bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neoplastic modulation of extracellular matrix: Stimulation of chondroitin sulfate proteoglycan and hyaluronic acid synthesis in co‐cultures of human colon carcinoma and smooth muscle cells
Authors: Iozzo Renato V. , Sampson Phyllis M. , Schmitt Gail K. 
Published: December 2004

AbstractPrevious studies have shown that human colon carcinomas contain elevated amounts of chondroitin sulfate proteoglycan (CS‐PG) and hyaluronic acid, and that the major site of synthesis of these products is the host mesenchyme surrounding the tumor. These findings have led to the proposal that the abnormal formation of the tumor stroma is modulated by the neoplastic cells. The experiments of this paper were designed to explore further this complex phenomenon in an in vitro system using co‐cultures of phenotypically stable human colon smooth muscle (SMC) and carcinoma cells (WiDr). The results showed a 3–5‐fold stimulation of CS‐PG and hyaluronic acid biosynthesis in the co‐cultures as compared to the values predicted from the individual cell type cultured separately. The increase in CS‐PG was not due to changes in specific activity of the precursor pool, but was rather due to a net increase in synthesis, inasmuch as it was associated with neither a stimulation of cell proliferation nor with an inhibition of intracellular breakdown. These biochemical changes were corroborated by ultrastructural studies which showed a marked deposition of proteoglycan granules in the co‐cultures. Several lines of evidence indicated that the SMC were responsible for the overproduction of CS‐PG: (i) SMC synthesized primarily CS‐PG when cultured alone, in contrast to the WiDr, which synthesized exclusively heparan sulfate proteoglycan; (ii) only the SMC in co‐culture stained with an antibody raised against the amino terminal peptide of a CS‐PG (PG‐40), structurally and immunologically related to that synthesized by the SMC; (iii) the stimulation of CS‐PG in SMC could be reproduced, though to a lesser extent, using medium conditioned by WiDr, whereas medium conditioned by SMC had no effects on WiDr. In conclusion this study has reproduced in vitro a tumor‐associated matrix with a proteoglycan composition similar to that observed in vivo and provides further support to the concept that production of a proteoglycan‐rich extracellular environment is regulated by specific tumor‐host cell interactions.
Cell type and tissue distribution of the fibroblast growth factor receptor
Authors: Olwin Bradley B. , Hauschka Stephen D. 
Published: December 2004

AbstractA receptor for fibroblast growth factor (aFGF, bFGF) was partially characterized in intact cell cultures, cell plasma membranes, and tissue plasma membrane preparations. Analysis of 24 different cell types from four species identified a 165‐kDa FGF receptor present on the cell surface of most mesodermal and neuroectodermal cells. Chemical crosslinking of 125I‐aFGF to its cell surface receptor was specifically blocked by a 100‐fold molar excess of either aFGF or bFGF. In contrast to the similar molecular weight of FGF receptors, different cell types exhibited significant variations in binding of 125I‐aFGF to intact cultures with low values of 8 pM and 700, to high values of 60 pM and 30,000, for the Kd and receptor number per cell, respectively. A binding assay was developed for quantitation of 125I‐aFGF binding to cell‐ and tissue‐derived membrane preparations. Membranes prepared from baby hamster kidney cells exhibited a Kd of 55 pM, while a similar Kd of 67 pM was determined for intact baby hamster kidney cells. Although ten different adult bovine tissue membrane preparations and human term placental membranes exhibited no specific binding of 125I‐aFGF, FGF receptor was detected in embryonic murine tissues (17 days gestation). These results support the existence, in a variety of cells, of either a common FGF receptor that binds both aFGF and bFGF or closely related FGF receptors that cannot be distinguished by molecular weight. The differential binding of FGF to its receptor in embryonic vs. adult tissues suggests a potentially broad role for FGF in embryonic development and a more restrictive role in the adult.
TGFβ gene transcription in normal and neoplastic liver growth
Authors: Carr Brian I. , Huang Ting H. , Itakura Keiichi , Noël Micheline , Marceau Normand 
Published: December 2004

