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Plasminogen activator and collagenase production by cultured capillary endothelial cells 总被引:16,自引:17,他引:16
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Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells. 相似文献
3.
Carla DB Fernandez Fernanda F Bellentani Glaura SA Fernandes Juliana E Perobelli Ana Paula A Favareto André F Nascimento Antonio C Cicogna Wilma DG Kempinas 《Reproductive biology and endocrinology : RB&E》2011,9(1):32
Background
Obesity is rapidly becoming a worldwide epidemic that affects children and adults. Some studies have shown a relationship between obesity and infertility, but until now it remains controversial. Thus, the aim of the present study was to investigate the effect of high-fat diet-induced obesity on male reproductive parameters. 相似文献4.
Christian Jogler Wei Lin Anke Meyerdierks Michael Kube Emanuel Katzmann Christine Flies Yongxin Pan Rudolf Amann Richard Reinhardt Dirk Schüler 《Applied and environmental microbiology》2009,75(12):3972-3979
In this report, we describe the selective cloning of large DNA fragments from magnetotactic metagenomes from various aquatic habitats. This was achieved by a two-step magnetic enrichment which allowed the mass collection of environmental magnetotactic bacteria (MTB) virtually free of nonmagnetic contaminants. Four fosmid libraries were constructed and screened by end sequencing and hybridization analysis using heterologous magnetosome gene probes. A total of 14 fosmids were fully sequenced. We identified and characterized two fosmids, most likely originating from two different alphaproteobacterial strains of MTB that contain several putative operons with homology to the magnetosome island (MAI) of cultivated MTB. This is the first evidence that uncultivated MTB exhibit similar yet differing organizations of the MAI, which may account for the diversity in biomineralization and magnetotaxis observed in MTB from various environments.Magnetotactic bacteria (MTB) synthesize magnetosomes, which are membrane-enclosed organelles comprising crystals of magnetite (Fe3O4) or, less commonly, greigite (Fe3S4) (3) that are aligned in intracellular chains along dedicated cytoskeletal structures (26, 36, 38). Magnetic alignment along the magnetic field lines of the earth facilitates navigation in the stratified environment within freshwater and marine sediments (3, 13). MTB do not form a coherent phylogenetic group, but the trait of magnetotaxis is found in species within different phylogenetic clades, including Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, and the Nitrospira phylum (1, 3, 10, 41). Different species produce magnetosome crystals with a multitude of different morphologies displaying a broad variety of intracellular arrangements, including one, two, or multiple chains (3, 14). The perfectly shaped magnetosome crystals and highly ordered chain structures cannot be synthesized by chemical methods as yet. Therefore, an understanding of the genetic mechanisms controlling magnetosome formation is also of great interest for the inorganic production of advanced magnetic nanomaterials (3, 13, 28).Most genes controlling magnetosome formation and magnetotaxis in Magnetospirillum gryphiswaldense and other freshwater magnetospirilla are clustered within four major operons (mamAB, mamGFDC, mms6, and mamXY) (18, 34, 37, 49) that are part of a large genomic magnetosome island (MAI) (49). It was recently shown that the MAI is also conserved in marine MTB, including the MV-1 magnetotactic vibrio strain and the MC-1 magnetic coccus strain. The homologous genomic regions display similar gene contents and, to a lesser extent, a conserved gene synteny (23). It has been suggested that the MAI was transferred horizontally between different MTB (37). However, the divergence between the MAI regions of strain MV-1, strain MC-1, and the magnetospirilla suggests that the events of horizontal gene transfer (HGT) did not occur very recently.Despite continued efforts by many laboratories, the majority of MTB are still not available in pure culture. In particular, the huge diversity of uncultivated species with respect to different morpho- and phylotypes and, in particular, magnetosome crystal shapes is not nearly fully represented by cultivated species. Thus, understanding of the genetic diversity of the magnetotaxis and magnetosome biosynthetic machinery has to rely on culture-independent techniques such as the metagenomic analysis of environmental MTB (24).It has been demonstrated that single genes and even entire operons can be cloned and functionally expressed from uncultivated soil or marine bacteria by using large insert libraries that provide contiguous sections from single organisms (4, 21, 22). The potential to identify and clone genes for metabolic pathways with relevance for biotechnological applications has already been demonstrated in metagenomic projects, such as the identification of polyketide synthase genes from microbial consortia of marine sponges (25) or other environmental samples (8, 31). The cost of sequencing and the challenges that are associated with the management of vast datasets, however, preclude comprehensive genomic studies of highly complex communities. Consequently, approaches that are based on the analysis of a group of bacteria with reduced species diversity are favored. This requires that the sample material is enriched for the target organisms before DNA preparation, for example, by flow sorting, centrifugation, or other physical enrichment techniques (32, 42) or by focusing on natural communities with reduced species diversity (48).Unlike other uncultivated bacteria, MTB exhibit magnetically directed swimming behavior, which enables their selective enrichment from environmental samples without the need of cultivation (16). This approach was utilized in a number of earlier studies uncovering the morphological and phylogenetic diversity of MTB found in environmental populations (10, 43, 45-47). However, these investigations were confined to PCR-based analysis of 16S rRNA genes, ultrastructural studies, and fluorescence in situ hybridization.In this study we used an improved magnetic collection technique to selectively harvest large numbers of uncultivated MTBs, which allowed the extraction of genomic DNA for the construction of large insert metagenomic libraries from different aquatic habitats. Large parts of the MAI from two uncultivated MTB were identified by hybridization using heterologous magnetosome gene probes and end sequencing. We demonstrate for the first time that uncultivated MTB exhibit a clustered organization of magnetosome genes which resembles that of cultivated species and yet displays variations that may account for the observed diversity in biomineralization and magnetotaxis in MTB from various environments. The levels of similarity between and synteny of magnetosome genes of uncultivated and cultivated MTB provide further evidence for HGT. 相似文献
5.
A common polygenic basis for quinine and PROP avoidance in mice 总被引:3,自引:2,他引:1
Inbred strains of mice (Mus musculus) differ greatly in ability to taste
various bitter compounds. For some compounds, the differences result from
allelic variation at a single locus. However, segregation patterns
incompatible with monogenic inheritance have been found for quinine
avoidance. The Soa bitter sensitivity locus exerts some influence on this
phenotype, but an unknown number of other loci also contribute. Relative
avoidance patterns for quinine sulfate in panels of naive inbred strains
resembled avoidance patterns for 6-n-propyl-2- thiouracil (PROP),
suggesting a common genetic basis. In particular, C57BL/6J mice strongly
avoided both 0.1 mM quinine sulfate and 1 mM PROP in two-bottle preference
tests, whereas C3H/HeJ mice were indifferent to both. Therefore, 12 BXH/Ty
recombinant inbred strains, derived from these strains, were tested with
both solutions to begin identification of the unknown bitter loci. Naive
mice were tested for four consecutive days with each compound (order
counterbalanced). Some BXH/Ty strain means resembled those of the parent
strains, but others were intermediate. This indicated recombination among
loci affecting avoidance, and therefore polygenic inheritance. The strain
means were highly correlated across compounds (r = 0.98), suggesting that
the same polygenes controlled both phenotypes. The BXH/Ty means for both
compounds were then compared with the strain genotypes at 212 chromosome
position markers distributed throughout the genome. Eight markers on five
chromosomes (3, 6, 7, 8 and 9) yielded significant correlations. Six of the
markers were correlated with both phenotypes, again suggesting common
polygenic inheritance. The marker with the highest correlation was Prp,
tightly linked to Soa on chromosome 6. The correlated marker regions likely
contain quantitative trait loci affecting bitter avoidance. The phenotypic
similarity of PROP to quinine, rather than to phenylthiourea, apparently
stemming from a common polygenic basis, indicates a difference between mice
and humans in gustatory organization related to bitters.
相似文献
6.
