摘要:

AbstractPrevious studies have shown that glycosaminoglycans in the extracellular matrix accelerate the inactivation of target proteases by certain protease inhibitors. It has been suggested that the ability of the matrix of certain cells to accelerate some inhibitors but not others might reflect the site of action of the inhibitors. Previous studies showed that fibroblasts accelerate the inactivation of thrombin by protease nexin‐1, an inhibitor that appears to function at the surface of cells in extravascular tissues. The present experiments showed that endothelial cells also accelerate this reaction. The accelerative activity was accounted for by the extracellular matrix and was mostly due to heparan sulfate. Fibroblasts but not endothelial cells accelerated the inactivation of thrombin by heparin cofactor II, an abundant inhibitor in plasma. This is consistent with previous suggestions that heparin cofactor II inactivates thrombin when plasma is exposed to fibroblasts and smooth muscle cells. Neither fibroblasts nor endothelial cells accelerated the inactivation of C1s by plasma C1‐inhibi

ISSN:0730-2312    

DOI:10.1002/jcb.240360302

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

年代:1988

数据来源: WILEY

摘要:

AbstractIncubation of Swiss mouse 3T3 cells at 37°C with bovine brain‐derived growth factor (BDGF) decreased the cell surface125I‐EGF binding activity of these cells by 70–80%. This down‐modulation of the EOF receptor by BDGF was time, temperature, and dose dependent Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high‐affinity EGF receptors. The BDGF‐induced down‐modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF.BDGF down‐modulated the EGF receptor in phorbol myristic acetate (PMA)‐pretreated cells, as well as in control cells. Furthermore, PMA‐pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA‐pretreated cells. This BDGF‐induced down‐modulation of the EGF receptor in PMA‐desensitized cells suggests that BDGF down‐regulates the EGF receptor by a mechanism distinct from that of PMA.Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down‐modulation induced either by BDGF or by platelet‐derived growth factor (PDGF) but had no effect on the PMA‐induced down‐modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF‐induced down‐modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down‐modulation of the EGF receptor by increasing the internalization of cell surface high‐affinity receptors and that the internalization process may not

ISSN:0730-2312    

DOI:10.1002/jcb.240360303

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

年代:1988

数据来源: WILEY

摘要:

AbstractDesmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS‐PAGE. These preparations were extracted in 9 M urea, 10 mM Tris‐HCl (pH 9), and 25 mM B‐mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea‐soluble and insoluble fractions were analyzed by SDS‐PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin‐like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris‐HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane‐associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoprot

ISSN:0730-2312    

DOI:10.1002/jcb.240360304

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

年代:1988

数据来源: WILEY

摘要:

AbstractA prepro‐gastrin‐releasing peptide (GRP) gene was introduced into Swiss 3T3 mouse embryo fibroblasts by DNA transfection in an attempt to establish autocrine growth stimulation. Clonal transfectants expressed varying amounts of GRP encoding mRNA. They synthesized and secreted a ∼ 15‐kd pro‐GRP hormone but not fully processed 2.8‐kd GRP. Accordingly, no changes in growth properties were associated with GRP gene expression. We postulate that Swiss 3T3 fibroblasts lack the enzymes necessary to process significantly pro‐GRP into biologically active peptides and that this deficiency may be responsible for the failure to establish autocrine growth stimulation in the tran

ISSN:0730-2312    

DOI:10.1002/jcb.240360305

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

年代:1988

数据来源: WILEY

摘要:

AbstractMutants unable to regulate intracellular pH through the Na+:H+antiport system were found to evolve tumors less frequently than wild‐type CCL39 hamster lung fibroblasts, after transplantation in athymic nude mice. When rare tumors arose, they comprised cells which were transformed in vitro, but which upon retransplantation grew at a lower rate than tumor cells originating from CCL39 cells. Both parental and mutant cells became transformed after transfection of the activated Harveyrasoncogene, but transfectants derived from the mutants had a weaker tumorigenic potential. These results suggest that transformed characteristics can be acquired independently from the Na+:H+antiporter. However, the presence of this system provides a selective growth advantage when cells are confronted with natural environments, as it occurs during the expansion of tumors in a hos

ISSN:0730-2312    

DOI:10.1002/jcb.240360306

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

年代:1988

数据来源: WILEY

摘要:

AbstractProducts ofrasgenes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttranslational modification by fatty acid. In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification ofrasproteins prior to the fatty acid acylation. The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel. The fatty acid acylation does not contribute to this mobility shift. This modification is affected by thedpr1mutation which has recently been shown to affect the processing of yeastRASproteins. To further characterize the nature of the modification event, we have clonedDPR1gene from the DNA ofSaccharomyces cerevisiae. The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb. Genes related to theDRP1appear to be present in a distantly related yeast,Schizosaccharomyces pombeas well as in guinea pig and human cells.

