A block in virus-like particle maturation following assembly of murine leukaemia virus in insect cells

Expression of the murine leukaemia virus (MLV) major Gag antigen p65(Gag) using the baculovirus expression system leads to efficient assembly and release of virus-like particles (VLP) representative of immature MLV. Expression of P180(Gag-Pol), facilitated normally in mammalian cells by readthrough of the p65(Gag) termination codon, also occurs efficiently in insect cells to provide a source of the MLV protease and a pattern of p65(Gag) processing similar to that observed in mammalian cells. VLP release from P180(Gag-Pol) expressing cells however remains essentially immature with disproportionate levels of the uncleaved p65(Gag) precursor when compared to the intracellular Gag profile. Changing the p65(Gag) termination codon altered the level of p65(Gag) and p180(Gag-Pol) within expressing cells but did not alter the pattern of released VLP, which remained immature. Coexpression of p65(Gag) with a fixed readthrough p180(Gag-Pol) also led to only immature VLP release despite high intracellular protease levels. Our data suggest a mechanism that preferentially selects uncleaved p65(Gag) for the assembly of MLV in this heterologous expression system and implies that, in addition to their relative levels, active sorting of the correct p65(Gag) and p180(Gag-Pol) ratios may occur in producer cells. (C) 2003 Elsevier Inc. All rights reserved.

Indirect population dynamic benefits of altered life-history trade-offs in response to egg harvesting

Variations in demographic rates due to differential resource allocation between individuals are important considerations in the development of accurate population dynamic models. Systematic harvesting can alter age structure and/or reduce population density, conferring indirect positive benefits on the source population as a result of a consequent redistribution of resources between the remaining individuals. Independently of effects mediated through changes in density and competition, demographic rates can also be influenced by within-individual competition for resources. Harvesting dependent life stages can reduce an individual's current reproductive costs, allowing increased investment in its future fecundity and survival. Although such changes in demographic rates are well known, there has been little exploration of the potential impact on population dynamics. We use empirical data collected from a successfully reintroduced population of the Mauritius kestrel Falco punctatus to explore the population consequences of manipulating reproductive effort through harvesting. Consequent increases in an individual's future fecundity and survival allow source populations to withstand longer and more intensive harvesting regimes without being exposed to an increase in extinction risk, increasing maximum sustainable yields. These effects may also buffer populations against the impacts of stochastic events, but directional shifts in environmental conditions that increase reproductive costs may have detrimental population-level effects.

Change in the effect of constant photoperiods on the rate of sexual maturation in modern genotypes of domestic pullet

1. Data for modern egg-type hybrids reared on constant daylengths show that, as expected, they mature more quickly than earlier genotypes. However, the constant photoperiod which gives earliest sexual maturity has not changed as a result of selection and is 10 h for both early and modern genotypes. 2. Further analysis showed that the rate of delay in sexual maturity for constant photoperiods above 10 h is similar for modern and for early hybrids ( +0.29 d for each incremental one hour of photoperiod), the response of modern hybrids below 10 h ( +4.22 d for each one-hour reduction in photoperiod) is more than double that of early hybrids ( +1.71 d/h).

Seed development, maturation and storage behaviour of Mimusops elengi L

Mass maturity (end of the seed-filling phase) occurred at about 72 days after flowering (DAF) in developing seeds of Mimusops elengi, at which time seed moisture content had declined to about 55%. The onset of ability to germinate was detected at 56 DAF and seeds showed 98% germination by 84 DAF. Tolerance of desiccation to 10% moisture content was first detected at 70 DAF and was maximal by 84 DAF. Delaying collection by a further 14 days to 98 DAF, when fruits began to be shed, reduced seed viability, particularly for seeds first dried to 10% moisture content. Hence the best time for seed collection appears to be about 14 days before fruits shed. In a separate investigation with six different seed lots, desiccation below about 8-12% moisture content reduced viability (considerably in some lots). The viability of dry seeds (below about 10% moisture content) stored hermetically was reduced at cool temperatures (5 degrees C and below), and none survived storage at sub-zero temperatures. The results suggest that Mimusops elengi shows intermediate seed storage behaviour and that the optimal hermetic seed storage environment is about 10% moisture content at 10 degrees C, while short-term, moist, aerated storage at high (40%) moisture content is also feasible.