Diversity and vertical distribution of magnetotactic bacteria along chemical gradients in freshwater microcosms 总被引:4,自引:0,他引:4
Flies CB Jonkers HM de Beer D Bosselmann K Böttcher ME Schüler D 《FEMS microbiology ecology》2005,52(2):185-195
The vertical distribution of magnetotactic bacteria along various physico-chemical gradients in freshwater microcosms was analyzed by a combined approach of viable cell counts, 16S rRNA gene analysis, microsensor profiling and biogeochemical methods. The occurrence of magnetotactic bacteria was restricted to a narrow sediment layer overlapping or closely below the maximum oxygen and nitrate penetration depth. Different species showed different preferences within vertical gradients, but the largest proportion (63-98%) of magnetotactic bacteria was detected within the suboxic zone. In one microcosm the community of magnetotactic bacteria was dominated by one species of a coccoid "Alphaproteobacterium", as detected by denaturing gradient gel electrophoresis in sediment horizons from 1 to 10 mm depth. Maximum numbers of magnetotactic bacteria were up to 1.5 x 10(7) cells/cm3, which corresponded to 1% of the total cell number in the upper sediment layer. The occurrence of magnetotactic bacteria coincided with the availability of significant amounts (6-60 microM) of soluble Fe(II), and in one sample with hydrogen sulfide (up to 40 microM). Although various trends were clearly observed, a strict correlation between the distribution of magnetotactic bacteria and individual geochemical parameters was absent. This is discussed in terms of metabolic adaptation of various strains of magnetotactic bacteria to stratified sediments and diversity of the magnetotactic bacterial communities. 相似文献
7.
Whitehouse DB; Tomkins J; Lovegrove JU; Hopkinson DA; McMillan WO 《Molecular biology and evolution》1998,15(4):456-462
The expanding molecular database provides unparalleled opportunities for
characterizing genes and for studying groups of related genes. We use
sequences drawn from the database to construct an evolutionary framework
for examining the important glycolytic enzyme phosphoglucomutase (PGM).
Phosphoglucomutase plays a pivotal role in the synthesis and utilization of
glycogen and is present in all organisms. In humans, there are three
well-described isozymes, PGMI, PGM2, and PGM3. PGM1 was cloned 5 years ago;
however, repeated attempts using both immunological approaches and
molecular probes designed from PGM1 have failed to isolate either PGM2 or
PGM3. Using a phylogenetic strategy, we first identified 47 highly
divergent prokaryotic and eukaryotic PGM-like sequences from the database.
Although overall amino acid identity often fell below 20%, the relative
order, position, and sequence of three structural motifs, the active site
and the magnesium-- and sugar-binding sites, were conserved in all 47
sequences. The phylogenetic history of these sequences was complex and
marked by duplications and translocations; two instances of transkingdom
horizontal gene transfer were identified. Nonetheless, the sequences fell
within six well-defined evolutionary lineages, three of which contained
only prokaryotes. Of the two prokaryotic/eukaryotic lineages, one contained
bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human
PGM1 and the second contained likely homologs to human PGM2. Indeed, an
amino acid sequence, derived from a partial human cDNA, that fell within
the second cross-kingdom lineage bears several characteristics expected for
PGM2. A third lineage may contain homologs to human PGM3. On a general
level, our phylogenetic-based approach shows promise for the further
utilization of the extensive molecular database.
相似文献
8.
Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration 总被引:14,自引:4,他引:10
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The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout. 相似文献
9.
Both magnetic collection and "race track" purification techniques were highly effective for selective enrichment of magnetotactic bacteria (MTB) from complex communities, as suggested by amplified ribosomal DNA restriction analysis and denaturing gradient gel electrophoresis combined with sequence analysis of 16S rRNA genes. Using these purification methods, the occurrence and diversity of MTB in microcosms from various marine and freshwater environments were assayed by using a combined microscopic, molecular, and cultivation approach. Most microcosms were dominated by magnetotactic cocci. Consistently, the majority of retrieved 16S RNA sequences were affiliated with a distinct cluster in the Alphaproteobacteria. Within this lineage the levels of sequence divergence were <1 to 11%, indicating genus-level diversity between magnetotactic cocci from various microcosms, as well as between MTB from different stages of succession of the same microcosms. The community composition in microscosms underwent drastic succession during incubation, and significant heterogeneities were observed between microcosms from the same environmental sources. A novel magnetotactic rod (MHB-1) was detected in a sediment sample from a lake in northern Germany by fluorescence in situ hybridization. MHB-1 falls into the Nitrospira phylum, displaying 91% 16S rRNA sequence similarity to "Magnetobacterium bavaricum." In extensive cultivation attempts, we failed to isolate MHB-1, as well as most other MTB present in our samples. However, although magnetotactic spirilla were not frequently observed in the enrichments, 10 novel isolates of the genus Magnetospirillum which had not routinely been isolated in pure culture before were obtained. 相似文献
10.