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  • 1
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Previous results from our laboratory demonstrated the existence of two subpopulations of porcine somatotropes of low- (LD) and high density (HD) that exhibit differences in ultrastructure and respond in an opposite manner to somatostatin (SRIF) in vitro. In LD cells, SRIF did not affect basal growth hormone (GH) release but partially blocked the stimulatory effect induced by GH-releasing factor (GRF). Conversely, SRIF paradoxically stimulated the secretory activity of HD somatotropes. Here, we have analysed in detail the basic parameters that characterize this differential response. To this end, the time- and dose-dependent effects of SRIF-14 were evaluated on separate monolayer cultures of both subpopulations. Likewise, the direct effect of the peptide on individual somatotropes from each subset was assessed by cell immunoblot assay. Finally, we compared the effects of SRIF-14 and SRIF-28 on cultures of LD and HD cells. SRIF-14 (10−7 M) induced a rapid (30 min) and sustained (4 h) 2-fold increase in GH release from HD cells, whereas it did not affect GH secretion from LD somatotropes. Surprisingly, a low dose of SRIF (10−15 M) stimulated GH release from both LD (154.1±8.2% of basal, P〈0.05) and HD (337.2±55.5% of basal, P〈0.05) subpopulations, even more effectively than higher doses of the peptide. Results from cell blotting showed that SRIF stimulatory effects were exerted directly upon individual somatotropes. Finally, SRIF-28 elicited similar responses to those observed for SRIF-14 in both somatotrope subpopulations, yet 10−15 M SRIF-28 was less potent than the same dose of SRIF-14 in stimulating GH release from HD cells. Our present findings demonstrate that SRIF can function as a true GH-releasing factor in cultures of porcine pituitary cells by acting specifically and directly upon somatotropes. Furthermore, together with previous observations, these results strongly suggest that SRIF is not merely an inhibitor of GH release in pigs, but might play a dual modulatory role. Heterogeneity of the somatotrope population contributes greatly to this divergent effect of SRIF.
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  • 2
    ISSN: 1365-2826
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Previous studies from our laboratory have demonstrated that porcine somatotropes can be separated into two subpopulations of low (LD) and high density (HD) by centrifugation in a Percoll gradient. The two subsets are present throughout porcine postnatal growth, although their relative proportions vary with age. In prepubertal animals, HD cells exhibit higher secretory granule content and release more GH than LD cells under basal culture conditions. In the present study, we analysed the ultrastructure of separated LD and HD cells from neonate and mature female pigs, and quantified cell size as well as the relative abundance of several subcellular organelles on immunoidentified somatotropes. Subsequently, GH release under basal conditions was assessed for cultures of unseparated cells and also for LD and HD somatotropes obtained at different stages of postnatal development. Results from the morphometric study demonstrated that LD somatotropes were significantly smaller in size, contained less secretory granules and displayed a more developed endoplasmic reticulum than their HD counterparts, regardless of the age of the pituitary donors. In terms of secretory ability, a significant age-associated decrease in GH release was observed in monolayer cultures of unseparated cells from prepubertal and mature pigs compared to neonates. A similar decline in GH-releasing ability was detected for cultures of HD cells. For LD cells, GH secretion only decreased significantly in mature animals. In spite of the divergent pattern followed by both subpopulations during growth, HD somatotropes released significantly more GH than LD somatotropes at the three ages studied. Taken together, our findings demonstrate that the population of porcine somatotropes is mainly composed of two subtypes, LD and HD, which differ in density, morphology and basal secretory activity. These differences are essentially maintained during porcine postnatal development. The progressive reduction in the secretory capacity of HD and LD somatotropes, coupled to the decrease in the relative abundance reported for the HD subpopulation, provides the cytological basis for a better understanding of the decline in GH release associated with age in pigs.
