Independence of H-2 and viral antigens on the cell surface and absence of H-2 antigens on murine leukemia virus and mouse mammary tumor virus particles

By indirect immunoelectron microscopy we tested for the presence of H-2 antigens on murine mammary tumor virus (MMTV) and murine leukemia virus (MuLV) particles. The association of H-2 antigens and viral antigens on the virus-infected cell surface was investigated with antibody-induced redistribution. Mammary tumor cells and leukemia cell lines with different H-2 genotypes and carrying different MuMTV or MuLV were used. No H-2 antigens could be demonstrated on the envelope of MMTV and MuLV particles, even after the permeabilization of their envelopes with saponin. On the surface of virus-infected cells antibody-induced patching or capping of the viral antigens did not result in copatching or cocapping of the H-2 antigens. In the reciprocal tests no co-redistribution of viral antigens with H-2 antigens was seen. Our experiments failed to show any physical association between H-2 antigens and MMTV or MuLV antigens on the cell surface.Abbreviations used in this paper MMTV mammary tumor virus - MuLV murine leukemia virus - MHC major histocompatibility complex - IEM immunelectron microscopy     

Extended working hours and health

Statistical analyses of the relation between the amount of working hours and impairments to health, based on data from a European survey on working conditions in 2000, clearly reveal that there is a substantial correlation between the number of working hours per week and the frequencies of health complaints. This applies to both musculo-skeletal disorders as well as to psycho-vegetative complaints. The relationship of the duration of the exposure to working conditions to health impairments is moderated by a great number of individual (e.g., age) and situational (e.g., shift-work) variables, showing additive or interactive effects for which selected examples have been presented. In general, however, there is a consistent functional relationship between the number or working hours and their effects on the workers that holds over a great variety of conditions. It is argued that requests for extending working hours should thus be handled with care.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.     

Purified protein S contains multimeric forms with increased APC-independent anticoagulant activity

Protein S, the cofactor of activated protein C (APC), also expresses anticoagulant activity independent of APC by directly inhibiting prothrombin activation via interactions with factor Xa, factor Va, and phospholipids. In different studies, however, large variations in APC-independent anticoagulant activities have been reported for protein S. The investigation presented here shows that within purified protein S preparations different forms of protein S are present, of which a hitherto unrecognized form (<5% of total protein S) binds with high affinity to phospholipid bilayers (K(d) < 1 nM). The remaining protein S (>95%) has a low affinity (K(d) = 250 nM) for phospholipids. Using their different affinities for phospholipids, separation of the forms of protein S was achieved. Native polyacrylamide gel electrophoresis demonstrated that the form of protein S that binds to phospholipids with low affinity migrated as a single band, whereas the high-affinity protein S exhibited several bands that migrated with reduced mobility. Size-exclusion chromatography revealed that the slower-migrating bands represented multimeric forms of protein S. Multimeric protein S (<5% of total protein S) appeared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of protein S. Comparison of purified protein S preparations that exhibited a 4-fold difference in APC-independent anticoagulant activity showed that the ability to inhibit prothrombin activation correlated with the content of multimeric protein S. Multimeric protein S could not be identified in normal human plasma, and it is therefore unlikely that this form of protein S contributes to the APC-independent anticoagulant activity of protein S that is observed in plasma.     

Cultivation of Walsby's square haloarchaeon

The square haloarchaea of Walsby (SHOW group) dominate hypersaline microbial communities but have not been cultured since their discovery 25 years ago. We show that natural water dilution cultures can be used to isolate members of this group and, once in pure culture, they can be grown in standard halobacterial media. Cells display a square morphology and contain gas vesicles and poly-beta-hydroxybutyrate (PHB) granules. The 16S rRNA gene sequence was >99% identical to other SHOW group sequences. They prefer high salinities (23-30%), and can grow with a doubling time of 1-2 days in rich media. The ability to culture SHOW group organisms makes it possible to study, in a comprehensive way, the microbial ecology of salt lakes.     

Liquid serial dilution is inferior to solid media for isolation of cultures representative of the phylum-level diversity of soil bacteria

Representatives of only four well-characterized bacterial phyla were isolated from a pasture soil by using liquid serial dilution culture. In contrast, members of Acidobacteria, Verrucomicrobia, and Gemmatimonadetes and of other poorly represented bacterial lineages were isolated in earlier experiments with solidified versions of the same media. We conclude that, contrary to expectation, liquid serial dilution culture is inferior to culturing on solid media for isolating representatives of many bacterial phyla from soil.     

Regulation of T-cell death-associated gene 51 (TDAG51) expression in human T-cells

T-cell death-associated gene 51 (TDAG51) has been described to regulate T-cell receptor/CD3-dependent induction of CD95/Fas and subsequent activation-induced cell death (AICD) in a murine T-cell hybridoma. Using well-defined pharmacological inhibitors, we investigated the regulation of TDAG51 expression in human T-cells and the correlation with cell death. TDAG51 was induced in resting T-cells, lymphoid cell lines and AICD-susceptible as well as AICD-resistant T-cell clones, and induction was inhibited by MAP-kinase inhibitors and PKC inhibitor G?6983. No correlation between the effects of inhibitors on TDAG51 expression and cell death was observed. The constitutive TDAG51 expression in five pancreatic carcinoma cell lines was reduced by MAP-kinase inhibitors but not by G?6983. Furthermore, the inducible overexpression of TDAG51 in TetOn Jurkat cells did not modulate cellular proliferation, phorbolester/ionomycin-induced growth arrest, or the expression of various cell surface molecules. Our results indicate that the expression of TDAG51 in human T-cells does not correlate with AICD.     

KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 microM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.     

Measuring principles of frictional coefficients in cartilaginous tissues and its substitutes

The frictional properties of cartilaginous tissues, such as the hydraulic permeability, the electro-osmotic permeability, the diffusion coefficients of various ions and solutes, and the electrical conductance, are vital data to characterise the extracellular environment in which chondrocytes reside. This paper analyses one-dimensional measurement principles of these coefficients. Particular attention is given to the deformation dependence of them and the highly deformable nature of the tissues. A suggested strategy is the combination of a diffusion experiment using radiotracer methods, an electro-osmotic flow experiment and an electro-osmotic pressure experiment at low electric current.