Sterol content analysis suggests altered eburicol 14 alpha-demethylase (CYP51) activity in isolates of Mycosphaerella graminicola adapted to azole fungicides

The recent decline in the effectiveness of some azole fungicides in controlling the wheat pathogen Mycosphaerella graminicola has been associated with mutations in the CYP51 gene encoding the azole target, the eburicol 14 alpha-demethylase (CYP51), an essential enzyme of the ergosterol biosynthesis pathway. In this study, analysis of the sterol content of M. graminicola isolates carrying different variants of the CYP51 gene has revealed quantitative differences in sterol intermediates, particularly the CYP51 substrate eburicol. Together with CYP51 gene expression studies, these data suggest that mutations in the CYP51 gene impact on the activity of the CYP51 protein.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Immunohistochemical localization of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in bovine oocytes during in vitro maturation and fertilization

The aim of this study was to evaluate the distribution of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in immature oocytes and in matured oocytes before and after IVF. Denuded oocytes were submitted to a whole-mount immunofluorescence procedure. Specimens were imaged and fluorescent intensities quantified by scanning laser confocal microscopy. Immunoreactivity for inhibin alpha subunit (both alpha(C) and pro-alpha. regions), abundant in the ooplasm of immature oocytes, decreased after maturation (a 68% and 88% decrease, respectively; P < 0.001), but increased after IVF by 2- and 5.7-fold, respectively (P < 0.01). Intense staining for PA was detected in immature oocytes (predominantly in the outer ooplasm and zona pellucida) but after maturation and fertilization it was localized mainly in the zona pellucida, perivitelline space and oolemma. Immunoreactivity for RA in the ooplasm decreased by 58% after maturation (P < 0.001) but increased again by 75% after fertilization (P < 0.01). Immunoreactivity for beta(B) was localized mainly in the zona pellucida and did not change after maturation. However, immurloreactivity for beta(B) was not detected in the zona pellucida after fertilization, but remained unchanged in unfertilized oocytes. Immunoreactivity for follistatin was detected in the ooplasm and zona pellucida of immature oocytes but decreased progressively in the ooplasm after maturation (a 63% decrease; P < 0.001) and did not change after IVF. Examination of partially denuded cumulus-oocyte complexes confirmed abundant expression of alpha(C), pro-alpha, beta(A) and follistatin immunoreactivity in cumulus cells, whereas beta(B) subunit staining was weak or absent in cumulus cells, but intense in the zona pellucida. In conclusion, the present study shows that qualitative and quantitative changes in the distribution of inhibin/activin subunits and follistatin accompany oocyte maturation and fertilization. The possibility, indicated by these observations, that activin A and activin B may play distinct roles in bovine oocyte maturation and fertilization warrants further study.

Fecal microbiota in patients receiving enteral feeding are highly variable and may be altered in those who develop diarrhea

Background: The pathogenesis of diarrhea in patients receiving enteral feeding includes colonic water secretion, antibiotic prescription, and enteropathogenic colonization, each of which involves an interaction with the gastrointestinal microbiota. Objective: The objective was to investigate temporal changes in the concentrations of fecal microbiota and short-chain fatty acids (SCFAs) in patients starting 14-d of enteral feeding and to compare these changes between patients who do and do not develop diarrhea. Design: Twenty patients starting exclusive nasogastric enteral feeding were monitored for 14 d. Fecal samples were collected at the start, middle, and end of this period and were analyzed for major bacterial groups by using culture independent fluorescence in situ hybridization and for SCFAs by using gas-liquid chromatography. Results: Although no significant changes in fecal microbiota or SCFAs were observed during enteral feeding, stark alterations occurred within individual patients. Ten patients (50%) developed diarrhea, and these patients had significantly higher concentrations of clostridia (P = 0.026) and lower concentrations (P = 0.069) and proportions (P = 0.029) of bifidobacteria. Patients with and without diarrhea had differences in the proportion of bifidobacteria (median: 0.4% and 3.7%; interquartile range: 0.8 compared with 4.3; P = 0.035) and clostridia (median: 10.4% and 3.7%; interquartile range: 14.7 compared with 7.0; P = 0.063), respectively, even at the start of enteral feeding. Patients who developed diarrhea had higher concentrations of total fecal SCFAs (P = 0.044), acetate (P = 0.029), and butyrate (P = 0.055). Conclusion: Intestinal dysbiosis occurs in patients who develop diarrhea during enteral feeding and may be involved in its pathogenesis. Am J Clin Nutr 2009; 89: 240-7.