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  • 3
    ISSN: 1600-065X
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Summary: A multigene family of immunoglobulin superfamily (Ig-SF) killer cell inhibitory receptors (KIRs) specifically recognize HLA class I molecules, while the interaction with H-2 products is mediated by members of the murine Ly49 C-type lectin family. A common structural feature of these receptors with inhibitory function is the presence of cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that couple them to SHP phosphatases. Strong support for the involvement of the CD94 C-type lectin receptor complex in NK cell-mediated recognition of Bw6+ HLA-B, HLA A and HLA-C alleles has been obtained. The cloned CD94 molecule covalently assembles with at least two different glyco-proteins (43 kDa and 39 kDa) to form functional receptors. NK cells inhibited upon HLA recognition express the CD94/p43 dimer, whose specificity for HLA molecules partially overlaps the Ig-SF receptor system. By contrast. NK clones bearing the homologous CD94/p39 receptor are triggered upon its ligation by CD94-specific mAbs. Remarkably, a set of Ig-SF receptors (p50) homologous to p58 KIRs also display an activating function. CD94-associated molecules belong to the NKG2 family of C-type lectins; the NKG2-A gene encodes for the p43 subunit. which contains cytoplasmic ITIMS. Expression of the different CD94 heterodimeric receptors will enable precise analysis of their putative interaction with HLA class I molecules.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd, UK
    Molecular microbiology 29 (1998), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Escherichia coli loses its rod shape by inactivation of PBP2 (penicillin-binding protein 2), target of the β-lactam mecillinam. Under these conditions, cell division is blocked in rich medium. Division in the absence of PBP2 activity is restored (and resistance to mecillinam is conferred) when the three cell division proteins FtsQ, FtsA and FtsZ are overproduced, but not when only one or two of them are overproduced. Division in the absence of PBP2 activity is also restored by a doubling in the ppGpp pool, as in the argS201 mutant. However, the nucleotide ppGpp, a transcriptional regulator of many operons, does not govern any of the five promoters of the ftsQAZ operon, as shown by S1 mapping of ftsQAZ mRNA 5′ ends in exponentially growing wild-type cells in the mecillinam-resistant argS201 mutant (intermediate ppGpp level) or during the stringent response elicited by isoleucine starvation (high ppGpp level). Furthermore, the concentration of FtsZ protein is not increased in exponentially growing mecillinam-resistant argS201 cells. These results show that the ftsQAZ operon is not the ppGpp target responsible for mecillinam resistance. We are currently trying to identify those targets that, at intermediate ppGpp levels, allow cells to divide as spheres in the absence of PBP2.
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  • 5
    ISSN: 1573-5028
    Keywords: cyanobacteria ; ferredoxin ; gltB ; gltS ; glutamate synthase ; Synechocystis sp. PCC 6803
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The first two genes of ferredoxin-dependent glutamate synthase (Fd-GOGAT) from a prokaryotic organism, the cyanobacterium Synechocystis sp. PCC 6803, were cloned in Escherichia coli. Partial sequencing of the cloned genomic DNA, of the 6.3 kb Hind III and 9.3 kb Cla I fragments, confirmed the existence of two different genes coding for glutamate synthases, named gltB and gltS. The gltB gene was completely sequenced and encodes for a polypeptide of 1550 amino acid residues (M r 168 964). Comparative analysis of the gltB deduced amino acid sequence against other glutamate synthases shows a higher identity with the alfalfa NADH-GOGAT (55.2%) than with the corresponding Fd-GOGAT from the higher plants maize and spinach (about 43%), the red alga Antithamnnion sp. (42%) or with the NADPH-GOGAT of bacterial source, such as Escherichia coli (41%) and Azospirillum brasilense (45%). The detailed analysis of Synechocystis gltB deduced amino acid sequence shows strongly conserved regions that have been assigned to the 3Fe-4S cluster (CX5CHX3C), the FMN-binding domain and the glutamine-amide transferase domain. Insertional inactivation of gltB and gltS genes revealed that both genes code for ferredoxin-dependent glutamate synthases which were nonessential for Synechocystis growth, as shown by the ferredoxin-dependent glutamate synthase activity and western-blot analysis of the mutant strains.
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  • 6
    ISSN: 1573-6881
    Keywords: Plasma membrane ; ascorbate stabilization ; coenzyme Q ; cytochrome b 5 reductase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ10 and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ10 reductase activity and its internal sequence being identical to cytochrome b 5 reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ10 content in isolated plasma membranes. We show here the role of CoQ10 and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b 5-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ10 and its NADH-dependent reductase.
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