Nitration of manganese superoxide dismutase during ocular inflammation.

Reactive nitrogen species, in particular, peroxynitrite (ONOO(-)) have been proposed to play an important role in the pathogenesis of endotoxin-induced uveitis (EIU). Tyrosine nitration by ONOO(-) has been shown in other model systems to inhibit the activity of the superoxide anion quenching enyzme, manganese superoxide dismutase (MnSOD), perhaps contributing to progression of disease. In this study, it is confirmed through immunoanalysis that nitrated proteins are produced during EIU, and furthermore, that MnSOD is a target of nitration during the inflammatory response. In addition, through microsequencing analyses, nitrated albumin--apparent in both control and EIU eyes--was identified. Positive immunostaining of nitrated proteins was seen in the ciliary epithelium, inflammatory cells, and protein exudate of eyes from rats injected with endotoxin. Incubation of nitrotyrosine immunoprecipitates from the iris and ciliary body (ICB) with a polyclonal antibody against MnSOD revealed that nitrated MnSOD was present only in the ICB of EIU rats. When the total activity of the enzyme was examined, it was observed that despite the presence of nitrated MnSOD, activity was increased relative to control. Analysis of MnSOD mRNA and protein from the ICB of both groups demonstrated an increase in mRNA expression and consequently a three- to five-fold increase in MnSOD protein in EIU rats as compared to control rats. Further examination of MnSOD protein expression through immunohistochemistry noted enhanced immunostaining in the ciliary epithelium of eyes of EIU rats. Additional investigation of a 70 kDa band apparent in nitrotyrosine immunoprecipitates from the ICB of control and EIU rats revealed that the plasma protein albumin is nitrated as well. This protein is present as a result of the breakdown of the blood-aqueous barrier during inflammation. In summary, two endogenous nitration targets, albumin and MnSOD, were identified. Nitrated MnSOD appears to be specifically targeted to the ICB during inflammation, underscoring the importance of the interface in EIU. Furthermore, the expression and activity of the enzyme is increased in the ICB during EIU, perhaps regulating reactive nitrogen species produced within the cells. This study implicates ONOO(-) in the pathogenesis of EIU and imparts the putative role MnSOD plays in disease resolution.

Pulmonary infection with Serratia marcescens.

A case of pulmonary infection, presenting with fever and productive cough (pseudohaemoptysis) was diagnosed as having infection with Serratia marcescens on performing culture and sensitivity tests. The organism was confirmed upto species level using the standard biochemical tests.

Pathophysiology and treatment of psoriasis.

The pathogenesis and treatment of psoriasis are reviewed. Psoriasis is characterized by defects in the normal cycle of epidermal development that lead to epidermal hyperproliferation, altered maturation of skin cells, and vascular changes and inflammation. The condition typically manifests as areas of thickened, flaky, silvery white and reddened skin that may hurt, itch, and bleed. Biochemical markers of psoriasis are changes in levels of keratins, keratinocyte transglutamase, migration inhibitory factor-related protein, skin-derived antileukoproteinase, involucrin, small protein rich protein 2, filaggrin, and cytokines. Types of psoriasis that may be clinically encountered include plaque psoriasis, guttate psoriasis, erythrodermic psoriasis, and pustular psoriasis. Psoriasis is believed to be genetically linked but can also be triggered by mechanical, ultraviolet, and chemical injury; various infections; prescription drug use; psychological stress; smoking; and other factors. Topical treatment of psoriasis is usually the first line of therapy. Topical treatments consist of emollients and keratolytic agents, anthralin, coal tar, corticosteroids, vitamin D3 analogues, topical retinoids, and topical psoralens plus ultraviolet A (UVA) light. In patients who do not respond adequately to topical therapy, oral or injectable therapy, such as oral retinoids, methotrexate, cyclosporine, tacrolimus, and oral psoralens plus UVA light, may be warranted. Patients receiving systemic treatments should be carefully monitored for adverse effects and drug-drug interactions. Drug therapy is the mainstay of the treatment of psoriasis. The potential adverse effects and interactions necessitate vigilant monitoring.

