PubMed Health⌕ Search

Biomedical subjects

C N Rao

Publications and source records attributed to C N Rao.

At least 19 recordsLinked to original sources
PubMed1997

Interactions of the amino-terminal noncollagenous (NC1) domain of type VII collagen with extracellular matrix components. A potential role in epidermal-dermal adherence in human skin.

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous domains, NC1 and NC2. The NC1 domain contains multiple submodules with homology to known adhesive molecules including fibronectin type III-like repeats and the A domain of von Willebrand factor. In this study, we produced the entire NC1 domain of human type VII collagen in the stably transfected human kidney 293 cell clones and purified large quantities of the recombinant NC1 protein from serum-free culture media. The recombinant NC1 formed interchain disulfide-bonded dimers and trimers and was N-linked glycosylated. Tunicamycin inhibited the cellular secretion of NC1, suggesting that N-linked glycosylation may play a role in NC1 secretion. The recombinant NC1 was indistinguishable from the authentic NC1 obtained from human amnions or WISH cells with respect to N-linked sugar content, electrophoretic mobility, rotary shadow imaging, and binding affinity to type IV collagen. Purified recombinant NC1, like authentic NC1, also bound specifically to fibronectin, collagen type I, and a laminin 5/6 complex. Both monomeric and trimeric forms of NC1 exhibited equal affinity for these extracellular matrix components, suggesting that the individual arms of NC1 can function independently. The multiple interactions of NC1 with other extracellular matrix components may support epidermal-dermal adhesion.

Amnion↗
PubMed1996

Extracellular matrix-associated serine protease inhibitors (Mr 33,000, 31,000, and 27,000) are single-gene products with differential glycosylation: cDNA cloning of the 33-kDa inhibitor reveals its identity to tissue factor pathway inhibitor-2.

Recently, we reported the identification and partial characterization of three serine protease inhibitors (M(r) 33,000, 31,000, and 27,000) from the extracellular matrix (ECM) of human umbilical vein endothelial cells and skin cells. Here, we report that a full-length cDNA clone for the 33-kDa inhibitor from SV-40 transformed human skin fibroblasts (t12FB) is identical to a recombinant trypsin/tissue factor pathway inhibitor called TFPI-2 from placenta. By immunoblotting, the three inhibitors from ECM and cell lysates demonstrated cross-reactivity with an antiTFPI-2 IgG. To further elucidate how these inhibitors are related, pulse-chase labeling of t12FB with [35S]methionine followed by immunoprecipitation with antiTFPI-2 IgG was performed on ECM and cytosolic proteins. A precursor-product relationship did not exist between the three inhibitors from ECM. In contrast, the various species of inhibitors from cytosolic fractions demonstrated a precursor-product relationship. Within the cytosolic fraction, 26-, 29-, and 30-kDa inhibitors were detected in the early chases (0 and 15 min) but they form precursors to the synthesis of the 33-kDa inhibitor which accumulated in the later chases (30 min to 1 h). When pulse-chase experiments were performed in the presence of tunicamycin, synthesis as well as sequestration of the three inhibitors into ECM was completely inhibited. In the presence of tunicamycin, the cells synthesized and sequestered a single 25.5-kDa inhibitor into ECM. Peak quantities of the 25.5-kDa inhibitor appeared in the ECM after 6 h chase while they were 1 h for the 27- and 31-kDa inhibitors and 3 h for the 33-kDa inhibitor. To further support that the three inhibitors are related but only differ in the extent of glycosylation, the 33-kDa inhibitor from the t12FB ECM was deglycosylated with N-glycosidase F and the products were identified by immunoblotting with antiTFPI-2 IgG. The enzyme released the 31-, 27-, and 25.5-kDa inhibitors from the 33-kDa inhibitor. Collectively, these results demonstrate that the ECM-associated 33-, 31-, and 27-kDa inhibitors are biosynthetic products of a single gene with differential glycosylation. The 25.5-kDa inhibitor is unglycosylated, whereas 27- and 30- to 31-kDa inhibitors are partially glycosylated and the 33-kDa inhibitor is fully glycosylated. Inhibition of glycosylation significantly retarded the rate of secretion of the inhibitor but did not prevent its association with ECM. Quantitation of the inhibitors with cell-conditioned medium and ECM fractions reveals that 70-75% were ECM-associated and 25-30% cell-associated. None or very little of the inhibitors (0-2%) remained in a conditioned medium. Because they are primarily associated with ECM, the inhibitors may play a major role in ECM remodeling and turnover.

