摘要:

AbstractThe ability of murine Steel factor to promote the in vitro production of granulocyte‐macrophage progenitor cells (CFU‐GM) was examined in short‐term liquid cultures. Bone marrow from C57BL/6J or Sl/Sldmice was placed in culture for seven days with either Steel factor alone or in the presence of IL‐3. CFU‐GM responsive to GM‐CSF, IL‐3, and CSF‐1 were measured in the input population and again after 3 or 7 days in culture. Steel factor alone increased the number of all CUF‐GM types as early as 3 days after culture initiation, with further increases at day 7. This effect was protentiated by the addition of IL‐3. Production of CFU‐GM by C57BL/6J orSl/Sldmarrow was comparable except for enhanced production of CSF‐1 responsive progenitors bySl/Sldmarrow. A recombinant Sldprotein was also shown to be equivalent to the wild‐type protein in its capacity to promote CFU‐GM production from normal bone mar

ISSN:0730-2312    

DOI:10.1002/jcb.240500302

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

年代:1992

数据来源: WILEY

摘要:

AbstractDuring the past several years, numerous laboratories have reported isolation and purification of proteinase inhibitors from human urine. Many of these molecules were incompletely characterized and some of them may have been artifacts in part because of harsh procedures used for their isolation. Consequently, there is disagreement and confusion regarding the biochemical characteristics of these inhibitors. We previously reported the isolation of a proteinase inhibitor, EDC1, from the urine of a leukemic patient. This molecule, Mr30 kDa, was antigenically related to plasma inter‐α‐trypsin inhibitor (IATI) and inhibited the growth of a virally transformed B cell line. Immunoreactive EDC1 was also the major component of low molecular weight proteinuria observed in cancer patients. We now report a new method for the isolation EDC1 from urine of patients with adenocarcinomas of colon and lung and melanoma and compare its partial amino acid sequence with that of HI 30, a proteinase inhibitor previously isolated from pooled normal urine by Hochstrasser et al. [Hoppe‐Seyler's Z physiol Chem 357:153–162, 1976]. Our method involves (i) a batchwise cation exchange, (ii) gel filtration chromatography, (iii) anion exchange chromatography on FPLC, and (iv) reverse phase C18 chromatography on HPLC. This method is mild and results in an over all yield of 0.4 to 1.2 mg of EDC1/liter urine. On the basis of the partial N‐terminal amino acid sequence of its N terminal (38 residues) and middle regions (29 residues), EDC1 appears to be identical with H130. Surprisingly, during this isolation procedure, another proteinase inhibitor, Mr22 kDa, which cross‐reacted with antisera to EDC1 and IATI, was also isolated. The 22 kDa molecule was a major component of the IATI related urinary molecules and was identical with the 30 kDa EDC1 in which first the 15 N terminal residues were clipped. The lower Mrinhibitor was not an artifact formed during storage or isolation procedure because the Western blot analysis of fresh cancer and normal urine revealed the 30 and 22 kDa molecules. Thus, both the 30 kDa EDC1 (HI30) and its clipped variant, the 22 kDa molecule, are physiologic components of IATI related urinary proteinase inhibitors and excretion of both forms may be increased in patients with advanced cancer. © 1992 Wi

ISSN:0730-2312    

DOI:10.1002/jcb.240500303

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

年代:1992

数据来源: WILEY

摘要:

AbstractVinculin is a cytoskeletal protein believed to be involved in linking microfilaments to the cell membrane. it is a substrate for the Ca2+‐ and phospholipid‐dependent protein kinase C. We show here that when human platelets attach and spread on a solid surface, the α isoforms of vinculin become phosphorylated at serine and/or threonine residues. Phosphorylation is dependent on adhesion to a surface, since suspended, unattached platelets can produce filopodia but no phosphorylation of vinculin. Phosphorylation is also dependent on actin polymerization, as it does not occur when platelets had been pretreated with cytochalasin B. Most likely, protien kinase C is responsible for the phosphorylation of vinculin, since phosphorylation also occurs when platelets are treated with a phorbol ester, which activates protein kinase C, and is blocked by treatment with a staurosporine derivative which inhibits this enzyme. These results suggest that phosphorylation plays a role in anchoring vinculin at sites of microfilament‐membrane interaction. © 1992 Wiley‐

ISSN:0730-2312    

DOI:10.1002/jcb.240500304

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe have previously shown that vimentin is a growth‐regulated gene whose mRNA levels increase after srum stimulation of quiescent hamster fibroblasts. In this study, the control of the growth‐regulated expression of vimentin was determined in ts13 cells induced to proliferate by serum. Both transcriptional and post‐transcriptional mechanisms of regulation were exmined by determining transcriptional rates, cytoplasmic transcript abundance, transcript stability, and protein abundance. We observed a fourfold increase in vimentin transcripts in the cytoplasm of serum‐stimulated ts13 cells. Since transcripts are stable in both quiescent and stimulated cells, this induction of vimentin expression is a result of a fivefold increase in vimentin‐specific transcriptional activity. As a result of this increased transcript availability, the abundance of polymerized vimentin protein increased following serum stimulation of quiescent fibroblasts. Overall, the induction of vimentin expression in fibroblasts by serum is a consequence of increased vimentin‐specific transcriptional activity. The significance of this with regard to cytoskeletal organization and cell division is discussed. © 1992 Wil