Emergence of variants with altered survival properties in stationary phase cultures of Campylobacter jejuni

During the stationary phase of Campylobacter jejuni NCTC 11351 viable numbers fluctuate in a characteristic fashion. After reaching the maximum cell count (ca. 2 X 10(9) CFU/ml) in early stationary phase (denoted phase 1), viable numbers subsequently decrease to about 10(6) CFU/ml after 48 h and then increase again to about 10(8) CFU/ml (denoted phase 2) before decreasing once more to a value intermediate between the previous maximum and minimum values. To investigate whether the increase in viable numbers following the initial decline was due to the emergence of a new strain with a growth advantage in stationary phase analogous to the 'GASP' phenotype described in Escherichia coli [Science 259 (1993) 1757], we conducted mixed culture experiments with cells from the original culture and antibiotic-resistant marked organisms isolated from the re-growth phase. In many experiments of this type, strains isolated from phase 2 failed to out-compete the original strain and we have thus been unable to demonstrate a convincing GASP phenotype. However, strains isolated from phase 2 showed a much lower rate of viability loss in early stationary phase and a small increase in resistance to aeration, peroxide challenge and heat, indicating that the emergent strain was different from the parent. These results support the view that dynamic population changes occur during the stationary phase of C jejuni that may play a role in the survival of this organism. (C) 2003 Published by Elsevier B.V.

Effect of altered dietary n-3 fatty acid intake upon plasma lipid fatty acid composition, conversion of [C-13]alpha-linolenic acid to longer-chain fatty acids and partitioning towards beta-oxidation in older men

The effect of increased dietary intakes of alpha-linolenic acid (ALNA) or eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for 2 months upon plasma lipid composition and capacity for conversion of ALNA to longer-chain metabolites was investigated in healthy men (52 (SD 12) years). After a 4-week baseline period when the subjects substituted a control spread, a test meal containing [U-C-13]ALNA (700 mg) was consumed to measure conversion to EPA, docosapentaenoic acid (DPA) and DHA over 48 h. Subjects were then randomised to one of three groups for 8 weeks before repeating the tracer study: (1) continued on same intake (control, n 5); (2) increased ALNA intake (10 g/d, n 4); (3) increased EPA+DHA intake (1.5 g/d, n 5). At baseline, apparent fractional conversion of labelled ALNA was: EPA 2.80, DPA 1.20 and DRA 0.04%. After 8 weeks on the control diet, plasma lipid composition and [C-13]ALNA conversion remained unchanged compared with baseline. The high-ALNA diet resulted in raised plasma triacylglycerol-EPA and -DPA concentrations and phosphatidylcholine-EPA concentration, whilst [C-13]ALNA conversion was similar to baseline. The high-(EPA+DHA) diet raised plasma phosphatidylcholine-EPA and -DHA concentrations, decreased [C-13]ALNA conversion to EPA (2-fold) and DPA (4-fold), whilst [C-13]ALNA conversion to DHA was unchanged. The dietary interventions did not alter partitioning of ALNA towards beta-oxidation. The present results indicate ALNA conversion was down-regulated by increased product (EPA+DHA) availability, but was not up-regulated by increased substrate (ALNA) consumption. This suggests regulation of ALNA conversion may limit the influence of variations in dietary n-3 fatty acid intake on plasma lipid compositions.

Effect of temperature and salt on the maturation of white-salted cheese

White-salted cheeses were prepared from ultrafiltered (UF) cows' milk and salted to give final salt-in-moisture (SM) levels of 2.5, 3.2 and 4.0%. The cheeses were stored at 5degreesC and 10degreesC for up to 15 weeks. The microflora was dominated by lactic acid bacteria (LAB) but some mould growth was evident within 15 weeks at all SM levels and both temperatures. Levels of water-soluble nitrogen (WSN), attributed to chymosin activity, increased significantly with time, the rate being inversely proportional to the SM level and increasing with storage temperature. Similar effects were noted for trichloroacetic acid-soluble nitrogen (TCA-SN) and free amino acid (FAA) levels, both of which would also be affected by bacterial protease activity. The proteolytic activity was reflected by changes in the hardness and fracturability of the cheeses.