Base Sequence↗
PubMed1995

Partial characterization of novel serine proteinase inhibitors from human umbilical vein endothelial cells.

Serine proteinase inhibitors play major roles in physiological and pathophysiological processes such as angiogenesis, intravascular fibrinolysis, wound healing, and tumor invasion, and metastasis. Here, we report that human umbilical vein endothelial cells (HUVEC) synthesize three inhibitors (33,000, 31,000, and 27,000 Da) which inhibit degradation of gelatin or casein by trypsin, elastase, plasmin, and alpha-chymotrypsin. The 33- and 31-kDa inhibitors were constitutively found in the cell lysate and extracellular matrix, but not in the conditioned medium of HUVEC. Following treatment with phorbol 12-myristate-13-acetate (PMA), all the three inhibitors were expressed in the CM and increased activity was found in cell lysates and extracellular matrix. The inhibitors were not detected in PMA-treated cells in the presence of cycloheximide or actinomycin D. The inhibitors specifically bound to trypsin and were recovered from trypsin affinity column as smaller-sized inhibitors without loss of antitrypsin activity. Polyclonal antibodies to inter-alpha-trypsin inhibitor did not cross-react with the 33-, 31-, and 27-kDa inhibitors. These results suggest that HUVEC synthesize and secrete novel 33-, 31-, and 27-kDa serine proteinase inhibitors.

Alpha-Globulins↗
PubMed1995

Novel extracellular matrix-associated serine proteinase inhibitors from human skin fibroblasts.

Serine proteinase inhibitors play a major role in the turnover of connective tissues. In this study, we isolated and determined partial amino-terminal amino acid sequence of trypsin/elastase/plasmin inhibitors (M(r) 33,000 and 31,000) from the extracellular matrix of SV40-transformed human skin fibroblasts. The antitrypsin activity of the inhibitors was monitored by substrate reverse zymography. Polyclonal antisera to alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-antiplasmin, inter-alpha-trypsin inhibitor, plasminogen activator inhibitors-1 and -2, and a monoclonal antibody to protease nexin-1 did not label the 33-, 31-, and 27-kDa inhibitors. A computer search for amino acid sequence homology indicated that the 31-kDa inhibitor is novel. In contrast, the sequence of the 33-kDa inhibitor shared 70 to 90% homology with the amino-terminal sequence of a recently characterized 32-kDa trypsin/tissue factor inhibitor called tissue factor pathway inhibitor-2. The 33- and 31-kDa inhibitors bind to heparin-Sepharose and were recovered from the affinity beads as well as from the t12 FB extracellular matrix with 1 M NaCl. Based on these results, we propose that the extracellular matrix of human mesenchymal cells sequester a family of novel serine proteinase inhibitors.

Amino Acid Sequence↗
PubMed1995

Alpha 1-antitrypsin is degraded and non-functional in chronic wounds but intact and functional in acute wounds: the inhibitor protects fibronectin from degradation by chronic wound fluid enzymes.

