Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis, TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CNBr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J. H., Leavis, P. C., & Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nature of the trifluoperazine binding sites on calmodulin and troponin-C

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.     

Identification and characterization of 10 polymorphic microsatellite loci in the German cockroach,Blattella germanica

Primer sequences and initial characterization are presented for 10 microsatellite loci isolated from the German cockroach, Blattella germanica. In a sample of 30 individuals from a single population sample, all loci were polymorphic with two to 12 alleles segregating per locus and levels of observed heterozygosity ranging from 0.27 to 0.92. One locus showed a deficit of heterozygotes. Experimental conditions are described for polymerase chain reaction multiplexing, which enables the genotyping of eight loci in three electrophoretic runs consisting of one set of three and two sets of two markers. Seven primer sets cross‐amplify in the related Blattella asahinai.     

Inhibition of PKCalpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide

Receptor-coupled contraction of smooth muscle involves recruitment to the plasma membrane of downstream effector molecules PKCalpha and rhoA but the mechanism of this signal integration is unclear. Caveolins, the principal structural proteins of caveolar plasma membrane invaginations, have been implicated in the organization and regulation of many signal transducing molecules. Thus, using laser scanning confocal immunofluorescent microscopy, we tested the hypothesis that caveolin is involved in smooth muscle signaling by investigating caveolin isoform expression and localization, together with the effect of a peptide inhibitor of caveolin function, in intact differentiated smooth muscle cells. All three main caveolin isoforms were identified in uterine, stomach, and ileal smooth muscles and assumed a predominantly plasma membranous localization in myometrial cells. Cytoplasmic introduction of a peptide corresponding to the caveolin-1 scaffolding domain-an essential region for caveolin interaction with signaling molecules--significantly inhibited agonist-induced translocation of both PKCalpha and rhoA. Translocation was unimpaired by a scrambled peptide and was unaltered in sham-treated cells. The membranous localization of caveolins, and direct inhibition of receptor-coupled PKCalpha and rhoA translocation by the caveolin-1 scaffolding domain, supports the concept that caveolins can regulate the integration of extracellular contractile stimuli and downstream intracellular effectors in smooth muscle.     

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.     

Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance

Vancomycin-resistant Enterococcus faecium spread dramatically in hospital settings in the USA in the 1990s and reached endemicity at the turn of the century. Similarly, rising prevalence rates are currently observed in several European countries, with prevalence rates of greater than 10% reported in seven of these. On the basis of multilocus sequence typing (MLST), the population structure of E. faecium was elucidated and the existence of a distinct high-risk enterococcal clonal complex, designated clonal complex-17 (CC17), which is associated with the majority of hospital outbreaks and clinical infections in five continents, was revealed. This complex is correlated with ampicillin and quinolone resistance and with the presence of a putative pathogenicity island. Preliminary MLST data suggest that similar hospital-adapted complexes might also exist in E. faecalis.     

Reshaping of sandstone surfaces by cryptoendolithic cyanobacteria: bioalkalization causes chemical weathering in arid landscapes

We report a novel weathering mechanism in South African sandstone formations, where cryptoendolithic cyanobacteria induce weathering by substrate alkalization during photosynthesis. As a result, the upper rock part is loosened and then eroded away by physical forces such as wind, water, trampling. This special type of ‘exfoliation’ is widely distributed and affects the geomorphology of whole sandstone mountain ranges and outcrops across several biomes. We show, that this weathering type is initiated by bioalkalization because of the photosynthesis of cryptoendolithic (i.e. those organisms living in small tight open spaces between the sand grains) cyanobacteria causing pH values high enough to enhance silica solution in the cryptoendolithic zone. As modern cyanobacteria are the initial photoautotrophic colonizers of bare rocks in arid and semiarid landscapes, it is possible that they may also have played a significant role in shaping sandstone landscapes in the geological past.