摘要:

AbstractAdipocyte precursors from the stromal vascular fraction of human adipose tissue were allowed to differentiate in serum‐free defined medium, whereafter their catecholamine stimulated lipolytic response was compared to that of mature isolated human adipocytes. Seventy‐five to ninety percent of the fibroblast‐like cells accumulated lipid droplets and glycerol‐3‐phosphate dehydrogenase activities of 1,000–2,800 mU/mg protein were measured in cell homogenates of differentiated cells. Lipolysis could be stimulated by both isoproterenol and norepinephrine in both differentiated preadipocytes as well as mature adipocytes. The results obtained with β‐adrenergic agents suggested the presence of a higher affinity receptor in differentiated preadipocytes as compared to mature adipocytes. Mature adipocytes responded well to β‐adrenergic agents, but no antilipolytic α2‐adrenergic response was observed in the differentiated preadipocytes. The presence of Gi proteins in the differentiated preadipocytes was suggested by the antilipolytic effect of adenosine as well as the lipolytic activity generated by pertussis toxin. In conclusion, our medium supported the differentiation of a very high percentage of human preadipocytes which developed a sensitive β‐adrenergic lipolytic response but which lacked an α2‐adrener

ISSN:0730-2312    

DOI:10.1002/jcb.240540102

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

年代:1994

数据来源: WILEY

摘要:

AbstractCurrent evidence suggest an important role for increased repair of drug‐induced DNA damage as one of the major mechanisms involved in tumor cell resistance to cis‐DDP. In this study, we examined the DNA repair capacity and the activities of three DNA repair related proteins, namely, DNA polymerases α and β, and total DNA ligase in cells of a malignant oligodendroglioma obtained from a patient before therapy and compared it with those of a specimen of the tumor acquired after the patient had failed cis‐DDP therapy. DNA repair capacity was quantitated as the extent of reactivation of the chloramphenicol‐O‐acetyltransferase (CAT) gene in a eukaryotic expression vector that has been damaged and inactivated by prior treatment with cis‐DDP and then transfected into the tumor cells. The extent of DNA‐platinum adduct formation in the expression vector was determined by flameless atomic absorption spectrometry. The level of cis‐DDP resistance of cells of the two tumors was determined with the capillary tumor stem cell assay. We observed a 2.8‐fold increased capacity to repair Pt‐DNA adducts and reactivate the CAT gene in cells of the tumor obtained after cis‐DDP therapy, compared to cells of the untreated tumor. This was associated with increases of 9.4‐fold and a 2.3‐fold, respectively, in DNA polymerase β and total DNA ligase activities in cells of the treated tumor. At 5 μM cis‐DDP, there was a 5.9‐fold increase in the in vitro cis‐DDP resistance of post‐therapy tumor cells relative to cells of the untreated tumor. No significant difference in DNA polymerase α activity was observed between the two tumors. These data suggest that the enhanced ability to repair cis‐DDP induced DNA damage, mediated, in part, by increased tumor DNA polymerase β and DNA ligase activities, plays an important role in the in vivo acquisition of cis‐DDP resistance in human malignant gliomas, and that these proteins and/or their encoding genes may represent critical targets for strate

ISSN:0730-2312    

DOI:10.1002/jcb.240540103

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

年代:1994

数据来源: WILEY

摘要:

AbstractUsing degenerate oligonucleotide primers derived from conserved regions in the catalytic domains of protein kinases, we have identified transcripts of the protein kinase families inTrypanosoma bruceiby the polymerase chain reaction technique. From the cDNAs synthesized from poly(A)+RNA purified from the bloodstream form of the pathogen, we have obtained seven distinct partial cDNA sequences. Deduced amino acid sequences of these seven clones contain conserved regions characteristic of catalytic domains of eukaryotic protein serine/threonine kinases, DNA gel blots showed that one of the clones, TbPK‐A4 is most likely a member of a subfamily in the protein kinase gene family, whereas the other six are probably each encoded by a single gene in the genome ofT. brucei. The full‐length cDNA of TbPK‐A1 was cloned, sequenced, and found to encode an open reading frame of 350 amino acid residues. Its gene (designatedKFR1) demonstrated high sequence similarity toKSS1andFUS3fromSaccharomyces cerevisiaeand rat MAP kinase at the amino acid level. There are a 3‐ to 4‐fold higher level ofKFR1transcript and a 2‐fold increase ofKFR1protein in the bloodstream form when compared with the insect form ofT. brucei. This preferential expression ofKFR1in the bloodstream form ofT. bruceimay play a role in controlling the cell cycle and thus the growth rate of t

ISSN:0730-2312    

DOI:10.1002/jcb.240540104

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

年代:1994

数据来源: WILEY

摘要:

AbstractWe have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion‐exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases α and δ, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52‐cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T‐antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalianMultiprotein DNAReplicationComplex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co‐purify with th

ISSN:0730-2312    

DOI:10.1002/jcb.240540105

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

年代:1994

数据来源: WILEY

摘要:

AbstractInterrelationships between proliferation and expression of cell growth as well as bone cell‐related genes were examined from two standpoints. First, the consequence of downregulating proliferation by DNA synthesis inhibition on expression of a cell cycle‐regulated histone gene and genes associated with development of the bone cell phenotype (type I collagen, alkaline phosphatase, osteopontin, and osteocalcin) was investigated. Second, the requirement for stringent growth control to support functional relationships between expression of proliferation and differentiation‐related genes was explored. Parameters of cell growth and osteoblast‐related gene expression in primary cultures of normal diploid osteoblasts, that initially express proliferation‐dependent genes and subsequently postproliferative genes associated with mature bone cell phenotypic properties, were compared to those operative in ROS 17/2.8 osteosarcoma cells that concomitantly express cell growth and mature osteoblast phenotypic genes. Our findings indicate that in both normal diploid osteoblasts and osteosarcoma cells, expression of the cell cycle regulated histone genes is tightly coupled with DNA synthesis and controlled predominantly at a posttranscriptional level. Inhibition of proliferation by blocking DNA synthesis with hydroxyurea upregulates a subset of developmentally expressed genes that postproliferatively support progressive establishment of mature osteoblast phenotypic properties (e.g., alkaline phosphatase, type I collagen, and osteopontin). However, the osteocalcin gene, which is expressed during the final stage of osteoblast differentiation when extracellular matrix mineralization occurs, is not upregulated. Variations in the extent to which inhibition of proliferation in normal diploid osteoblasts and in ROS 17/2.8 osteosarcoma cells selectively affects transcription and cellular levels of mRNA transcripts from bone cell‐related genes (e.g., osteocalcin) may reflect modifications in proliferation/differentiation interrelationships when stringent growth control i

ISSN:0730-2312    

DOI:10.1002/jcb.240540106

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

年代:1994

数据来源: WILEY

摘要:

AbstractVascular endothelial growth factor (VEGF) is a newly identified growth and permeability factor with a unique specificity for endothelial cells. Recently the flt‐encoded tyrosine kinase was characterized as a receptor for VEGF. A novel tyrosine kinase receptor encoded by the KDR gene was also found to bind VEGF with high affinity when expressed in CMT‐3 cells. Screening for flt and KDR expression in a variety of species and tissue‐derived endothelial cells demonstrates that flt is predominantly expressed in human placenta and human vascular endothelial cells. Placenta growth factor (PIGF), a growth factor significantly related to VEGF, is coexpressed with flt in placenta and human vascular endothelial cells. KDR is more widely distributed and expressed in all vessel‐derived endothelial cells. These data demonstrate that cultured human endothelial cells isolated from different tissues express both VEGF receptors in relative high levels and, additionally, that all investigated nonhuman endothelial cells in culture are also positive for KDR gene exp

ISSN:0730-2312    

DOI:10.1002/jcb.240540107

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

年代:1994

数据来源: WILEY

摘要:

AbstractClose correlation between tissue transglutaminase (tTG) induction and growth regulation and/or cell death processes has been suggested in many cell lineages. In this study, the regulation of the tTG levels by various growth and differentiation factors and its relation to growth rate and cell death processes were investigated in two rat hepatoma cell lines, McA‐RH7777 and McA‐RH8994, using a monoclonal antibody against liver tTG. Transforming growth factor‐β1 (TGF‐β1) and retinoic acid (RA) each increased tTG to the level of 8‐ to 32‐fold above that of control cultures in both cell lines after 72‐h treatment. Dexamethasone (DEX) induced a 16‐ to 32‐fold of tTG in McA‐RH8994 cells while it did not change the enzyme level in McA‐RH7777 cells. Simultaneous addition of DEX and RA increased the tTG level to more than 50‐fold in McA‐RH7777 cells as well as McA‐RH8994 cells. Other factors, such as TGF‐α, hepatocyte growth factor, dimethyl sulfoxide, and protein kinase C activator, did not show significant increases of the tTG levels. Although tTG induction by TGF‐β1 or DEX appeared to be correlated with their growth suppressive effects, RA increased the tTG level without suppressing the growth rate of hepatoma cells. TGF‐β1 was also shown to induce cell death in both cell lines. Our results demonstrate that RA and DEX are capable of modulating the TGF‐β1‐induced cell death processes independent of the tTG levels. We present evidence here that tTG induction by itself is not the direct cause of growth suppres

ISSN:0730-2312    

DOI:10.1002/jcb.240540108

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

年代:1994

数据来源: WILEY

摘要:

AbstractExpression of the proto‐oncogene c‐myc increases in the hemodynamically overloaded heart, but expression by cardiac myocytes has not been shown. To address this issue, right ventricular overload was induced in cats by pulmonary artery banding. Expression of c‐myc and α‐skeletal actin mRNA were determined by Northern analysis. Immuno‐reactive Myc protein was identified by histochemical staining.Steady state levels of c‐myc mRNA peaked within 2 h after banding. Levels of α‐skeletal actin mRNA were maximally increased 48 h–1 week after banding and were still elevated at 1 month. Prominent staining of myocyte nuclei for immunoreactive Myc protein was detected 48 h after banding although a few interstitial nuclei were also positive.These studies show that c‐myc and α‐skeletal actin gene expression are upregulated in a large animal model of hemodynamic overload. The localization of the immunoreactive Myc protein to right ventricular myocyte nuclei after pulmonary artery banding supports the hypothesis that c‐myc induction is part of a general response in cardiac hypertrophy that is common t

ISSN:0730-2312    

DOI:10.1002/jcb.240540109

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

年代:1994

数据来源: WILEY

摘要:

AbstractType II collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin‐1 (IL‐1) and interferon‐γ (IFN‐γ) are capable of decreasing the levels of α1 (II) procollagen mRNA and suppressing the synthesis of type II collagen in cultured human chondrocytes. Data reported here show that these effects of IL‐1 and IFN‐γ on the expression of the human type II collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three types of experimental evidence: (1) in nuclear run‐off assays, preincubation of chondrocytes with either IL‐1 or IFN‐γ decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB) indicated that the suppression of α1 (II) procollagen mRNA by IL‐1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT‐B/4.0) containing 4.0 kb of 5′‐flanking sequences of COL2A1 (−577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT‐B/4.0 was observed in human chondrocytes incubated with an insulin‐containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT‐B/4.0 in these cells was inhibited by either IL‐1 or IFN‐γ. Furthermore, expression of pCAT‐B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL‐1

ISSN:0730-2312    

DOI:10.1002/jcb.240540110

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

年代:1994

数据来源: WILEY

摘要:

AbstractCharacteristic changes in vimentin were studied in 9L rat brain tumor cells treated at 45°C. During heat‐shock treatment, vimentin molecules were rapidly phosphorylated and reorganized from a filamentous form into a perinuclear higher‐order structure that was less extractable by nonionic detergent. These effects were found to be highly transient, peaked at 30 min after the onset of heat‐shock treatment, and subsided thereafter. Simultaneously, the solubility of the constitutively expressed heat‐shock protein70 (HSC70) was also temporarily decreased and the kinetics was identical to that of vimentin. The results indicated that HSC70 and vimentin were co‐insolubilized during the heat‐shock treatment. We propose that the reorganization of the intermediate filaments resulted from enhanced phosphorylation of vimentin leads to the concurrent association of HSC70 to the intermediate filaments. This process may play an essential role in regulating heat

ISSN:0730-2312    

DOI:10.1002/jcb.240540111

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

年代:1994

数据来源: WILEY