Fluid obtained from chronic and acute wounds were examined for the presence of fibronectin, alpha 1-antitrypsin, and proteinases capable of degrading both proteins. Immunoblot analysis of fluids from ten chronic wounds revealed that fibronectin and alpha 1-antitrypsin were degraded in nine of ten samples. In contrast, both fibronectin and alpha 1-antitrypsin were intact in acute wound fluids. The degradation of the inhibitor and fibronectin occurred in the same wound fluids, and these two events correlated perfectly. Chronic or acute wound fluid proteins were coupled to benzamidine Sepharose 6B beads and incubated with fibronectin or alpha 1-antitrypsin. Chronic wound fluid proteins degraded fibronectin in the presence of ethylenediaminetetraacetate, leupeptin, cystatin, and pepstatin but not in the presence of phenylmethylsulfonyl fluoride. Acute wound fluids and normal human serum did not contain enzymes capable of degrading fibronectin. These data suggest that serine proteinases are responsible for fibronectin degradation in chronic wound fluids. Chronic wound fluids that contained degraded alpha 1-antitrypsin also contain proteinases capable of degrading alpha 1-antitrypsin from human serum. Acute wound fluids and normal human serum did not contain enzymes capable of degrading alpha 1-antitrypsin. The inhibitor from acute wound fluids bound to one of its targets, trypsin. In contrast, the fragment(s) of alpha 1-antitrypsin from chronic wound fluids did not bind trypsin. Chronic wounds associated with degraded fibronectin and the inhibitor contained ten- to forty-fold more elastase activity than acute wounds. The degradation of fibronectin by chronic wound fluid enzymes was inhibited by alpha 1-antitrypsin in a dose-dependent manner. Collectively, these results demonstrate that there are enzymes in chronic wounds that perturb the function of alpha 1-antitrypsin and allow fibronectin degradation by uninhibited serine proteinases.

Acute Disease↗
PubMed1995

Partial characterization of matrix-associated serine protease inhibitors from human skin cells.

Serine protease inhibitors have important regulatory roles in angiogenesis, intravascular fibrinolysis, wound healing, and cell migration. In this study, the extracellular matrix secreted by cultured human keratinocytes, foreskin fibroblasts, and SV-40-transformed human skin fibroblasts was analyzed for serine protease inhibitors by substrate reverse zymography. We found that the extracellular matrix deposited by these cells contained three inhibitors (M(r) 33,000, 31,000, and 27,000). These inhibitors protected the degradation of gelatin by trypsin and elastase, and of casein by plasmin. In contrast, the gelatinolytic activities of thermolysin and papain were not inhibited. Compared to untreated cells, cells treated with phorbol 12-myristate 13-acetate showed a two- to 10-fold increase in the expression of these inhibitors. Cycloheximide and actinomycin D decreased the cellular expression of these inhibitors, suggesting the involvement of de novo protein and mRNA synthesis. Antitrypsin activity of these inhibitors was resistant to heat and sodium dodecylsulfate, but was lost after reduction of disulfide bonds. The inhibitors bound specifically to trypsin and could be eluted from a trypsin column in active form. Collectively, these data suggest that the extracellular matrix deposited by keratinocytes and dermal fibroblasts contains active serine protease inhibitors.

Cell Line, Transformed↗
PubMed1991

The high affinity murine laminin receptor is a member of a multicopy gene family.

The high affinity laminin receptor is differentially expressed in metastasis. We now report that there are multiple copies (6 +/- 1) of the laminin receptor gene in the murine genome of normal diploid cells as well as in cell lines derived from cancer cells. We have analyzed three distinct cDNA clones isolated from an Okayama-Berg cDNA library of transformed mouse fibroblasts that may represent transcripts of three different laminin receptor genes. Polymorphic changes include insertion of bases at the 5' terminus, a base substitution within the coding region resulting in an amino acid change from phenylalanine to leucine, a base substitution obliterating a polyadenylation signal, as well as changes in the length of the 3' untranslated domains. The discovery of multiple transcripts of laminin receptor genes suggests that there is a strong selective pressure to maintain laminin receptor expression in murine cells.

Animals↗
PubMed1991

Changes in the subunit composition of laminin during the increased tumorigenesis of mouse A9 cells.

