This difference in k_CHO reflects a reduced CHO oxidation at a given BLC at 100 than at 50 RPM. At a low exercise intensity, a higher cycling cadence can substantially reduce the reliance on CHO at a given metabolic rate and/or BLC.     

Pancreatic lipase inhibition activity of trilactone terpenes of Ginkgo biloba

The prevalence of obesity is increasing at an alarming rate, but, unfortunately, only a few drugs are currently available on the market. In the present study, the methanolic extract of Ginkgo biloba L. (Ginkgoaceae) was investigated as an inhibitor of pancreatic lipase (PL) in an attempt to explain its hypolipidaemic activity. In vitro assay of G. biloba leaves extract revealed a substantial PL inhibition activity (IC(50)?=?16.5 μg/mL). Further investigation was performed by employing theoretical docking simulations and experimental testing to uncover the active constituents responsible for G. biloba anti-lipase activity. Virtually, terpene trilactones, including ginkgolides and bilobalide, were found to fit within the binding pocket of PL via several attractive interactions with key amino acids. Experimentally, ginkgolides A, B, and bilobalide were found to inhibit PL significantly (IC(50)?=?22.9, 90.0, and 60.1 μg/mL, respectively). Our findings demonstrated that the hypolipidaemic effects of G. biloba extract can be attributed to the inhibition of PL by, at least in part, terpene trilactones. In conclusion, this work can be considered a new step towards the discovery of new natural safe hypolipidaemic PL inhibitors.     

Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.     

Patterns of CCR5, CXCR4, and CCR3 Usage by Envelope Glycoproteins from Human Immunodeficiency Virus Type 1 Primary Isolates

Coreceptor usage by Envs from diverse primary human immunodeficiency virus type 1 isolates was analyzed by a vaccinia virus-based expression and assay system. Usage of recombinant CCR5 and CXCR4 correlated closely with fusogenicity toward macrophages and T-cell lines expressing endogenous coreceptors. Surprisingly, recombinant CCR3 was utilized by most primary and T-cell-line-adapted Envs. Endogenous CXCR4 in macrophages was functional as a coreceptor.     

Late Maternal Folate Supplementation Rescues from Methyl Donor Deficiency-Associated Brain Defects by Restoring Let-7 and miR-34 Pathways

The micronutrients folate and vitamin B12 are essential for the proper development of the central nervous system, and their deficiency during pregnancy has been associated with a wide range of disorders. They act as methyl donors in the one-carbon metabolism which critically influences epigenetic mechanisms. In order to depict further underlying mechanisms, we investigated the role of let-7 and miR-34, two microRNAs regulated by methylation, on a rat model of maternal deficiency. In several countries, public health policies recommend periconceptional supplementation with folic acid. However, the question about the duration and periodicity of supplementation remains. We therefore tested maternal supply (3 mg/kg/day) during the last third of gestation from embryonic days (E) 13 to 20. Methyl donor deficiency-related developmental disorders at E20, including cerebellar and interhemispheric suture defects and atrophy of selective cerebral layers, were associated with increased brain expression (by 2.5-fold) of let-7a and miR-34a, with subsequent downregulation of their regulatory targets such as Trim71 and Notch signaling partners, respectively. These processes could be reversed by siRNA strategy in differentiating neuroprogenitors lacking folate, with improvement of their morphological characteristics. While folic acid supplementation helped restoring the levels of let-7a and miR-34a and their downstream targets, it led to a reduction of structural and functional defects taking place during the perinatal period. Our data outline the potential role of let-7 and miR-34 and their related signaling pathways in the developmental defects following gestational methyl donor deficiency and support the likely usefulness of late folate supplementation in at risk women.     

Effect of lead (Pb) on the systemic movement of RNA viruses in tobacco (Nicotiana tabacum var. Turkish)

Effect of various lead (Pb) concentrations on the systemic movement of RNA viruses was examined in tobacco plants. Prior to inoculation, plants were grown hydroponically for 6 days in Hoagland’s solution supplemented with five concentrations of lead nitrate [Pb(NO₃)₂]: 0.0 (control), 10, 15, 50, and 100 μM. Four different RNA viruses with different cell-to-cell movement mechanisms were used. Two weeks after inoculation lower and upper leaves of each treatment were harvested and examined for the presence of viral coat protein. In plants inoculated with Tobacco mosaic virus, Potato virus X, and Tobacco etch virus, TEM images and western blot assays confirmed the presence of viral coat proteins in the upper leaves of all lead treatments. However, in plants inoculated with Turnip vein-clearing virus (TVCV), no signs of viral particles were detected in the upper leaves of plants treated with 10 μM or 15 μM lead nitrate. In contrast, plants treated with high concentrations of lead nitrate (50 μM or 100 μM) showed viral particles in their upper leaves. In plants treated with 10 μM or 15 μM lead nitrate, callose accumulation was the same as in control plants. This suggests that non-toxic concentrations of lead nitrate may trigger the production of putative cellular factors in addition to callose that interfere with the TVCV systemic movement. In contrast, plants treated with 100 μM lead nitrate showed less callose as compared to control plants, facilitating the systemic movement of TVCV.     

Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene.

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.     

Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk.

BACKGROUND: CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus-1 (HIV-1). Recently, an inactive CCR5 allele (designated here as CCR5-2) was identified that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. The reports conflict on the effect of heterozygous CCR5-2 on HIV-1 susceptibility, and race and risk levels have not yet been fully analyzed. Here we report our independent identification of CCR5-2 and test its effects on HIV-1 pathogenesis in individuals with contrasting clinical outcomes, defined race, and quantified risk. MATERIALS AND METHODS: Mutant CCR5 alleles were sought by directed heteroduplex analysis of genomic DNA from random blood donors. Genotypic frequencies were then determined in (1) random blood donors from North America, Asia, and Africa; (2) HIV-1+ individuals; and (3) highly exposed-seronegative homosexuals with quantified risk. RESULTS: CCR5-2 was the only mutant allele found. It was common in Caucasians, less common in other North American racial groups, and not detected in West Africans or Tamil Indians. Homozygous CCR5-2 frequencies differed reciprocally in highly exposed-seronegative (4.5%, n = 111) and HIV-1-seropositive (0%, n = 614) Caucasians relative to Caucasian random blood donors (0.8%, n = 387). This difference was highly significant (p < 0.0001). By contrast, heterozygous CCR5-2 frequencies did not differ significantly in the same three groups (21.6, 22.6, and 21.7%, respectively). A 55% increase in the frequency of heterozygous CCR5-2 was observed in both of two cohorts of Caucasian homosexual male, long-term nonprogressors compared with other HIV-1+ Caucasian homosexuals (p = 0.006) and compared with Caucasian random blood donors. Moreover, Kaplan-Meier estimates indicated that CCR5-2 heterozygous seroconvertors had a 52.6% lower risk of developing AIDS than homozygous wild-type seroconvertors. CONCLUSIONS: The data suggest that homozygous CCR5-2 is an HIV-1 resistance factor in Caucasians with complete penetrance, and that heterozygous CCR5-2 slows the rate of disease progression in infected Caucasian homosexuals. Since the majority (approximately 96%) of highly exposed-seronegative individuals tested are not homozygous for CCR5-2, other resistance factors must exist. Since CCR5-2 homozygotes have no obvious clinical problems, CCR5 may be a good target for the development of novel antiretroviral therapy.     

CCR5Delta32 protein expression and stability are critical for resistance to human immunodeficiency virus type 1 in vivo

Human immunodeficiency virus type 1 (HIV-1) infection of individuals carrying the two alleles of the CCR5Delta32 mutation (CCR5(-/-)) has rarely been reported, but how the virus overcomes the CCR5Delta32 protective effect in these cases has not been delineated. We have investigated this in 6 infected (HIV(+)) and 25 HIV(-) CCR5(-/-) individuals. CD4(+) T lymphocytes isolated from HIV(-) CCR5(-/-) peripheral blood mononuclear cells (PBMCs) showed lower levels of CXCR4 expression that correlated with lower X4 Env-mediated fusion. Endogenous CCR5Delta32 protein was detected in all HIV(-) CCR5(-/-) PBMC samples (n = 25) but not in four of six unrelated HIV(+) CCR5(-/-) PBMC samples. Low levels were detected in another two HIV(+) CCR5(-/-) PBMC samples. The expression of adenovirus 5 (Ad5)-encoded CCR5Delta32 protein restored the protective effect in PBMCs from three HIV(+) CCR5(-/-) individuals but failed to restore the protective effect in PBMCs isolated from another three HIV(+) CCR5(-/-) individuals. In the latter samples, pulse-chase analyses demonstrated the disappearance of endogenous Ad5-encoded CCR5Delta32 protein and the accumulation of Ad5-encoded CCR5 during the chase periods. PBMCs isolated from CCR5(-/-) individuals showed resistance to primary X4 but were readily infected by a lab-adapted X4 strain. Low levels of Ad5-encoded CCR5Delta32 protein conferred resistance to primary X4 but not to lab-adapted X4 virus. These data provide strong support for the hypothesis that the CCR5Delta32 protein actively confers resistance to HIV-1 in vivo and suggest that the loss or reduction of CCR5Delta32 protein expression may account for HIV-1 infection of CCR5(-/-) individuals. The results also suggest that other cellular or virally induced factors may be involved in the stability of CCR5Delta32 protein.