ISSN:0730-2312    

DOI:10.1002/jcb.240500305

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe human erythroleukemic cell line, K562, can be induced to differentiate by the addition of activin A, a newly purified protein belonging to the TGF‐β1family. The present studies used flow cytometric cell cycle analysis, indirect immunofluorescence staining of the proliferating cell nuclear antigen (PCNA), and thymidine incorporation assay of cell proliferation to study the effects of activin A on the cell cycle during differentiation in K652 cells. Activin phase. The latter can be observed after approximately 24 hr of incubation with activin A and then disappears after this early stage of induction of differentiation. Cell cycle kinetics analysis using synchronized K562 cells also confirms that in the presence of activin A, K562 cells progress normally through various phases of cell, except that there is prolongation of the G1phase between 10 to 24 hr of culture. Furthermore, this transient arrest in G1is correlated with dephosphorylation of a nucleoprotein, the RB gene product, which occurs within 9–24 hr of incubation with activin A; and phosphorylation of RB protein then develops afterward. In addition, these cell cycle‐related events are observed to occur earlier than the accumulation of hemoglobins in K562 cells. It is concluded that transient dephosphorylation of RB protein and prolongation of G1phase of cell cycle precede and accompany erythroid differentiation caused by activin A and chemical inducers, thus constituting part of the mechanism for induction of differentiation in the erythroleukemia cells. © 1992 Wiley‐

ISSN:0730-2312    

DOI:10.1002/jcb.240500306

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe expression of vasuclar smooth muscle (VSM) α‐actin mRNA during BC3H1 myogenic cell differentiation is specifically stimulated by conditions of high cell density. Non‐proteolytic dissociation of cell‐cell and cell‐matrix contacts in post‐confluent cultures of BC3H1 myocytes using EDTA promotes loss of the differentiated morphological phenotype. EDTA‐dispersed myocytes exhibit an undifferentiated fibroblastoid appearance and contained reduced levels of both VSM and skeletal α‐actin mRNA. Muscle α‐actin mRNA levels in EDTA‐dispersed myocytes were not restored to that observed in confluent myocyte preparations by experimental manipulation of cell density conditions. Pulse‐labeling techniques using L‐[35S] cysteine to identify muscle actin biosynthetic intermediates revelated that EDTA‐dispersed myocytes expressed nascent forms of both the VSM and skeletal muscle α‐actin polypetide chains. However EDTA‐dispersed myocytes were less effieicent in the post‐translational processing of immautre VSM α‐actin compared to non‐dispersed myocytes. Simple cell‐to‐cell contact may mediate VSM α‐actin processing efficiency since high‐density preparations of EDTA‐dispersed myocytes processed more VSM α‐actin intermediate than myocytes plated at low density. The actin isoform selectivity of the response to modulation of intercellular contacts suggests that actin biosynthesis in BC3H1 myogenic cells involves mehcanisms capable of discriminating between different isoform classes of nas

ISSN:0730-2312    

DOI:10.1002/jcb.240500307

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

年代:1992

数据来源: WILEY

摘要:

AbstractHMG‐like chromosomal proteins fromTruypansoma cruziwere studies. Four HMG‐like proteins, designated HMG A, HMGA‐B, HMG‐C, and HMG‐E, were isolated and found to have molecular weights of 35.5 kd, 27.5 kd, 21.8 kd and 10.4 kd, respectively. Immunological relatedness was demonstrated between the mammalian HMG 1,2 and the HMG‐A and HMG‐B fromT. cruzi. The relative amount of HMG‐C and HMG‐E proteins vary inT. cruzidepending to the proliferative stage of the cells. HMG‐E protein is increased in proliferating cells when compared to its level in non‐proliferating cells. HMG‐C is increased in the non‐proliferating cells. Probably, the shifts observed in the relative amount of HMG‐like proteins are related to the proliferating cells of this flagellate. The results are consistent with those described for other lower eukaryotes where the HMG‐like proteins isolated are similar but not identical to HMG proteins from verteb