AbstractTGFβ is a potent, nontoxic inhibitor of mitogen‐induced DNA synthesis in primary cultures of adult rat hepatocytes. Using a cDNA probe, we investigated TGFβ gene expression in quiescent, regenerating, and neoplastic liver, and several hepatoma lines by Northern gel analysis. We found that regenerating liver had increased TGFβ gene transcripts beginning at about 8 h, with a broad peak of 48–120 h and return to normal after 9 days. Separation of the regenerating liver into its constituent cell types, followed by RNA extraction and reprobing, revealed that increased TGFβ gene transcripts were confined to the enriched endothelial‐cell population and not the hepatocytes. Increased hepatic TGFβ expression was also found in fetal liver and in rats immediately after birth. Elevated TGFβ mRNA levels were also found in primary cultures of oval cells and an established bile ductular cell line, as well as in carcinogen‐altered liver epithelial cell lines. Transcripts were undetectable in normal human liver but were abundant in the human hepatoma lines Hep G2, Hep 3B, PLC/PRF/5, and SK‐Hep‐1. Elevated levels were also found in the normal rat liver–derived lines BRL‐3A and clone 9 and the H4IIE rat hepatoma, but not in the HTC, MH1C1, and MH7777 rat hepatomas. The hepatocarcinogen diethylnitrosamine induced high transcript levels after single injections in a time‐ and dose‐dependent manner. These results suggest that the liver may be a paracrine organ with respect to TGFβ gene expression, which can be induced by carcinogens and by growth stimulation.
An inhibitor specific for the mouse T‐cell associated serine proteinase 1 (TSP‐1) inhibits the cytolytic potential of cytoplasmic granules but not of intact cytolytic T cells
Authors: Simon Markus M. , Prester Marlot , Kramer Michael D. , Fruth Uli 
Published: December 2004

AbstractWe have investigated a proteinase inhibitor, designed according to the preferred amino acid sequence that is cleaved by the murine T‐cell specific serine proteinase 1 (TSP‐1) for its effect on the cytolytic potential of cloned cytotoxic T‐cell lines (CTLL) and of cytoplasmic granules, derived from these cells. Pretreatment of effector cells with H‐D‐Pro‐Phe‐Arg‐chloromethyl‐ketone (PFR‐CK) prior to the cytotoxicity assay did not result in inhibition of cytolytic activity of three independent CTLL and did not effect their granule‐associated TSP‐1 activity after extraction with Triton X‐100. Furthermore, PFR‐CK did not interfere with cytolysis of target cells by CTLL when present for the entire incubation period. In contrast, PFR‐CK inhibited in a dose‐dependent manner both TSP‐1 activity and the hemolytic/cytolytic potential of isolated cytoplasmic granules after their pretreatment with high‐salt concentration. We interpret these results to mean that cytolysis of target cells by CTLL involves the granule‐associated proteinase TSP‐1, which probably becomes active upon exocytosis following effector‐target cell interactions.
Viruses and cytokines: Evidence for multiple roles in pancreatic beta cell destruction in type 1 insulin‐dependent diabetes mellitus
Authors: Campbell Iain L. , Harrison Leonard C. 
Published: December 2004

AbstractInsulin‐dependent (type 1) diabetes mellitus (IDDM) is due to the selective autoimmune‐mediated destruction of pancreatic beta cells possibly initiated by viruses. To elucidate the possible role of viruses and cytokines in the pathogenesis of IDDM, we have examined the effect of reovirus infection on beta cell major histocompatibility complex (MHC) expression and the effect of interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) on beta cell function in vitro. Infection of RIN‐m5F (rat insulinoma) cells with reovirus‐1 or reovirus‐3 was associated with a tenfold increase in class 1 MHC protein and mRNA expression. Reovirus infection did not induce the expression of class 11 MHC by RIN‐m5F cells. Exposure of reovirus to ultraviolet light almost completely abolished its ability to induce class 1 MHC protein expression on infected cells.Murine islets cultured for 3 days with IFN‐γ and/or TNF‐α had a significantly reduced insulin response to glucose, which was more marked with a combination of the cytokines. During 6 days of culture in IFN‐γ plus TNF‐α islets underwent noticeable degeneration associated with an 80% reduction in insulin content. These findings together with previous data suggest viruses and cytokines may have multiple roles in beta cell destruction, indirectly through enhanced MHC protein expression and directly through functional impairment and loss of viability.
Competence growth factors can cause modification in higher‐order chromatin structure in mouse embryo 3T3 fibroblasts
Authors: Ortega Jose Miguel , Meneghini Rogerio 
Published: December 2004

AbstractThe authors compared sedimentation rates of nucleoids from mouse embryo 3T3 fibroblasts cultured in the presence or absence of different cell growth factors. The results clearly showed that rapidly sedimenting nucleoids are obtained only when cells are supplied with any of the following competence growth factors: platelet‐derived growth factor (PDGF), fibroblast growth factor (FGF), or the product of the oncogene v‐sis (a peptide homologous to PDGF). The tumor promoter phorbol 12‐myristate 13‐acetate, an activator of protein kinase C and a partial mitogen, shares this property with the competence growth factors. Removal of these factors from the medium causes cells to enter Go and nucleoids to sediment at a slower rate. Protein synthesis is required for growth factor induction of change in nucleoid sedimentation, but inhibition of either DNA synthesis or DNA repair does not antagonize the effect of growth factors. Titration of nucleoids with ethidium bromide indicates that one possible mechanism for the nucleoid change is the unwinding of DNA in supercoiled loops. The results indicated that the nucleoid change constitutes a cell response to competence factors that might have an important role in cell proliferation.
Specific desensitization of the epidermal growth factor receptor by pp60v‐src
Authors: Gray George M. , Wei Chung‐Wen , Macara Ian G. 
Published: December 2004

AbstractBALB/c 3T3 cells infected with a temperature‐sensitive mutant (LA90) of RSV have been used to investigate possible heterologous interactions between the pp60v‐src tyrosyl kinase and the epidermal growth factor (EGF) and bradykinin receptors. The LA90 pp60v‐src exhibits a very rapid activation t1/2 (<5 min) of protein kinase activity on decreasing the temperature from 40°C to 35°C. This change in temperature was also found to induce a very rapid decrease in the affinity for 125I‐EGF of receptors on the RSV‐LA90‐infected cells but not of those on control parental cells. However, no significant changes were detected in the binding of 3H‐bradykinin to either cell line. Two separable processes control the desensitization of the EGF receptor by pp60v‐src, both of which are independent of protein kinase C. The first is rapid and transient, while the second is sensitive to cycloheximide and persists long after inactivation of pp60v‐src.
A phorbol ester and phospholipid‐activated, calcium‐unresponsive protein kinase in mouse epidermis: Characterization and separation from protein kinase C
Authors: Gschwendt M. , Kittstein W. , Horn F. , Leibersperger H. , Marks F. 
Published: December 2004

AbstractThe phosphorylation of an Mr 82,000 protein (p82) in the Triton X‐100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)‐catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82‐kinase system is down‐modulated after TPA treatment of mouse skin, with a half‐life of around 5 h. Down‐modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down‐modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction.
Phospholipase A2 engineering: Design, synthesis, and expression of a gene for bovine (pro)phospholipase A2
Authors: Noel Joseph P. , Tsai Ming‐Daw 
Published: December 2004

AbstractA gene coding for the (pro)phospholipase A2 (PLA2) from bovine pancreas has been designed, synthesized, and expressed in Escherichia coli. The gene was designed with a variety of restriction sites that will facilitate future mutagenesis studies. Codons occurring frequently in prokaryotic systems were chosen whenever possible. The total gene spans 404 base pairs and was divided into 33 oligonucleotides. The gene was constructed in two halves of 224 and 180 base pairs from the oligonucleotides by the shotgun ligation technique using pBSM13‐ as the cloning vehicle. The two fragments were then ligated and cloned into pBSM13‐ to complete the gene. The (pro)PLA2 gene was then verified by restriction site mapping and dideoxy sequencing. The gene was expressed to high levels from a high copy number vector, designated as pJPN, derived from the E. coli secretion vector pIN‐III‐ompA3. Although the protein failed to be excreted and was in the form of insoluble inclusion body, active PLA2 could be obtained by renaturation of the inclusion body pellet followed by tryptic activation, which removes the signal sequence and the pro‐peptide of proPLA2. The PLA2 thus obtained reacted with the antisera raised against the natural PLA2 purified from bovine pancreas, and the specific activity of the expressed PLA2 was identical to that of the natural PLA2. The shotgun ligation and synthetic gene approaches are simple and inexpensive and can be adapted to express most of the enzymes in the phospholipase A2 family.
Further characterization of four lipocortins from human peripheral blood mononuclear cells
Authors: Coméra Christine , Rothhut Bernard , Cavadore Jean‐Claude , Vilgrain Isabelle , Cochet Claude , Chambaz Edmond , Russo‐Marie Françoise 
Published: December 2004