ISSN:0730-2312    

DOI:10.1002/jcb.240360307

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

年代:1988

数据来源: WILEY

摘要:

AbstractIn order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeastCOX5agene (the structural gene for subunit Va of the inner membrane protein complex cytochromecoxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modifiedCOX5aso that the DNA sequences encoding the wild‐type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequenc

ISSN:0730-2312    

DOI:10.1002/jcb.240360308

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

年代:1988

数据来源: WILEY

摘要:

AbstractThis communication presents our recent studies on the biosynthesis of influenza virus hemagglutinin (HA) in a mammalian‐cell‐free system and its translocation across microsomal membranes. RNAs coding for wild‐type (full‐length) and mutant (truncated) forms of HA were generated by in vitro transcription by using bacteriophage T7 DNA‐dependent RNA polymerase. These RNAs were translated in a rabbit reticulocyte system that was supplemented with dog pancreas membranes, either before translation was initiated or after it had been artificially terminated with the antibiotic cycloheximide. All forms of HA could be cotranslationally translocated. However, only truncated molecules (83% of full length) could translocate after protein synthesis had been terminated. Posttranslational translocation was dependent on the presence of a functional N‐terminal signal sequence and occurred only in the presence of ribosomes. The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed based on the signal

ISSN:0730-2312    

DOI:10.1002/jcb.240360309

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

年代:1988

数据来源: WILEY

摘要:

AbstractAlterations in expression of major histocompatibility complex (MHC) antigens on tumor cells clearly correlate with the tumorgenicity and metastatic potential of those cells. These changes in the biological behavior of the tumor cells are presumably secondary to resulting changes in their susceptibility to immune recognition and destruction. Murine leukemia viruses (MuLV) exert regulatory effects on class I genes of the MHC locus. MuLV infection results in substantial increases in cell surface expression of all three class I MHC antigens. These viral effects on MHC antigen expression profoundly influence immune‐mediated interaction with the infected cells, as assessed by cytotoxic T lymphocyte recognition and killing. Control of class I MHC and beta‐2 microglobulin genes by MuLV takes place via atrans‐acting molecular mechanism. MuLV controls expression of widely separated endogenous cellular MHC genes, transfected xenogeneic class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to a bacterial reporter gene. These findings indicate that MuLV exerts its effects on MHC expression via atransmechanism. The MuLV‐responsive sequences on the MHC genes appear to lie within 1.2 kilobases upstream of the initiation codon for thos

ISSN:0730-2312    

DOI:10.1002/jcb.240360310

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

年代:1988

数据来源: WILEY

摘要:

AbstractMetastatic variants of the B16 melanoma displaying high experimental metastatic potential have been shown to express high levels of a 72,000‐dalton glycoprotein (Met‐72) on their cell surface (Kimura AK, Xiang J:J Nat Can Inst76:1247–1253, 1986). Monoclonal antibodies (MoAb) directed against the Met‐72 determinant have been used in this study as immunohistochemical reagents on preparations of fresh B16 melanoma tumors and their metastases. These immunohistochemical analyses have utilized frozen sections, impression smears, and cytospin preparations of fresh tumors harvested at various time points during tumor growth, to view the presence and location of Met‐72‐positive metastatic variants within tumor masses. Biotinylated anti‐Met‐72 MoAbs were reacted with freshly dissociated tumor cells from a B16 melanoma ovarian metastasis. These cells were then reacted with fluorescein isothiocyanate (FITC)‐streptavidin and analyzed by flow cytometry. A discrete population of positively staining cells was detected and isolated by cell sorting techniques. Met‐72‐positivc cells were then cloned and reanalyzed after several weeks ofin vitroexpansion and found to have high experimental metastatic potential to ovaries. Frozen sections of subcutaneous tumors and their metastases were analyzed by immunoperoxidase techniques. A consistent finding in these studies has been that the few tumor cells which showed high intensity of Met‐72 staining were positioned perivascularly and at the invading front

ISSN:0730-2312    

DOI:10.1002/jcb.240360311

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

年代:1988

数据来源: WILEY