ISSN:0730-2312    

DOI:10.1002/jcb.240500308

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

年代:1992

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐Beta (TGF‐β) is a potent growth inhibitor for several cell types including epithelial cells and hematopoietic progenitor cells. Using a human promonocytic leukemia cell line, THP‐1, we have shown that TGF‐β inhibits their proliferation and promotes differentiation into cells exhibiting macrophage‐like properties. Therefore, a key question is whether TGF‐β influences the expression of genes associated with proliferation and/or growth inhibition. TGF‐β treatment of THP‐1 cells results in downregulation of expression ofc‐myc. We also observe that TGF‐β1‐treated cells express reduced levels of the cell cycle regulated histone, H2B, but express elevated levels of an RNA splicing variant of this histone that has been observed to be upregulated in growth inhibited and terminally differentiated cells. In addition, a nuclear protein associated with senescence and withdrawal of cells from the cell cycle, statin, is also expressed by THP‐1 cells in response to TGF‐β1 ttreatment. These results suggest that TGF‐β1 is capable of inducing expression of specific nuclear proteins associated with differentiation and/or cessation of proliferation that may result in changes in nuclear organization and altered gene expression. Such changes in nuclear organization may be incompatible with continued proliferation of

ISSN:0730-2312    

DOI:10.1002/jcb.240500309

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

年代:1992

数据来源: WILEY

摘要:

AbstractThis study examines the effect of Mitomycin C, a fungal toxin which inhibits DNA synthesis, on the regeneration of partially denuded large vessel endothelium in vitro. Monolayers of bovine pulmonary artery endothelial cells were treated with Mitomycin C prior to or immediately following partial denudation and were incubated in the continuing presence of Mitomycin C; the effects of this treatment on monolayer repair, cell proliferation, and other aspects of endothelial phenotye were monitored. Cell proliferation, DNA, RNA, and protein synthesis were all reduced in a dose dependent manner in treated cultures. Incubation with Mitomycin C for 48 h or longer resulted in reduced cell spreading, and rounding up and loss of cells from both intact and partially denuded cultures. Effects were less severe with lower doses and shorter incubation times. However, significant reductions in monolayer regeneration occurred within 8 h of incubation, sufficiently early to suggest that Mitomycin C may affect aspects of the regeneration process independent of cell proliferation. Polarization/spreading of cells at the denudation edge was monitored by fluorescence staining for golgi with C5‐DMB‐ceramide, and for centrioles with antibodies to tubulin. Centrioles and golgi rapidly reoriented to a location at the putative leading edge of control cultures. Mitomycin C treatment had no effect on centriole reorientation, but caused a significant delay in golgi localization. These results suggest that Mitomycin C inhibits endothelial monolayer regeneration by mechanisms independent of cell proliferation and DNA synthesis, perhaps by interfering with cell spreading or translocation at the wound edge. © 1992 Wiley‐Lis

ISSN:0730-2312    

DOI:10.1002/jcb.240500310

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

年代:1992

数据来源: WILEY

摘要:

AbstractIntact nuclei derived from poorly or highly liver‐metastatic murine large‐cell lymphoma cell line RAW117 were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein (DNP/RNP) complexes withMsp‐1. The DNP/RNP complexes were composed of DNP/RNPs which were derived from the DNP/RNP complexes by incubation in the presence or absence of DNase‐1 and subsequent isolation by two‐dimensional [isoelectric focusing/sodium dodecylsulfate (SDS)] polyacrylamide gel electrophoresis (PAGE), electroelution from the gel, and removal of SDS. Approximately 450 DNP/RNPs in the two‐dimensional gels corresponding to discrete spots or in some cases streaks were analyzed for the presence ofv‐abl,p53,c‐neu, c‐H‐ras, β‐casein, 18s rDNA, and μ‐chain immunoglobulin genes using a hybridization technique. Ten DNP/RNP complexes contained tightly associatedp53DNA, whereas six contained c‐ or v‐abl, four contained μ‐chain gene, two contained c‐H‐ras, one contained dot‐blot β‐casein, two contained 18s rDNA, and c‐neuwas found in one of the DNP/RNPs. The DNP/RNPs were also analyzed for in vitro RNA polymerase and primase activities. To assess the potential transcription abilities of the isolated DNP/RNPs, individual DNP/RNPs or DNP/RNP mixtures (reconstituted after SDS‐PAGE separation) were examined for RNA polymerase initiation and synthesis. When RNA products were formed, these were purified by extracellulose chromatography and used as back‐hybridization probes for the genes of interest. The RNA products were also analyzed by RNA gel electrophoresis. RNA formation was inhibitable by actinomycin D, and the RNAs formed ranged in size from ∼ 80 kbp to ∼ 1 kbp. By mixing various DNP/RNP complexes together, different patterns of RNA synthesis were found. For example, one DNP/RNP of Mr∼ 140,000, isoelectric point(pl) ∼ 5.8 synthesized a high molecular weight RNA in vitro that hybridized with β‐casein cDNA, but β‐casein is not expressed in RAW117 cells, suggesting that the silencing of the β‐casein gene was negated by isolation of the DNP/RNP. Mixing this DNP/RNP with two other specific DNP/RNPs again inhibited the synthesis of β‐casein RNA, suggesting that interactions between DNP/RNP complexes can result in differential RNA expressio

ISSN:0730-2312    

DOI:10.1002/jcb.240500311

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

年代:1992

数据来源: WILEY