Biosynthesis of sulfated extracellular matrices by alveolar type II cells increases with time in culture.

The aim of this study was to determine the extent to which sulfate incorporated into biosynthesized basement membrane (BM) components increased as isolated type II cells progress toward a more type I cell-like phenotype from 7 to 21 days in culture. Specific sulfate cytochemistry, using high iron diamine, showed that type I-like cells in 21-day cultures deposited a more highly sulfated extracellular matrix. Biosynthetic labeling experiments using [35S]cysteine or [35S]sulfate as precursors confirmed the increased capacity of 21-day type I-like cells to biosynthesize sulfated BM components compared with type II-like cells in 7-day cultures, including a novel sulfated laminin. These biochemical changes in sulfation of BM components coincide with the established phenotypic transition from type II to type I cells during prolonged culture. More importantly, the data suggest that regulation of sulfation constitutes a potential mechanism by which type I and type II cells alter their environment in such a manner as to stabilize phenotype and modulate responses to growth factors.

Lectin binding characteristics of squamous cell carcinomas of the head and neck.

Lectins are polyvalent proteins of non-immune origin with exquisite carbohydrate binding specificity making them ideal for investigation of cell surface glycoprotein and glycolipid antigens. We examined the cell surface lectin binding phenotypes of 20 UM-SCC squamous cell carcinoma cell lines established from 17 patients with head and neck cancers using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of using a panel of fluorescein-conjugated lectins and inhibition by the appropriate monosaccharide to confirm specificity of binding. Conconavalin A (Con A) from Canavalia ensiformis and the peanut agglutinin (PNA) from Arachis hypogaea bound all SCC cell lines tested and wheat germ agglutinin (WGA) from Triticum vulgaris bound to 12 of 13 tumor cell lines. The blood group O specific lectin UEA 1 from Ulex europeus also bound to all cell lines regardless of the donor blood type. Lectins of Dolichos biflorus (DBA) and Griffonia simplicifolia (GS I-B4 or BSA I-B4) with binding specificity for glycoproteins associated with blood group A and B respectively, had reactivity that did not directly correlate with blood group antigen expression. In contrast to the other lectins in our panel which exhibited broad reactivity with SCC antigens, the BSA-II lectin from Griffonia simplicfolia, (GS II or BSA II) which has sugar binding specificity for terminal non-reducing GlcNAc, did not bind to any of the screened cell lines. Our results demonstrate a common pattern of lectin-defined carbohydrate expression on the cell surface of squamous cell carcinomas of head and neck that appears promising in defining the malignant cellular phenotype. Lectin binding profile may be useful in differentiating benign from malignant histopathology.

Assessment of sulfur mustard interaction with basement membrane components.

Bis-2-chloroethyl sulfide (sulfur mustard, HD) is a bifunctional alkylating agent which causes severe vesication characterized by slow wound healing. Our previous studies have shown that the vesicant HD disrupts the epidermal-dermal junction at the lamina lucida of the basement membrane. The purpose of this study was to examine whether HD directly modifies basement membrane components (BMCs), and to evaluate the effect of HD on the cell adhesive activity of BMCs. EHS laminin was incubated with [14C]HD, and extracted by gel filtration. Analysis of the [14C]HD-conjugated laminin fraction by a reduced sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SDS-PAGE) revealed the incorporation of radioactivity into both laminin subunits and a laminin trimer resistant to dissociation in reduced SDS-PAGE sample buffer, suggesting direct alkylation and cross-linking of EHS laminin by [14C]HD. Normal human foreskin epidermal keratinocytes were biosynthetically labeled with [35S]cysteine. 35S-labeled laminin isoforms, Ae.B1e.B2e. laminin and K.B1e.B2e. laminin (using the nomenclature of Engel), fibronectin, and heparan sulfate proteoglycan were isolated by immunoprecipitation from the cell culture medium, treated with HD or ethanol as control, and then analyzed by SDS-PAGE. On reduced SDS gels, these three BMCs not treated with HD showed the typical profile of dissociated subunits. However, HD treatment caused the appearance of higher molecular weight bands indicative of cross-linking of subunits within these BMCs. The HD scavengers sodium thiosulfate and cysteine prevented the cross-linking of BMC subunits by HD. Finally, tissue culture dishes coated with laminin or fibronectin were treated with HD or ethanol as a control, and human keratinocytes were plated on the BMC-coated surfaces. After 20 h of incubation, it was observed that cell adhesion was decreased significantly on the BMC-coated surfaces treated with HD. As expected, the preincubation of HD with cysteine diminished the HD inhibition of cell adhesion. Thus, HD alkylates adhesive macromolecules of the basement membrane zone and inhibits their cell adhesive activity. These findings support the hypothesis that the alkylation of basement membrane components by HD destabilizes the epidermal-dermal junction in the process of HD-induced vesication. The failure of the HD-alkylated BMCs to support the attachment of keratinocytes might also contribute to the slow reepithelialization of the wound site which is characteristic of HD-induced blistering.

