摘要:

AbstractMigrating cells degrade pericellular matrices and basement membranes. For these purposes cells produces a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an Mr72,000 protein was digested to an Mr62,000 form by human mast cell tryptase while the plasminogen activator inhibitor PAI‐1 was not affected. Cleavage of the Mr72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the Mr72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin‐Sepharose. The soluble form of the Mr72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact Mr72,000 and Mr62,000 degraded form of the protein process gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of Mr72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose‐dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices. © 1992 Wiley‐L

ISSN:0730-2312    

DOI:10.1002/jcb.240500402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractInfiltrating immune cells in 30 primary human epithelial breast tumours were studied using specific anti‐CD3 (T cells), anti‐CD68 (macrophages), anti‐CD57 (NK cells), and an anti‐pan‐B cell antibody (L26). The majority of tumour infiltrating inflammatory cell are T cells (40‐50%) and monocytes/macrophages (15–35%).The macrophage specific chemo‐attractant and growth factor CSF‐1 is detected by immunohistochemical techniques (IHC) at the level of invasive breast cancer cell in 46/50 tumours but not at the level of in‐ situ (pre‐invasive) cancer. A mosaic staining patterns was usually observed with a very high expression in areas of obvious stromal invasion (90%cells positive) and absent or trace staining in intraductal carcinoma. Macrophages and plasma cell are equally intensely positive. In‐situ‐hybridisation experiment confirm the production of CSF‐1 (mRNA) by tumour cells and show the same pattern of expression. Expression of the CSF‐1 receptor protein (fms) was also observed by IHC in 41/48 invasive tumours, albeit at weaker intensities than in tumour infiltrating monocytes/macrophages. A concomitant expression of both CSF‐1 andfmsin in‐situ carcinoma was never seen (n = 14). It is therefore proposed that the associated expression of CSF‐1 and its receptor may be linked to the invasive potential of breast cancer, the monocytic infiltrate being an indication of the quantitative importance of CSF‐1 production

ISSN:0730-2312    

DOI:10.1002/jcb.240500403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractIn human lymphoplastoid cell line (Z83) in which rDNA genes on chromosomes 22 are amplified but transcribed at a low level, immunocytological studies with antibodies to 5 methylcytidine provided evidence for hypermethylation of the rDNA. The extent of methylation of the 5′ flanking sequence of the ribosomal DNA was examined by comapring the size of restriciton fragments obtained by digestion of genomic DNA with EcoRI and Hpall or EcoRI and Mpsl. Southern blots indicated hypermethylation of the 5′ flanking sequences of many copies of rRNA genes in these cells, but not in a control lymphoblastoid cell line without rDNA amplification. Results obtained with somatic hybird human‐hamster cell line, in which the rRNA genes on the single human chromosomes 22 are inactive, showed that only a small fraction of the CCGG sites in the 5′ flanking sequences of the transcriptionally silent rRNA genes in this hybird were methylated. Since inactive rRNA genes can show such a minimal level of methylation, it is likely that the extreme hypermethylation of the apmlified rRNA genes in Z83 association with their inactivation rather than following it. © 1992 Wiley

ISSN:0730-2312    

DOI:10.1002/jcb.240500404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractArachidonic acid is mobilized from fetal membrane phospholipids at paturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometiral contractions. Phospholipid mobilization requires activation of phospholipase A2or C both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids thereby may alter phospholipase activity through two mechanism: modulation of intracellular free Ca2+concentration or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I–VI were identfied in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pool: a pool which only associated with the membrane in the presence of calcium (calcium‐dependent pool) and a calcium‐independent pool that remained membrane bound in the presence of calicium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium‐dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium‐dependent pool of annexin V increased in chorion; and calcium‐independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in totoal annexin I concentration with labor in amnion reflects a substantial decrease (80–90%) in the pool tightly bound to the membrane in a calcium‐independent manner. The striking change distinguishes annexin I as a potential candiate inhibitor which is specially downgregulated a parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostglandins production at labor. © 1992

ISSN:0730-2312    

DOI:10.1002/jcb.240500405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractAbstract a variant human H2B histone gene (GL 105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNA regulated differentially during the hela S3cell cycle. The H2B‐GL105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3′ end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non‐translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B‐GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B‐GL105 histone gene was localized to chromosome region 1q21‐1q23 by chromosomal in situ hybridization and by analysis of rodent‐human somatic cell hybrids using an H2B‐GL105 specific probe. The H2B‐GL105 gene is paired with a functional H2A histone gene and this H2B/H2B gene pair is seperated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF‐1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3′ end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication‐dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.

