Simian retrovirus-D serotype 1 (SRV-1) envelope glycoproteins gp70 and gp20: expression in yeast cells and identification of specific antibodies in sera from monkeys that recovered from SRV-1 infection.

The gp70 and transmembrane gp20 envelope proteins of simian retrovirus-D serotype 1 (SRV-1) were expressed in Saccharomyces cerevisiae as fusion proteins with human superoxide dismutase (SOD). Expression of the SOD-gp70 and SOD-gp20 sequences yielded fusion proteins of 52 and 29 kilodaltons, respectively. The yeast-expressed SRV-1 envelope proteins were used in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in the sera of rhesus macaques that recovered from SRV-1. Sera from 47 of 49 such monkeys tested positive for antibodies to the SOD-gp70 fusion protein, while 45 of 49 reacted positively to SOD-gp20. None of 26 SRV-1-nonexposed monkeys tested positive in either ELISA. Monkeys immunized with the recombinant SRV-1 gp20 and gp70 proteins made good ELISA and Western blot (immunoblot) antibodies to whole SRV-1. This antibody was not neutralizing in vitro, however.     

Disturbed nonlinear multispecies models in ecology.

We analyze a disturbed form of the general Lotka-Volterra model of an ecosystem with m interacting species. The disturbances act on the intrinsic growth rates of the species and are assumed to be bounded but otherwise unknown. We employ a Lyapunov technique and the concept of "reachable set" from control theory to estimate the set of all possible population densities that are attainable as a result of the disturbances. To calculate estimates for this reachable set, a number of numerical methods that entail the solution to one or more global optimization problems are developed. Specific examples involving two, three, and four species are solved. We also derive an explicit analytical expression that represents an estimate for the reachable set in the m-dimensional case. The estimate is conservative but can be evaluated without carrying out any optimization procedure. We show that methods developed in this paper can be applied to certain other types of nonlinear ecosystem models.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Immune response to a new hepatitis B vaccine in healthcare workers who had not responded to standard vaccine: randomised double blind dose-response study.

OBJECTIVE: To evaluate the immunogenicity and reactogenicity of a new triple S recombinant hepatitis B vaccine in a cohort of healthy people in whom currently licensed hepatitis B vaccines had persistently not induced an immune response. DESIGN: Single centre, randomised, double blind, dose-response study. SETTING: Research vaccine evaluation centre at a teaching hospital. SUBJECTS: 100 healthcare workers aged 18-70 years with a history of failure to seroconvert after at least four doses of a licensed hepatitis B vaccine containing the S component. INTERVENTION: Each subject was randomly allocated two doses of 5, 10, 20, or 40 micrograms of a new hepatitis B vaccine two months apart. MAIN OUTCOME MEASURES: Immunogenicity of the four doses. Seroconversion and seroprotection were defined as an antibody tire > 10 IU/l and > 100 IU/l respectively against an international antibody standard. RESULTS: 69 subjects seroconverted after a single dose of the vaccine. After the booster vaccination one other subject seroconverted, bringing the overall seroconversion rate to 70%. Fifteen subjects given 5 micrograms of vaccine, 19 given 10 micrograms, 16 given 20 micrograms, and 20 given 40 micrograms seroconverted. Seroconversion rates in the four antigen dose groups were 60% (15/25), 76% (19/25), 64% (16/25), and 80% (20/25). After the booster dose there was no significant dose-response effect on the overall seroconversion rate, although the small sample size meant that a clinically important dose-response could not be ruled out. CONCLUSION: A single dose of 20 micrograms of the vaccine was as effective as two doses of either 40 micrograms or 20 micrograms of this vaccine formulation in terms of seroconversion, seroprotection, and geometric mean titres.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

The Effect of Aerobic Exercise on Intrahepatocellular and Intramyocellular Lipids in Healthy Subjects

Background

Intrahepatocellular (IHCL) and intramyocellular (IMCL) lipids are ectopic lipid stores. Aerobic exercise results in IMCL utilization in subjects over a broad range of exercise capacity. IMCL and IHCL have been related to impaired insulin action at the skeletal muscle and hepatic level, respectively. The acute effect of aerobic exercise on IHCL is unknown. Possible regulatory factors include exercise capacity, insulin sensitivity and fat availability subcutaneous and visceral fat mass).

Aim

To concomitantly investigate the effect of aerobic exercise on IHCL and IMCL in healthy subjects, using Magnetic Resonance spectroscopy.

Methods

Normal weight, healthy subjects were included. Visit 1 consisted of a determination of VO_2max on a treadmill. Visit 2 comprised the assessment of hepatic and peripheral insulin sensitivity by a two-step hyperinsulinaemic euglycaemic clamp. At Visit 3, subcutaneous and visceral fat mass were assessed by whole body MRI, IHCL and IMCL before and after a 2-hours aerobic exercise (50% of VO_2max) using ¹H-MR-spectroscopy.

Results

Eighteen volunteers (12M, 6F) were enrolled in the study (age, 37.6±3.2 years, mean±SEM; VO_2max, 53.4±2.9 mL/kg/min). Two hours aerobic exercise resulted in a significant decrease in IMCL (−22.6±3.3, % from baseline) and increase in IHCL (+34.9±7.6, % from baseline). There was no significant correlation between the exercise-induced changes in IMCL and IHCL and exercise capacity, subcutaneous and visceral fat mass and hepatic or peripheral insulin sensitivity.

Conclusions

IMCL and IHCL are flexible ectopic lipid stores that are acutely influenced by physical exercise, albeit in different directions.

Trial Registration

ClinicalTrial.gov {"type":"clinical-trial","attrs":{"text":"NCT00491582","term_id":"NCT00491582"}}NCT00491582     

Eight new microsatellite markers in Atlantic cod (Gadus morhua L.) derived from an enriched genomic library

One hundred and fifty random clones from an enriched genomic library of Atlantic cod were sequenced. Primer pairs were designed for 15 microsatellites containing perfect di‐, tri‐, tetra‐ and hexanucleotide repeats and characterized in 96 unrelated fish. Eight markers were successfully amplified, with the number of alleles ranging from two to nine per locus and observed heterozygosity ranging from 0.341 to 0.977. Loci Gmo‐G13 and Gmo‐G14 had a significant excess of homozygotes. All loci conformed to the Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed no significant departure from the null hypothesis between any of the loci.