We compared the structure and subunit composition of laminin in less tumorigenic mouse A9 and highly tumorigenic mouse A9HT cells by pulse-chase studies. During a 15' pulse, the ratio of laminin B1 to B2 subunits in cell lysates is 1:1 in both the A9 and A9HT cells; however, after a 3 hr chase, this ratio changes to 6:1 and 2:1 in the A9 and A9HT cells, respectively. Analysis of mature laminin subunits in culture media after a 3 hr chase also showed a similar higher ratio of B1 to B2 in the A9 cells as compared to the A9HT cells. The higher ratio of B1 to B2 subunits in A9 cells was evident as early as after a 30' pulse. A comparative analysis of steady-state levels of mRNAs for the laminin subunits B1 and B2 between A9 and A9HT cells showed a ratio of 1:1 for B1 and a ratio of 1:1.65 for B2. The ratio of B1 to B2 mRNAs in A9 cells was 1:1.3 whereas in A9HT cells it was 1:2.5, suggesting changes in the processing of mRNA in the highly tumorigenic A9HT cells. These observations suggest that the processing of laminin B subunits is altered during the process of increased tumorigenicity, thus resulting in the synthesis and secretion of structurally different laminin in tumorigenic A9HT cells as compared to the parent and less tumorigenic A9 cells.

Animals↗
PubMed1990

Laminin "A" chain fragment of Mr 43 kilodalton contains PC12 cell attachment promoting site.

In the present study, the ability of a 43 K fragment, originating from the long arm of the 400 K laminin "A" chain (Rao and Kefalides, manuscript in press), to promote the attachment and de novo neurite outgrowth by the pheochromocytoma cell line, (PC12) was studied. Unprimed PC12 cells (not treated with nerve growth factor, NGF), just as the primed cells (treated with NGF), rapidly and efficiently attach to the laminin fragment but less efficiently to the intact laminin coated bacteriological plastic surfaces. The neurite outgrowth was continuous from primed cells attached to both laminin and laminin fragment substrates, when cultured for four days in the presence of NGF. On the other hand, no significant neurite outgrowth was observed from unprimed PC12 cells attached to the laminin fragment coated bacteriological dishes, in the absence of NGF. These data suggest that the 43 K laminin "A" chain fragment contains the PC12 cell attachment promoting site.

Adrenal Gland Neoplasms↗
PubMed1990

Sulfatide-binding domain of the laminin A chain.

A sulfatide-binding site on the globular end region of the long arm of laminin has been identified. Following proteolytic digestion with thermolysin, an intact fragment of the laminin A chain carboxyl-terminal domain exhibiting sulfatide-binding activity was isolated using gel filtration and heparin affinity chromatography. This fragment is composed of two peptides that are covalently linked by at least one disulfide bond and encompass the carboxyl-terminal 394 amino acids of the A chain. The clusters of charged residues in the primary structure of these fragments are sufficient for heparin-binding activity but not sulfatide binding since reduction and alkylation of the fragments abolished sulfatide binding under conditions in which heparin binding was retained. Thus, sulfatide binding requires an intact three-dimensional structure. The iodinated fragment bound to A2058 melanoma and T47D breast carcinoma cells and could be displaced by the unlabeled fragment. Based on incorporation of [35S] sulfate, both cell lines synthesize sulfated glycolipids that bind to laminin. In agreement with previous data that indicate a synergistic interaction of the sulfatide-binding domain with other laminin-binding sites on melanoma cells during attachment, the isolated sulfatide-binding fragment significantly inhibited interaction of labeled intact laminin with melanoma and breast carcinoma cells in direct binding assays.

Amino Acid Sequence↗
PubMed1990

Identification and characterization of a 43-kilodalton laminin fragment from the "A" chain (long arm) with high-affinity heparin binding and mammary epithelial cell adhesion-spreading activities.

