Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, <Emphasis Type="Italic">Butyrivibrio fibrisolvens</Emphasis>

Background  

Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA.     

ESR evidence for sequential divalent cation binding in higher plant cell walls

Electron spin resonance linewidth measurements have been made on intact cell walls exchanged with various combinations of Mn²⁺ and Ca²⁺. These experiments were performed to find the Mn²⁺ nearest-neighbor distance and thereby determine whether carboxylate-Mn²⁺ complexes potentiate ion association at adjacent sites on cell wall polyuronides. Our results show that as the fraction of available binding sites occupied by Mn²⁺ increased from 2% to 27%, the nearest-neighbor distance parameter decreased only from 14 to 11 Å. These distances are close to polyuronide interanionic spacings. The small change in the distance parameter with concentration is evidence for sequential rather than random binding. Competitive ion-exchange with Ca²⁺ was found to reduce the Mn²⁺ spin-spin line broadening at similar total bound Mn²⁺ concentrations. This is expected only if Ca²⁺ competes at adjacent sites. The data presented offer strong support for the hypothesis that carboxylate groups near already occupied sites have a greater affinity for divalent cations than other sites along the polyuronide main chain.     

Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier

The cornified envelope is assembled from transglutaminase cross-linked proteins and lipids in the outermost epidermal layers and is essential for skin barrier function. Involucrin, envoplakin, and periplakin form the protein scaffold on which the envelope assembles. To examine their combined function, we generated mice deficient in all three genes. The triple knockouts have delayed embryonic barrier formation and postnatal hyperkeratosis (abnormal accumulation of cornified cells) resulting from impaired desquamation. Cornified envelopes form but are ultrastructurally abnormal, with reduced lipid content and decreased mechanical integrity. Expression of proteases is reduced and the protease inhibitor, serpina1b, is highly upregulated, resulting in defective filaggrin processing and delayed degradation of desmoglein 1 and corneodesmosin. There is infiltration of CD4⁺ T cells and a reduction in resident γδ⁺ T cells, reminiscent of atopic dermatitis. Thus, combined loss of the cornified envelope proteins not only impairs the epidermal barrier, but also changes the composition of T cell subpopulations in the skin.     

Identification of protein and mannoprotein antigens of Candida albicans of relevance for the serodiagnosis of invasive candidiasis.

Antigens from Candida albicans blastoconidia and germ tubes were identified by two-dimensional electrophoresis and Western blotting and characterized by microsequencing, reactivity with concanavalin A, and a panel of human sera. Antigens identified included a polydispersed area in the acidic high-molecular-mass regions of blastoconidium and germ-tube extracts, and 16 antigens varying in molecular masses and isoelectric points (pIs). The majority of the detected antigens, especially those in the polydispersed region, showed mannosyl groups, as determined by concanavalin A reactivity. Antibodies present in sera from patients with invasive candidiasis showed high reactivity with a number of antigens not detected with sera from blood donors. Eight of the 16 antigens could be identified by reactivity with monoclonal antibodies or by microsequencing. Five antigens showed homology with five enzymes previously described as antigens in C. albicans: enolase, phosphoglycerate kinase, malate dehydrogenase, and two isoforms of the fructose biphosphate aldolase. However, to our knowledge, this is the first report of the immunogenic activity of a kexin precursor, a mitochondrial complex I chaperone, and a diacylglycerol kinase catalytic domain from C. albicans. Antigens described in this study may be of potential interest for the serodiagnosis of invasive candidiasis.     

Protection against tuberculosis in Eurasian wild boar vaccinated with heat-inactivated Mycobacterium bovis

Tuberculosis (TB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex continues to affect humans and animals worldwide and its control requires vaccination of wildlife reservoir species such as Eurasian wild boar (Sus scrofa). Vaccination efforts for TB control in wildlife have been based primarily on oral live BCG formulations. However, this is the first report of the use of oral inactivated vaccines for controlling TB in wildlife. In this study, four groups of 5 wild boar each were vaccinated with inactivated M. bovis by the oral and intramuscular routes, vaccinated with oral BCG or left unvaccinated as controls. All groups were later challenged with a field strain of M. bovis. The results of the IFN-gamma response, serum antibody levels, M. bovis culture, TB lesion scores, and the expression of C3 and MUT genes were compared between these four groups. The results suggested that vaccination with heat-inactivated M. bovis or BCG protect wild boar from TB. These results also encouraged testing combinations of BCG and inactivated M. bovis to vaccinate wild boar against TB. Vaccine formulations using heat-inactivated M. bovis for TB control in wildlife would have the advantage of being environmentally safe and more stable under field conditions when compared to live BCG vaccines. The antibody response and MUT expression levels can help differentiating between vaccinated and infected wild boar and as correlates of protective response in vaccinated animals. These results suggest that vaccine studies in free-living wild boar are now possible to reveal the full potential of protecting against TB using oral M. bovis inactivated and BCG vaccines.     

Endoplasmic reticulum-associated degradation-induced dissociation of class II invariant chain complexes containing a glycosylation-deficient form of p41

The quality control system in the secretory pathway can identify and eliminate misfolded proteins through endoplasmic reticulum-associated degradation (ERAD). ERAD is thought to occur by retrotranslocation through the Sec61 complex into the cytosol and degradation by the proteasome. However, the extent of disassembly of oligomeric proteins and unfolding of polypeptide chains that is required for retrotranslocation is not fully understood. In this report we used a glycosylation mutant of the p41 isoform of invariant chain (Ii) to evaluate the ability of ERAD to discriminate between correctly folded and misfolded subunits in an oligomeric complex. We show that loss of glycosylation at position 239 of p41 does not detectably affect Ii trimerization or association with class II but does result in a defect in endoplasmic reticulum export of Ii that ultimately leads to its degradation via the ERAD pathway. Although class II associated with the mutated form of p41 is initially retained in the endoplasmic reticulum, it is subsequently released and traffics through the Golgi to the plasma membrane. ERAD-mediated degradation of the mutant p41 is dependent on mannose trimming and inhibition of mannosidase I stabilizes Ii. Interestingly, inhibition of mannosidase I also results in prolonged association between the mutant Ii and class II, indicating that complex disassembly and release of class II is linked to mannosidase-dependent ERAD targeting of the misfolded Ii. These results suggest that the ERAD machinery can induce subunit disassembly, specifically targeting misfolded subunits to degradation and sparing properly folded subunits for reassembly and/or export.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.