AbstractFour calcium and phospholipid binding proteins purified from mononuclear cells were characterized for PKC and EGF phosphorylation, actin binding capacity, and partial tissue distribution. Those named 35K, 32K, and 73K are equivalent, respectively, to lipocortin III, endonexin II and the 67 kDa calelectrin; 36K is a fragment of 73K. After purification, 35K and 73K were phosphorylated by protein kinase C in vitro but 36K nor 32K were not. None were phosphorylated by the epidermal growth factor receptor kinase in vitro; 73K bound F‐actin in a calcium‐dependent manner, whereas 35K, 36K, and 32K did not. Using Western blotting analysis, 32K and 73K were detected in high amounts in human lymphocytes, monocytes, liver, and placenta and in rat adrenal medulla; but 32K was not detected in polymorphonuclear cells, and 36K and 35K were detected in high amounts only, respectively, in human blood lymphocytes and polymorphonuclear cells. Thus, 32K and 73K appear to have a wide tissue distribution, whereas 35K has a much more restricted distribution.
Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli
Authors: Sheng Zeqi , Vanderpuye Oluseyi A. , Hull Steven R. , Carraway Coralie A. Carothers , Carraway Kermit L. 
Published: December 2004

AbstractMembrane‐microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. These microvilli are covered by a sialomucin complex, composed of the sialomucin ascites sialoglycoprotein‐1 (ASGP‐1) and the associated concanavalin A (Con A)‐binding glycoprotein ASGP‐2. Limited proteolysis of the microvilli releases large, highly glycosylated fragments of ASGP‐1 from the microvilli and increases the association of ASGP‐2 with the Triton‐insoluble microvillar microfilament core (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988). To analyze the topography of ASGP‐2 in the membrane and its association with the microfilament core, microvilli were treated with proteinase K for timed intervals and centrifuged. The pelleted microvilli were extracted with Triton X‐100 for the preparation of microfilament cores and Triton‐soluble proteins or with 0.1 M carbonate, pH 11, for the preparation of microvillar membranes depleted of peripheral membrane proteins. These microvilli fractions were analyzed by dodecyl sulfate gel electrophoresis, lectin blotting with Con A and L‐phytohemagglutinin, and immunoblotting with anti‐ASGP‐2. The earliest major proteolysis product from this procedure was a 70 kDa membrane‐bound fragment. At longer times a 60 kDa released fragment, 30–40 kDa Triton‐soluble fragments, and 25–30 kDa membrane‐ and microfilament‐associated fragments were observed. Phalloidin shift analysis of microfilament‐associated proteins on velocity sedimentation gradients indicated that the 25–30 kDa fragments were strongly associated with the microfilament core. From these studies we propose that ASGP‐2 has a site for indirect association with the microfilament core near the membrane on a 15–20 kDa segment.
Effect of inhibitors of N‐linked oligosaccharide processing on the cell surface expression of a melanoma integrin
Authors: Spiro Robert C. , Laufer David M. , Perry Susan K. , Harper John R. 
Published: December 2004

AbstractThe role of trimming and processing of N‐linked oligosaccharides on the cell surface expression of the melanoma vitronectin receptor, a member of the integrin family of cell adhesion receptors, was examined by using specific glucosidase and mannosidase inhibitors. Inhibition of glucosidases I and II by castanospermine or N‐methylde‐oxynojirimycin delayed the vitronectin receptor α/β chain heterodimer assembly and α chain cleavage and resulted in a decrease in the level of expression cell surface receptor. Conversely, the vitronectin receptor synthesized in the presence of the mannosidase I and II inhibitors, 1‐deoxymannojirimycin and swainsonine, was transported normally to the cell surface with its α chain N‐linked oligosaccharides in an endoglycosidase H–sensitive from. In the presence of swainsonine, time course studies of the cell surface replacement of control, endoglycosidase H–resitant receptor with an endoglycosidase H–sensitive form demonstrated a vitronectin receptor half‐life of approximately 15–16 h. These studies provide evidence that the rates of assembly, proteolytic cleavage, and cell surface expression of the melanoma vitronectin receptor are dependent on the initial trimming of glucosyl residues from the α chain N‐linked oligosaccharides.
Binding properties of biotinylated epidermal growth factor to its receptor on cultured cells and tissue sections
Authors: Spitzer Eva , de Los Angeles Maria , Perez Rolando , Grosse Richard 
Published: December 2004