A recently described procedure of reduction and carboxymethylation followed by heparin-Sepharose chromatography [Arumugham et al. (1988) Connect. Tissue Res. 18, 135-147] was used to characterize high-affinity heparin binding fragments of the laminin "A" chain. Two laminin fragments of Mr 53K and 43K selectively bound to the heparin-Sepharose column from the chymotrypsin digest of laminin, indicating that these fragments originate from the "A" chain. Without reduction and carboxymethylation but in the presence of 2.0 M urea, the heparin-Sepharose-bound material from the chymotrypsin laminin digest contains all the attachment-promoting activity for normal mouse mammary epithelial cells. The reduced 200-kDa intact three short arm fragment, fragments of Mr 70K-160K obtained either from laminin or from the reduced 200-kDa three short arm fragment, and the 53-kDa heparin binding fragment were all inactive in promoting the adhesion of mouse mammary epithelial cells. The mammary epithelial cell adhesion and spreading properties of laminin are associated with the high-affinity heparin binding 43-kDa fragment. The mammary epithelial cells attach to the 43-kDa fragment substrate and synthesize laminin, collagen type IV, and desmoplankins I and II as are the cells attached to laminin substrate and to the cells grown on tissue culture dishes. The biologically active 43-kDa fragment is generated from laminin, but not from the three short arm fragment. These results suggest that normal mouse mammary epithelial cells interact with laminin through a single site which is present in the 43-kDa heparin binding fragment located on the long arm of the "A" chain.

Animals↗
PubMed1989

Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor.

The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7137-7141]. Primer extension experiments demonstrated that the clone contained the complete 5' sequence of the murine laminin receptor mRNA. RNA blot data demonstrated a single-sized laminin receptor mRNA, approximately 1400 bases long, in human, mouse, and rat. The nascent laminin receptor predicted from the cDNA sequence is 295 amino acids long, with a molecular weight of 33,000, and contains one intradisulfide bridge, a short putative transmembrane domain, and an extracellular carboxy-terminal region which has abundant glutamic acid residues and multiple repeat sequences. The precursor of the laminin receptor is apparently smaller than the 67-kilodalton protein isolated from tissue. The apparent molecular weight on SDS-polyacrylamide gels of the rabbit reticulocyte cell-free translation product of selectively hybridized laminin receptor mRNA is 37,000. Antisera to three different domains of the cDNA-predicted receptor were used to study the relationship between the 37- and 67-kilodalton polypeptides. Antisera to cDNA-deduced synthetic peptides of the receptor immunoprecipitated a 37-kilodalton band both from cell-free translation products and from pulse-labeled cell extracts. On immunoblots of cell extracts, one antisynthetic peptide antiserum recognized only the 67-kilodalton receptor, while another antiserum identified both 37- and 67-kilodalton polypeptides, suggesting a precursor-product relationship between the two polypeptides.

Amino Acid Sequence↗
PubMed1987

A role for the laminin receptor in leukocyte chemotaxis.

Previous studies on the mechanism of leukocyte traversal of basement membranes showed that rabbit peritoneal exudate polymorphonuclear cells (PMN) preferentially used laminin, a major constituent of basement membrane, to attach to another component, type IV collagen. PMN also responded chemotactically to nanomolar levels of laminin. We have now determined that PMN possess a receptor for laminin. Scatchard analysis using 125I laminin indicates a single class of saturable high affinity binding sites (kd = 6.15 nM/L) on PMN and 3.6 X 10(4) sites per cell. A chymotryptically derived fragment of laminin, C1, which lacks both the long arm and the globular end regions of the short arm (i.e., matrix binding sites) but retains the laminin receptor binding region, gave similar results. Immunoperoxidase studies using monoclonal antibodies to the laminin receptor (mAbLR) indicated the presence of the receptor on the surface of PMN. These cells responded chemotactically to nanomolar levels of C1 and laminin, a result consonant with binding data. PMN chemotaxis to a formyl peptide was markedly inhibited by mAbLR, suggesting that the laminin receptor may be required for PMN chemotaxis in general. Our results suggest that PMN extravasation across basement membranes is aided both by reversible attachment of the cells to laminin in the matrix and by chemotaxis to a gradient of soluble intact and possibly degraded laminin. These characteristics have much in common with those of highly metastatic tumor cells.