ISSN:0730-2312    

DOI:10.1002/jcb.240500406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe major histocompatibility complex‐unrestricted, cell‐mediated, constitutive anti‐tumor cytotoxic function of natural killer cells is highly preserved in healthy elderly. A study of the dynamics of expression of natural killer cell–associated phenotypes during immunosenescence shows that selective, bidirectional, and disproportionate changes in certain natural killer cell subset number and ratio take place during aging. The mean natural killer cell subset ratio (%CD16+CD57+over%CD56+CD57−)gradually increases from a young adult level of 0.7 to 4.6 with advancing age predominantly due to a tripling of %CD16+57+cells as opposed to a moderate decrease (−54%)in %CD56+57−phenotype. The parallel increase in natural killer phenotype ratio and cytotoxic activity might represent a shift in the maturity status of these cells. Based on these findings, a model of natural killer cell immunosenescence is proposed. It is concluded that not all immunosenescent changes need be detrimental; some may even improve the potential for survival and represent an adaptational immunosenescent change. © 1992 W

ISSN:0730-2312    

DOI:10.1002/jcb.240500407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have recently demonstrated that the iv administration of acidic fibroblast growth factor(a‐FGF) to rats for 6 days results in a marked increase in thyroid weight colloid accumulation and flat, quiescent follicular cells. Whereas a‐FGF administration consistently increases thyroid weight, there are only minor alterations in serum TSH and thyroid hormones, and no change in intrathyroidal metabolism of125l metabolism. In the present work, we studied the effects of 1 or 6 daily injections of a‐FGF (60 μ/kg BW) or vehicle on the mRNA levels for histone, c‐fos, actin, type I 5′ deiodinase (5′D‐1), thyroid peroxidase, and thyroglobulin and cathepsin D in the thyroid, liver and bone. Rats were sacrificed 0.5, 2, 4, 8 and 24 h after the 1st or the 6th a‐FGF injection and thyroid, liver and calvarium were removed. The relative amounts of mRNAs were determined by slot blot analysis. There was a 43% increase in thyroid weight in rats treated with a‐FGF for 6 days compared to vehicle‐treated rats. We observed an increase in c‐fos mRNA content in the thyroid gland 0.5 to 4 h after 1 or 6 injections of a‐FGF. In contrast, treatment with a‐FGF for 1 or 6 days did not affect histone mRNA content, a marker of proliferative activity or actin mRNA levels. Treatment with a‐FGF caused a marked decrease in thyorid 5′ D‐I mRNA content in the thyroid. The decrease was present 2 h after the first injection and reached a nadir 8 h. After 6 daily injections, the decrease in 5′ D‐I mRNA was present throughout the whole day. In the liver, there was a significant decrease in 5′ D‐1 mRNA only 2 and 4 h after the 6daily injection of a‐FGF. There was no effect of a‐FGF treatment on the mRNA content of thyorid peroxidase, thyroglobulin, or a marker of lysosomal activity, cathepsin D. These data indicate that a‐FGF induces colloid accumulation in the rat thyroid without changes in proliferative or lysosomal activites, or alteration in the regulation of the thyroid specific genes thyroid peroxidase and thyroglobulin. Modification in gene expression and induction are reflected by the upregulation of the early response gene c‐fos. The marked and persistent decrease in 5′ deiodinase mRNA content after a‐FGF treatment suggests that a‐FGF may be involved in the regulation of

ISSN:0730-2312    

DOI:10.1002/jcb.240500408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractEpidermal growth factor (EGF) is a ubiquitous fibroblast mitogen which also stimulates the synthesis of the extracellular matrix degrading metalloproteinases, collagenase, and stromelysin. Using primary cultures of human skin fiibroblassts, we show that these metalloproteinase mRNAs are coordinately up‐regrated by EGF; and that dexamethasone, a potent inhibitor of collagenase and stromelysin syntehse, cordinately down‐regulates these EGF‐induced mRNAs. Nuclear run‐on assays showed that EGF increased trasncription of collagenase and stromelysin ∼ 2‐fold over the untreated control, while repression by dexamethasone was difficult to detect. However, steady state mRNA levels were induced ∼ 10‐fold by EGF and co‐treatment with dexamethasone decreased them to below control levels, suggesting modulation of mRNA stability. Thus, we measured the half‐life of these mRNAs using “pulse‐chase” methodology. Typically, the half‐life of EGF‐induced collagenase and stromelysin mRNAs was ∼ 30 h, and co‐treatment inhibitor DRB stabilized EGF‐induced metalloproteinase mRNAs by 30–50%. Additionally, we found that the transcirption inhibitor DRB stabilitzed EGF‐induced metalloproteinase mRNAs, suggesting an mRNA degradation pathway which requires transcription. Thus our data demonstrate that collagenase and stromelysin are coordinately regulated by EGF and by dexamethasone, primarily at the level of metalloproteinase

ISSN:0730-2312    

DOI:10.1002/jcb.240500409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractIn humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto‐oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3(1,253) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functioal nuclear bound glucocorticoid receptors (range 6,000‐22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In arragement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OCgene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto‐oncogenes c‐myc, c‐fos, and c‐jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c‐myc, c‐fos, and c‐jun mRNAs in nonproliferating (confluent) hOB cells by 3.5‐, 10‐, and 2.0‐fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex‐induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cell

ISSN:0730-2312    

DOI:10.1002/jcb.240500410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractTo understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone‐like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid‐treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10‐7M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c‐fosexpression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000‐fold elevated in glucocorticoid‐differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGFβ was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run‐on assay to mRNA levels indicates that glucocorticoids exert both transcriptional and post‐transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoid promote changes in gene expression involved in cell‐cell and cell‐extracellular matrix signaling mechanism that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue‐like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.

ISSN:0730-2312    

DOI:10.1002/jcb.240500411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY