Profiling the surfacome of Staphylococcus aureus

Staphylococcus aureus is a widespread opportunistic pathogen that can cause a wide variety of life‐threatening diseases. Especially for the colonization of human tissues and the development of invasiveness, surface‐exposed proteins are of major importance. In the present studies, we optimized a proteolytic shaving approach to identify those surface‐exposed protein domains – the surfacome – of S. aureus that are accessible to extracellular bio‐macromolecules, for example in the host milieu. Subsequently, this approach was applied to define the surfacomes of four strains with different genetic backgrounds. This resulted in the identification of 96 different proteins. Surprisingly, the overlap between the surfacomes of the four different strains was below 10% and each strain displayed its own characteristic set of surface‐exposed proteins. The data were also evaluated at the peptide level and here we observed a similar phenomenon. From 190 unique peptides only five were commonly found in the four strains. Besides well known cell wall proteins, we also identified some essential proteins, several yet uncharacterized exported proteins and predicted intracellular proteins. These results show for the first time that the cell surface of different S. aureus strains is not only highly variable, but also that the displayed proteins are very heterogeneous.     

Genetic variation at the porcine MYF-5 gene locus. Lack of association with meat production traits

The number of muscle fibers at birth appears to determine the maximal lean meat growth capacity in pigs and in cattle. Development of muscle fibers is regulated by the MyoD gene family consisting of MyoD1, myf-5, myf-6, and myogenin. Myf-5 is expressed in proliferating myoblasts. Here we report the genomic sequence of the porcine myf-5 gene with three microsatellites and two RFLPs located close to the coding sequences. Two of the microsatellites are located in the promoter region. The allelic distribution differs between breeds and selection lines. In two GY selection lines, 1216 pigs of two-generation families were genotyped for the HinfI RFLP, which was segregating in the GY breed. The other polymorphic loci are physically linked to this RFLP locus, and therefore the results can be extrapolated to these loci. Statistical analysis revealed no association with birth weight, growth rate, weight at slaughter age, carcass meat weight, and backfat thickness. Thus, in this study myf-5 did not explain genetic variation in meat (muscle) development in pigs. Received: 9 July 1998 / Accepted: 22 September 1998     

Fatty acid desaturation: variations on an oxidative theme

Significant progress in our understanding of the mechanism of fatty acid desaturation has been achieved. The site of initial oxidation has been determined for several membrane-bound desaturases and a common cryptoregiochemical theme has been revealed. The results of several studies, including a detailed analysis of a soluble plant desaturase system, point to a close mechanistic relationship between dehydrogenation and hydroxylation pathways.     

An Extended Bidomain Framework Incorporating Multiple Cell Types

The muscular layers within the walls of the gastrointestinal tract contain two distinct cell types, the interstitial cells of Cajal and smooth muscle cells, which together produce rhythmic depolarizations known as slow waves. The bidomain model of tissue-level electrical activity consists of single intracellular and extracellular domains separated by an intervening membrane at all points in space and is therefore unable to adequately describe the presence of two distinct cell types in its conventional form. Here, an extension to the bidomain framework is presented whereby multiple interconnected cell types can be incorporated. Although the derivation is focused on the interactions of the interstitial cells of Cajal and smooth muscle cells, the conceptual framework can be more generally applied. Simulations demonstrating the feasibility of the proposed model are also presented.     

Delta11 desaturases of Trichoplusia ni and Spodoptera littoralis exhibit dual catalytic behaviour

The Delta(11) desaturases found in moths such as Spodoptera littoralis play a critical role in the biosynthesis of their sex pheromones. The ability to functionally express these enzymes in yeast has allowed one to study the transformation of long-chain fatty acyl substrates to their 11-ene products in greater mechanistic detail. In this article, we report on the detection and quantitation of a minor 11-hydroxylated byproduct (0.1% of total fatty acids), which is formed by the Delta(11) desaturases found in Trichoplusia ni and Spodoptera littoralis. The position of the hydroxyl group was determined by characteristic mass spectral fragmentation of the trimethylsilyl derivatives and is in accord with predictions based on previous mechanistic investigations of the Spodoptera Delta(11) desaturase. The level of 11-hydroxylation was insensitive to the mode of desaturase expression (constitutive vs. induced) and the presence or absence of a b5-fusion domain. Our findings suggest that in future, a search for hydroxylated products should be included in functional analyses of insect desaturase genes.     

Metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine CCL2 in the central nervous system

Inflammatory leukocytes infiltrate the CNS parenchyma in neuroinflammation. This involves cellular migration across various structures associated with the blood-brain barrier: the vascular endothelium, the glia limitans, and the perivascular space between them. Leukocytes accumulate spontaneously in the perivascular space in brains of transgenic (Tg) mice that overexpress CCL2 under control of a CNS-specific promoter. The Tg mice show no clinical symptoms, even though leukocytes have crossed the endothelial basement membrane. Pertussis toxin (PTx) given i.p. induced encephalopathy and weight loss in Tg mice. We used flow cytometry, ultra-small superparamagnetic iron oxide-enhanced magnetic resonance imaging, and immunofluorescent staining to show that encephalopathy involved leukocyte migration across the glia limitans into the brain parenchyma, identifying this as the critical step in inducing clinical symptoms. Metalloproteinase (MPs) enzymes are implicated in leukocyte infiltration in neuroinflammation. Unmanipulated Tg mice had elevated expression of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase (MMP)-10, and -12 mRNA in the brain. PTx further induced expression of tissue inhibitor of metalloproteinase-1, metalloproteinase disintegrins-12, MMP-8, and -10 in brains of Tg mice. Levels of the microglial-associated MP MMP-15 were not affected in control or PTx-treated Tg mice. PTx also up-regulated expression of proinflammatory cytokines IL-1beta and TNF-alpha mRNA in Tg CNS. Weight loss and parenchymal infiltration, but not perivascular accumulation, were significantly inhibited by the broad-spectrum MP inhibitor BB-94/Batimastat. Our finding that MPs mediate PTx-induced parenchymal infiltration to the chemokine-overexpressing CNS has relevance for the pathogenesis of human diseases involving CNS inflammation, such as multiple sclerosis.