排序方式: 共有41条查询结果,搜索用时 15 毫秒
1.
2.
Plasminogen activator and collagenase production by cultured capillary endothelial cells 总被引:33,自引:17,他引:16 下载免费PDF全文
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.
Brookes S Rowe J Ruas M Llanos S Clark PA Lomax M James MC Vatcheva R Bates S Vousden KH Parry D Gruis N Smit N Bergman W Peters G 《The EMBO journal》2002,21(12):2936-2945
The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species. 相似文献
4.
Xiaohong R. Yang Melissa Rotunno Yanzi Xiao Christian Ingvar Hildur Helgadottir Lorenza Pastorino Remco van Doorn Hunter Bennett Cole Graham Joshua N. Sampson Michael Malasky Aurelie Vogt Bin Zhu Giovanna Bianchi-Scarra William Bruno Paola Queirolo Giuseppe Fornarini Johan Hansson Rainer Tuominen Laurie Burdett Belynda Hicks Amy Hutchinson Kristine Jones Meredith Yeager Stephen J. Chanock Maria Teresa Landi Veronica Höiom Håkan Olsson Nelleke Gruis Paola Ghiorzo Margaret A. Tucker Alisa M. Goldstein 《Human genetics》2016,135(11):1241-1249
5.
Francisca C. Reyes Beimeng Sun Hena Guo Darren Gruis Marisa S. Otegui 《Plant physiology》2010,153(2):624-631
Developing maize (Zea mays) endosperms can be excised from the maternal tissues and undergo tissue/cell-type differentiation under in vitro conditions. We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors. We show that both aleurone and starchy endosperm cells can be successfully transformed using a short cocultivation with A. tumefaciens cells. The highest transformation rates were obtained with the A. tumefaciens EHA101 strain and the pTF101.1 binary vector. The percentage of aleurone cells transformed following this method varied between 10% and 22% whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells. Cultured endosperms undergo normal cell type (aleurone and starchy endosperm) differentiation and storage protein accumulation, making them suitable for cell biology and biochemical studies. In addition, transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.The endosperm is a unique plant tissue that arises from a second fertilization event between a male gamete and the central cell. Its main function is to provide nutrients to the embryo either during seed development or during germination. In cereals, the endosperm consists of three main cell types: the starchy endosperm cells, which constitute the bulk of the endosperm and accumulate large quantities of storage proteins and starch; the epidermal aleurone cells; and the transfer cells, which are in contact with the maternal vascular tissues (Olsen, 2004). The cereal endosperm is important as a model system to study plant development, cell differentiation, programmed cell death, and synthesis, trafficking, and accumulation of storage compounds. In addition, it is a major source of carbohydrate and proteins for human and animal nutrition.In spite of its importance, cell biology studies on the cereal endosperm using modern imaging approaches such as expression of fluorescent subcellular markers are very scarce because: (1) the endosperm is deeply immersed in maternal tissues and therefore, not readily available for imaging analysis and (2) the long time required for transformation and regeneration of stable transgenic plants. Although several approaches for culturing maize (Zea mays) endosperm in vitro have been reported in the past years (Shimamoto et al., 1983), only recently a novel method developed by Odd-Arne Olsen and colleagues (Gruis et al., 2006) has proven to be successful in retaining endosperm tissue and cell type identity in in vitro conditions. Cultures derived from transgenic maize lines in which endosperm cell types are identified by the activity of specific promoters have shown that aleurone and starchy endosperm cell identity continues to be established in vitro (Gruis et al., 2006).Although Agrobacterium tumefaciens is not a natural pathogen of most monocots (Cleene, 1985; Binns and Thomashow, 1988), it has been successfully used to transform many cereals, including maize, wheat (Triticum aestivum), Sorghum, barley (Hordeum vulgare), and rice (Oryza sativa; Grimsley et al., 1989; Gould et al., 1991; Chan et al., 1993; Ishida et al., 1996, 2007; Gurel et al., 2009; Harwood et al., 2009; Hensel et al., 2009). In the case of maize, stable transgenic plants can be obtained by A. tumefaciens-mediated transformation using either super-binary or standard-binary vectors (Frame et al., 2002; Mohanty et al., 2009a, 2009b). However, transformation of isolated maize endosperms have been only possible using transient transformation approaches such as biolistic methods (Torrent et al., 1997; Gruis et al., 2006) and protoplast transfection (Gallie and Young, 1994). Unfortunately, these two methods are not always ideal for cell biology studies. On one hand, biolistic methods often result in high-copy number transgenic events and on the other, protoplasts are usually highly stressed cells not suitable for detailed protein localization studies. A. tumefaciens-mediated transformation methods circumvent these disadvantages by resulting in a low-copy number of transgenes in intact tissues.We have developed a method to transform in vitro-grown endosperms using a brief incubation time with A. tumefaciens cells carrying standard binary vectors. We present here a detailed explanation of the method and quantitative information on the transformation efficiency using different A. tumefaciens strains, culture density, and incubation time. We also provide evidence that the in vitro-differentiated aleurone and starchy endosperm cells are comparable to the corresponding cell types differentiated in planta and therefore, suitable for cell biology studies. In addition, we show that transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling imaging techniques. 相似文献
6.
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
7.
Storage protein accumulation in the absence of the vacuolar processing enzyme family of cysteine proteases 总被引:4,自引:0,他引:4 下载免费PDF全文
The role(s) of specific proteases in seed protein processing is only vaguely understood; indeed, the overall role of processing in stable protein deposition has been the subject of more speculation than direct investigation. Seed-type members of the vacuolar processing enzyme (VPE) family were hypothesized to perform a unique function in seed protein processing, but we demonstrated previously that Asn-specific protein processing in developing Arabidopsis seeds occurs independently of this VPE activity. Here, we describe the unexpected expression of vegetative-type VPEs in developing seeds and test the role(s) of all VPEs in seed storage protein accumulation by systematically stacking knockout mutant alleles of all four members (alphaVPE, betaVPE, gammaVPE, and deltaVPE) of the VPE gene family in Arabidopsis. The complete removal of VPE function in the alphavpe betavpe gammavpe deltavpe quadruple mutant resulted in a total shift of storage protein accumulation from wild-type processed polypeptides to a finite number of prominent alternatively processed polypeptides cleaved at sites other than the conserved Asn residues targeted by VPE. Although alternatively proteolyzed legumin-type globulin polypeptides largely accumulated as intrasubunit disulfide-linked polypeptides with apparent molecular masses similar to those of VPE-processed legumin polypeptides, they showed markedly altered solubility and protein assembly characteristics. Instead of forming 11S hexamers, alternatively processed legumin polypeptides were deposited primarily as 9S complexes. However, despite the impact on seed protein processing, plants devoid of all known functional VPE genes appeared unchanged with regard to protein content in mature seeds, relative mobilization rates of protein reserves during germination, and vegetative growth. These findings indicate that VPE-mediated Asn-specific proteolytic processing, and the physiochemical property changes attributed to this specific processing step, are not required for the successful deposition and mobilization of seed storage protein in the protein storage vacuoles of Arabidopsis seeds. 相似文献
8.
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9.
10.
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