AbstractA biotinylated derivative of murine epidermal growth factor (EGF) was prepared by covalent attachment of the terminal amino group of EGF to N‐biotinyl‐ε‐aminocaproyl‐N‐hydroxysuccinimide. The stoichiometry of biotin incorporation was in the range of one biotin moiety per EGF molecule. The biotinylated EGF (biotinyl‐ε‐caproyl‐EGF, BioEGF) binds to EGF receptors on intact Ehrlich ascites carcinoma (EAC) cells with an affinity similar to that of native EGF and displays the same mitogenic activity as EGF in a soft agar test system with normal rat kidney (NRK) cells. BioEGF was visualized on cultured cells and tissue sections of a head and neck tumour by commercial streptavidin/avidin detection systems. Cytochemical analyses of certain tumour forms can be easily performed using the BioEGF probe.
Biochemical and structural studies of tenascin/hexabrachion proteins
Authors: Taylor Hope C. , Lightner Virginia A. , Beyer Wayne F. , McCaslin Darrell , Briscoe Gina , Erickson Harold P. 
Published: December 2004

AbstractTenascin is a large, disulfide‐bonded glycoprotein of the extracellular matrix. The predominant form of tenascin observed by electron microscopy is a six‐armed oligomer, termed a hexabrachion. We have determined the molecular mass of the native human hexabrachion to be 1.9 × 106 Da by sedimentation equilibrium analysis and by electrophoresis on non‐reducing agarose gels. On reducing polyacrylamide gel electrophoresis (SDS‐PAGE), human tenascin showed a single prominent band at 320 kDa and minor bands of 220 and 230 kDa. The molecular weight of the native human hexabrachion is thus consistent with a disulfide‐bonded hexamer of the 320 kDa subunits.Upon treatment with neuraminidase, the apparent molecular weights of all human and chicken tenascin subunits on reducing SDS‐PAGE were decreased by about 10 kDa. Prolonged incubation with α‐mannosidase, however, caused no apparent change in the apparent molecular weight of tenascin subunits. Sedimentation in a cesium chloride gradient gave a higher buoyant density for human tenascin than for fibronectin, suggesting that it has a higher degree of glycosylation. The far‐UV circular dichroism spectrum indicates a predominance of β‐structure and a lack of collagen‐like or α‐helical structure.When human hexabrachions were reduced and acetylated, the resulting fragments were single arms which sedimented at 6 S in glycerol gradients and migrated at 320 kDa on non‐reducing gels. Treatment of tenascin with trypsin and α‐chymotrypsin also produced large fragments which were fractionated by gradient sedimentation and analyzed by non‐reducing SDS‐PAGE and electron microscopy. We present a structural model for the assembly of the observed fragments into the elaborate native hexabrachion.
Changes in gap junction protein (connexin 32) gene expression during rat liver carcinogenesis
Authors: Fitzgerald D. James , Mesnil Marc , Oyamada Masahito , Tsuda Hiroyuki , Ito Nobuyuki , Yamasaki Hiroshi 
Published: December 2004

AbstractA rat liver gap junction (GJ) cDNA probe that detects mRNA encoding the 32 Kd GJ‐protein (connexin 32) was employed to study GJ‐protein gene expression in rat liver tumors induced by a single exposure to diethylnitrosamine (DEN) followed by exposure to 2‐acetylaminofluorene (AAF)/CCL4/AAF or induced by systemic administration of N‐ethyl‐N‐hydroxyethylnitrosamine (EHEN). All carcinomas generated by these carcinogens showed markedly reduced levels of GJ‐protein mRNA. This may indicate that GJ‐protein levels and gap‐junctional intercellular communication (GJIC) capacity are also severely compromised. Moreover, all hyperplastic nodules also showed a reduced level of GJ‐protein mRNA. Taken together with our earlier finding that the liver tumor promoter phenobarbital inhibits GJ‐protein gene expression, these results suggest that deranged GJIC is a relatively early event in liver multistage carcinogenesis. A range of other cDNA probes was also used to characterize gene expression in the DEN‐induced tumors. Induction of expression was seen for glutathione S‐transferase (placental form) (GST‐P), γ‐glutamyltranspeptidase (GGT), and c‐raf but not for c‐Ha‐ras or c‐myc.