Human Brain Capillary Endothelium: Modulation of K+ Efflux and K+, Ca2+ Uptake by Endothelin

This report describes K⁺ efflux, K⁺ and Ca²⁺ uptake responses to endothelins (ET-1 and ET-3) in cultured endothelium derived from capillaries of human brain (HBEC). ET-1 dose dependently increased K⁺ efflux, K⁺ and Ca²⁺ uptake in these cells. ET-1 stimulated K⁺ efflux occurred prior to that of K⁺ uptake. ET-3 was ineffective. The main contributor to the ET-1 induced K⁺ uptake was ouabain but not bumetanide-sensitive (Na⁺-K⁺-ATPase and Na⁺-K⁺-Cl^– cotransport activity, respectively). All tested paradigms of ET-1 effects in HBEC were inhibited by selective antagonist of ET_A but not ET_B receptors and inhibitors of phospholipase C and receptor-operated Ca²⁺ channels. Activation of protein kinase C (PKC) decreased whereas inhibition of PKC increased the ET-1 stimulated K⁺ efflux, K⁺ and Ca²⁺ uptake in HBEC. The results indicate that ET-1 affects the HBEC ionic transport systems through activation of ET_A receptors linked to PLC and modulated by intracellular Ca²⁺ mobilization and PKC.     

Mitochondrial DNA structure and colony expansion dynamics of New Zealand fur seals (Arctocephalus forsteri) around Banks Peninsula

New Zealand fur seals are one of many pinniped species that survived the commercial sealing of the eighteenth and nineteenth centuries in dangerously low numbers. After the enforcement of a series of protection measures in the early twentieth century, New Zealand fur seals began to recover from the brink of extinction. We examined the New Zealand fur seal populations of Banks Peninsula, South Island, New Zealand using the mitochondrial DNA control region. We identified a panmictic population structure around Banks Peninsula. The most abundant haplotype in the area showed a slight significant aggregated structure. The Horseshoe Bay colony showed the least number of shared haplotypes with other colonies, suggesting a different origin of re-colonisation of this specific colony. The effective population size of the New Zealand fur seal population at Banks Peninsula was estimated at approximately 2500 individuals. The exponential population growth rate parameter for the area was 35, which corresponds to an expanding population. In general, samples from adjacent colonies shared 4.4 haplotypes while samples collected from colonies separated by between five and eight bays shared 1.9 haplotypes. The genetic data support the spill-over dynamics of colony expansion already suggested for this species. Approximate Bayesian computations analysis suggests re-colonisation of the area from two main clades identified across New Zealand with a most likely admixture coefficient of 0.41 to form the Banks Peninsula population. Approximate Bayesian computations analysis estimated a founder population size of approximately 372 breeding individuals for the area, which then rapidly increased in size with successive waves of external recruitment. The population of fur seals in the area is probably in the late phase of maturity in the colony expansion dynamic.     

Purification and characterization of Dolichos lablab lectin

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin- Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.      

Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

Introduction  

Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan.     

Genetic ablation of caveolin-2 sensitizes mice to bleomycin-induced injury

Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1^−/−) and Cav-2-deficient mice (Cav-2^−/−) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2^−/− mice, but not Cav-1^−/− mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2^−/− mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2^−/− mice. Since Cav-1^−/− mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.     

The Relationship between P Elements and Male Recombination in Drosophila Melanogaster

P element dysgenesis associated male recombination in Drosophila was examined with a selective system focused upon 5% of the standard female genetic map divided into eight recombination segments. We found no correspondence between P element mobilization events and recombination in males in the intervals monitored. We defined two adjacent short genetic and molecular regions, one devoid of male recombination and the other acting as a "hot spot" for exchange in the absence of supporting P element insertion and excision activity. These data suggest that, even in the presence of mobilizing P elements, transposase may be active at non-P element sites, and that the genome may harbor sequences ranging from highly responsive to completely unresponsive to transposase action. A viewpoint is presented wherein P elements, with sequences that bind transposase, serve to focus the recombination action of transposase to encompass a region of DNA radiating outward from the initial binding site. We suggest that this region is measured in terms of chromosomal segments rather than limited to P element sequences.     

Two distinct conformational states define the interaction of human RAD51‐ATP with single‐stranded DNA

An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 k_BT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.     

The HLA-A mutation assay: improved technique and normal results

The HLA-A assay for detection of mutant lymphocytes has been modified, principally by incorporation of an overnight-incubation step which has resulted in improved immunoselection. For 83 estimations on cells from 21 normal individuals with a mean age of 35, the in vivo mutant frequency was 2.99 X 10(-5) +/- 1.48 X 10(-5) (mean +/- 1 SD). For 11 estimations on cells from 10 elderly individuals with a mean age of 78, the mutant frequency was significantly greater, being 7.16 X 10(-5) +/- 4.06 X 10(-5). Similar results were obtained when HLA-A2 or HLA-A3 mutants were enumerated. Mutant frequency measured at the HLA-A locus is almost an order of magnitude greater than that measured at the hypoxanthine phosphoribosyl transferase locus, but at least the major part of this difference is likely to be due to factors other than a difference in genetic stability between the two loci.