Production of extracellular enzymes and aflatoxins in solid substrate fermentation with aflatoxigenic<Emphasis Type="Italic">Aspergillus</Emphasis> spp.

AflatoxigenicAspergillus flavus andAspergillus parasiticus were subjected to solid substrate fermentation process for 6 days to determine the formation of aflatoxins and production of extracellular enzymes (amyloglucosidase, cellulase, invertase and proteinase). Both organisms produced enzymes which generally increased with fermentation.Aspergillus flavus produced four enzymes whereasA. parasiticus produced three with no proteinase activity.Aspergillus parasiticus produced aflatoxins B₁, B₂ and G₁ but no G₂ andA. flavus produced aflatoxins B₁ and B₂. Invertase showed the highest activity withA. parasiticus and that corresponded with the highest total toxin produced. The enzyme activities were higher withA. parasiticus thanA. flavus although total toxins produced byA. parasiticus were lower than total toxins produced byA. flavus under the same environmental conditions.     

Molecular dynamics simulations and free energy calculations of base flipping in dsRNA

The family of adenosine deaminases acting on RNA (ADARs) targets adenosines in RNA that is mainly double stranded. Some substrates are promiscuously deaminated whereas others, such as the mammalian glutamate receptor B (gluR-B) pre-mRNA, are more selectively deaminated. Many DNA/RNA-base modification enzymes use a base flipping mechanism to be able to reach their target base and it is believed that ADARs function in a similar way. In this study we used molecular dynamics (MD) simulations to describe two sites on the gluR-B pre-mRNA, the selectively targeted R/G site and the nontargeted 46 site, in an attempt to explain the substrate specificity. We used regular MD and also a forced base flipping method with umbrella sampling to calculate the free energy of base opening. Spontaneous opening of the mismatched adenosine was observed for the R/G site but not for the 46 site.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Genetic diversity of eleven European pig breeds

A set of eleven pig breeds originating from six European countries, and including a small sample of wild pigs, was chosen for this study of genetic diversity. Diversity was evaluated on the basis of 18 microsatellite markers typed over a total of 483 DNA samples collected. Average breed heterozygosity varied from 0.35 to 0.60. Genotypic frequencies generally agreed with Hardy-Weinberg expectations, apart from the German Landrace and Schwäbisch-Hällisches breeds, which showed significantly reduced heterozygosity. Breed differentiation was significant as shown by the high among-breed fixation index (overall F_ST = 0.27), and confirmed by the clustering based on the genetic distances between individuals, which grouped essentially all individuals in 11 clusters corresponding to the 11 breeds. The genetic distances between breeds were first used to construct phylogenetic trees. The trees indicated that a genetic drift model might explain the divergence of the two German breeds, but no reliable phylogeny could be inferred among the remaining breeds. The same distances were also used to measure the global diversity of the set of breeds considered, and to evaluate the marginal loss of diversity attached to each breed. In that respect, the French Basque breed appeared to be the most "unique" in the set considered. This study, which remains to be extended to a larger set of European breeds, indicates that using genetic distances between breeds of farm animals in a classical taxonomic approach may not give clear resolution, but points to their usefulness in a prospective evaluation of diversity.     

Clinical and angiographic results with the ACS MULTI-LINK DUET trade mark Coronary Stent System - the DUET Study

BACKGROUND: The DUET Study is a multicenter prospective efficacy and safety evaluation of the ACS MULTI-LINK DUET coronary stainless steel balloon-expandable stent. AIMS: The primary objective was to determine the one-month incidence of MACE (major adverse cardiac events). The secondary objectives were the acute success rate, the restenosis and reocclusion rates (assessed by quantitative coronary angiography (QCA)) at six months and the occurrence of MACE in hospital and at six months. METHODS: Two hundred and ten patients were enrolled between February and June 1998 in 18 European centers. Successful stent placement was achieved in 209 patients. All patients were treated with ticlopidine 500 mg/day for one month and with aspirin >/=100 mg/day. To allow the investigators to gain familiarity with the stent system, the first one to three patients per center formed a separate lead-in population leaving an intention-to-treat population of 157 patients. The majority of the intention-to-treat population were male (79%); 28% had unstable angina, 69% had stable angina, 44% had had a previous myocardial infarction, 15% had had a previous percutaneous transluminal coronary angioplasty, and 3% had a history of stroke. The target vessel was 38.5% left anterior descending artery, 20.5% left circumflex artery and 41.0% right coronary artery. RESULTS: All but one of the intention-to-treat patients were effectively stented (17 required multiple stents). Six-month angiographic follow-up was available in 90% of the intention-to-treat population. Minimal lumen diameter (MLD) postprocedure was 2.61 +/- 0.33 mm, with a residual diameter stenosis of 16%. Six-month follow-up data showed an MLD of 1.87 +/- 0.56 mm with a residual diameter stenosis of 36%. The binary restenosis rate (>/=50% residual stenosis) was 15.6%. Up to one month following the procedure 94.9% of the population was MACE-free, with two subacute occlusions. At six months all patients were alive, of whom 82.8% were MACE-free, and 73% were free of anginal complaints. CONCLUSION: The results observed in the current DUET registry are comparable to data of other balloon-expandable-stent trials, with a low incidence of clinical events at follow-up.     

