Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands

InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion.     

Analysis of the collapsibility of the upper airway in a spectrum of sleep-disordered breathing: a modelling approach

Using a simplified model of the upper airways with two independent collapsible elements (nostrils and hypo-pharynx), we calculated the cross-sectional area of these two elements, taking into account pressure drops. We experimentally measured flow and pressure in the fossa and hypo-pharynx in various syndromes. This allowed us to compare the behaviour of the area supplied by our model with the aerodynamic resistance that is often used to analyse upper airway flow limitation events. We showed that nostril and hypo-pharyngeal areas are better correlated than the resistance values and thus concluded that the pressure divided by the square of the flow is a better parameter for analysing flow limitation in upper airways than resistance. Owing to its simplicity, our model is able to supply the area of the collapsible element in real time, which is impossible with more sophisticated models.     

Effect of native phosphocaseinate on the in vitro preservation of fresh semen

The fertilization capacity of goat sperm stored in milk extenders is approximately 12-24h. Long-term storage of goat sperm (up to 3 days) is desirable as it would confer greater flexibility to breeding farms. The aim of this study was to evaluate in vitro motility parameters of buck spermatozoa for up to 7 days of storage using skim milk or chemically defined extender supplemented with native phosphocaseinate (NPPC). Four experiments were conducted to determine optimum temperature (4 or 15 degrees C) and storage conditions (aerobic versus anaerobic), the effect of seminal plasma on sperm survival, the optimal concentration of NPPC and the effect of beta lactoglobulin (BL). Both skim milk and NPPC were found to be more efficient for preserving goat sperm at 4 degrees C than at 15 degrees C (P<0.01). Furthermore, when sperm was stored at 4 degrees C, no detrimental effects of seminal plasma were observed. Our results showed that motility parameters can be maintained with success until Day 4. However, NPPC-based extenders extend the in vitro survival to 7 days of storage. The optimal concentration of NPPC for the preservation of sperm cells for 4 days of storage was 81g/l and for 7 days of storage was 81 and 54g/l. No effect of the supplementation of the NPPC extender with BL was found.     

Projection structure of full length connexin 43 by electron cryo-crystallography

We previously used electron cryo-crystallography to determine the three-dimensional structure of recombinant gap junction channels formed by a C-terminal truncation mutant of Cx43 (11). The dodecameric channel is formed by the end-to-end docking of two hexameric connexons, each comprised of 24 transmembrane alpha-helices. We have now generated two-dimensional crystals of the recombinant, full-length channel, as well as crystals in which the C-tail has been completely removed by trypsin digestion. Projection density maps at 7.5 A resolution closely resemble our previous analysis of the C-terminal truncation mutant (9). A difference map between the full length and trypsin-treated channels suggests that there are small but significant shifts in protein density upon removal of the C-tail.     

Negative regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90

The release of cytochrome c from mitochondria results in the formation of an Apaf-1-caspase-9 apoptosome and induces the apoptotic protease cascade by activation of procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of purified Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome c-mediated activation of procaspase-9. We also show that Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90-Apaf-1 complex and relieves the inhibition of procaspase-9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade.     

Parasites and host life-history traits: implications for community ecology and species co-existence

Most of the evidence for a key role of parasites in structuring communities is based on the idea of a differential susceptibility of host species to infection and its consequences. Recent advances in community ecology suggest that life-history traits of free-living species can be an important determinant of their co-existence within communities. On the other hand, parasites have the potential to indirectly alter the life-history traits of their hosts, such as developmental time or dispersal. We discuss the idea that these indirect effects could influence the structure of free-living and parasite communities. We explore this idea in relation to related concepts including 'parasitic arbitration' and engineering processes.     

Genetic and morphological heterogeneity in small rodent whipworms in southwestern Europe: characterization of Trichuris muris and description of Trichuris arvicolae n. sp. (Nematoda: Trichuridae)

Genetic and morphological variability of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae), parasites of small rodents in southwestern Europe, was studied. Isozyme patterns of natural populations of nematodes parasitizing rodent species of the Muridae (Apodemus sylvaticus, Apodemus flavicollis, Mus musculus) and Arvicolidae (Clethrionomys glareolus, Microtus agrestis, Microtus arvalis) were analyzed at 6 putative loci. Two diagnostic loci were found in T. muris from Muridae and from Arvicolidae. Thus, the existence of 2 species of Trichuris restricted to different host families was indicated. They included Trichuris muris Schrank, 1788, originally described as being from mice, and Trichuris arvicolae n. sp., parasitizing the above species of Arvicolidae. The morphological variability of both species was compared. Although ranges of all morphological characters of the new species overlapped with those of T. muris, stepwise discriminant analysis yielded a 100% accurate classification of females when using vagina length and egg size. Males of T. muris and T. arvicolae cannot be separated entirely. A set of 6 variables yielded 95.7% discrimination; the most discriminating variables were spicule size and body width.