Animals↗
PubMed1987

Laminin expression by human pancreatic carcinoma cells in the nude mouse and in culture.

The abilities of 4 established human pancreatic tumor cell lines (PANC-1, CAPAN-1, AsPc-1, and BxPc-3) to synthesize and to maintain laminin-containing basement membranes when grown in the nude mouse have been examined and compared with synthesis of the glycoprotein laminin by these tumor cells when grown in culture. Immunohistochemical visualization of basement membrane integrity within the transplanted tumors employed a monoclonal antibody specific for human laminin to clearly distinguish between human tumor-produced laminin and murine basement membranes. This technique demonstrated strikingly different degrees of basement membrane formation and laminin distribution for the 4 biologically diverse pancreatic tumors. Basement membranes were present within the differentiated, less invasive tumors, whereas structure basement membranes were absent in the poorly differentiated, invasive tumors. Regardless of their propensity to produce basement membranes in vivo, all 4 pancreatic tumor cell lines continued to synthesize laminin in culture, as was determined by immunologic assays. The most invasive cell line, AsPc-1, released large quantities of soluble laminin into conditioned culture medium. The less invasive, well-differentiated tumor cells did not release laminin into the medium, but they retained the laminin produced by them within the cell layer. The combination of in vivo and in vitro studies indicates that the extent of basement membrane loss by these human pancreatic tumors is not due to an inherent inability of the tumor cells to synthesize the structural component laminin.

Animals↗
PubMed1986

Comparison of the specificities of laminin, thrombospondin, and von Willebrand factor for binding to sulfated glycolipids.

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfated glycolipids. These three glycoproteins differ, however, in their sensitivity to inhibition of binding by sulfated monosaccharides and polysaccharides. Heparin strongly inhibits binding of thrombospondin but only weakly inhibits binding of laminin and von Willebrand factor. Fucoidan strongly inhibits binding of both laminin and thrombospondin but not of von Willebrand factor. Laminin shows significant specificity for inhibition by monosaccharides, whereas thrombospondin does not. Thus, specific spacial orientations of sulfate esters may be primary determinants of binding for the three proteins. Laminin, thrombospondin, and von Willebrand factor also differ in their relative binding affinities for purified sulfated glycosphingolipids. The three proteins strongly prefer terminal-sulfated lipids and bind only weakly to sulfated gangliotriaosyl ceramide with a sulfate ester on the penultimate galactose. Thrombospondin binds with highest affinity to galactosyl sulfatide but only weakly to more complex sulfatides, whereas von Willebrand factor prefers galactosyl sulfatide but binds with moderate affinity to various sulfated glycolipids. Laminin also is less selective than thrombospondin but is less sensitive for detection of low sulfatide concentrations. Galactosyl sulfatide at 1-5 pmol can be detected by staining of lipids separated on high performance TLC with 125I-thrombospondin or 125I-von Willebrand factor. 125I-von Willebrand factor was examined as a reagent for detecting sulfated glycolipids in tissue extracts. Rat kidney lipids contain 5 characterized sulfated glycolipids: galactosyl ceramide I3-sulfate, lactosyl ceramide II3-sulfate, gangliotriaosyl ceramide II3-sulfate, and bis-sulfated gangliotriaosyl and gangliotetraosyl ceramides. von Willebrand factor detects all of these lipids as well as several additional minor sulfated lipids. Complex monosulfated lipids are detected in several human tissues including kidney, erythrocytes, and platelets by this technique.

Blood Platelets↗