Tesaglitazar, a dual PPAR{alpha}/{gamma} agonist, ameliorates glucose and lipid intolerance in obese Zucker rats

Insulin resistance, impaired glucose tolerance, high circulating levels of free fatty acids (FFA), and postprandial hyperlipidemia are associated with the metabolic syndrome, which has been linked to increased risk of cardiovascular disease. We studied the metabolic responses to an oral glucose/triglyceride (TG) (1.7/2.0 g/kg lean body mass) load in three groups of conscious 7-h fasted Zucker rats: lean healthy controls, obese insulin-resistant/dyslipidemic controls, and obese rats treated with the dual peroxisome proliferator-activated receptor alpha/gamma agonist, tesaglitazar, 3 mumol.kg(-1).day(-1) for 4 wk. Untreated obese Zucker rats displayed marked insulin resistance, as well as glucose and lipid intolerance in response to the glucose/TG load. The 2-h postload area under the curve values were greater for glucose (+19%), insulin (+849%), FFA (+53%), and TG (+413%) compared with untreated lean controls. Treatment with tesaglitazar lowered fasting plasma glucose, improved glucose tolerance, substantially reduced fasting and postload insulin levels, and markedly lowered fasting TG and improved lipid tolerance. Fasting FFA were not affected, but postprandial FFA suppression was restored to levels seen in lean controls. Mechanisms of tesaglitazar-induced lowering of plasma TG were studied separately using the Triton WR1339 method. In anesthetized, 5-h fasted, obese Zucker rats, tesaglitazar reduced hepatic TG secretion by 47%, increased plasma TG clearance by 490%, and reduced very low-density lipoprotein (VLDL) apolipoprotein CIII content by 86%, compared with obese controls. In conclusion, the glucose/lipid tolerance test in obese Zucker rats appears to be a useful model of the metabolic syndrome that can be used to evaluate therapeutic effects on impaired postprandial glucose and lipid metabolism. The present work demonstrates that tesaglitazar ameliorates these abnormalities and enhances insulin sensitivity in this animal model.     

A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab). The purified huMab had an affinity of 5 nM and effectively mediated tumor cell killing in in vitro and in vivo assays. These experiments show that nonimmunized phage antibody display libraries can be used to obtain high-affinity, functional, and clinically applicable huMabs directed against a tumor-associated antigen.     

Signal Transduction Pathways in Mycorrhizal Associations: Comparisons with the Rhizobium-Legume Symbiosis

A number of genera of soil fungi interact with plant roots to establish symbiotic associations whereby phosphate acquired by the fungus is exchanged for fixed carbon from the plant. Recent progress in investigating these associations, designated as mycorrhizae (sing., mycorrhiza), has led to the identification of specific steps in the establishment of the symbiosis in which the fungus and the plant interact in response to various molecular signals. Some of these signals are conserved with those of the Rhizobium-legume nitrogen-fixing symbiosis, suggesting that the two plant-microbe interactions share a common signal transduction pathway. Nevertheless, only legume hosts nodulate in response to Rhizobium, whereas the vast majority of flowering plants establish mycorrhizal associations. The key questions for the future are: what are the signal molecules produced by mycorrhizal fungi and how are they perceived by the plant? Copyright 1998 Academic Press.     

Sterigmatocystin — incidence, fate and production byA versicolor in ras cheese

Sterigmatocystin (STG) is a toxic metabolite produced by severalAspergillus species. Because of its toxic and carcinogenic properties the occurrence of STG in food is considered to represent a potential hazard to man. The present study was designed to investigate following points:

Primary structure of a precursor to the aspartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen

In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The deduced amino acid sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acid residues with a total molecular weight of 38,701. Clusters of identities around the active site cleft support the assumption that these proteinases have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.