Laminin in the cutaneous basement membrane as a potential target in lewisite vesication.

The epidermal-dermal junction has a complex molecular architecture, with numerous components playing key roles in adhesion of the epidermis to the dermis. The purpose of this study was to examine structural components of the epidermal-dermal junction as potential targets for toxicity by lewisite (dichloro(2-chlorovinyl)arsine). This was accomplished by (1) immunocytochemical mapping of laminin, type IV collagen, and bullous pemphigoid antigen (BPA) in lewisite-treated isolated perfused porcine skin flaps (IPPSF), (2) evaluation of protease activity in IPPSF blister fluid against laminin substrate from murine EHS tumor and human keratinocytes, and (3) examination of human keratinocyte laminin for direct chemical modification by lewisite. Lewisite-induced epidermal-dermal separation was localized to the lamina lucida. Localization of the separation suggested that laminin, a cysteine-rich and highly protease-sensitive adhesive glycoprotein, is a potential target for lewisite action. It was hypothesized that chemical modification of laminin directly (via chemical alkylation of laminin thiols by the arsenical) or indirectly (due to lewisite-induced cytotoxic release of proteases) could result in blister formation. Employing sensitive methodology, no evidence of proteolytic activity against EHS tumor laminin or human keratinocyte laminin was identified in the blister fluid. In addition, no evidence for direct chemical modification of laminin by lewisite was demonstrated. However, up to 36% of the thiol groups in human keratinocyte laminin immunoprecipitates was potentially available for reaction with alkylating agents. While these studies did not demonstrate a lewisite-induced chemical modification of laminin, they do not rule out the possibility that other adhesive molecules of the basement membrane are targets for lewisite action. Further evaluation of the molecular role that these binding modalities play in vesicant-induced separation may provide new insights into therapeutic and prophylactic strategies against the toxicity of such compounds and contribute to a better understanding of basement membrane biochemistry.

Expression of laminin chains during myogenic differentiation.

Expression of two Ae-related chains of the extracellular matrix glycoprotein laminin was induced as multipotent C3H10T1/2 mouse embryo fibroblasts differentiated into myoblasts and myofibers. C3H10T1/2 fibroblasts expressed the B1e (M(r) = 215,000) and B2e (M(r) = 205,000) laminin chains based on metabolic radiolabeling, immunoprecipitation, peptide mapping, and mRNA analysis. In contrast, myoblasts derived from C3H10T1/2 fibroblasts treated with DNA demethylating agents or transfected with the cDNA encoding MyoD expressed the Ae (M(r) = 400,000) and a novel Ae-related laminin chain (designated Ac3h, M(r) = 350,000) in addition to the B1e and B2e chains. Expression of the Ae and Ac3h chains paralleled the capacity for myofiber formation in six additional C3H10T1/2 myoblast clones with varied potentials for terminal differentiation and coincided with a switch in laminin isoforms from those of M(r) = 850,000 synthesized by C3H10T1/2 fibroblasts to those of M(r) = 900,000-950,000 synthesized by C3H10T1/2 myoblasts and myofibers. Cultures of mouse C2C12, mouse BC3H1, rat L6, and primary mouse myoblasts also synthesized the Ae, Ac3h, B1e, and B2e laminin chains. The results demonstrate that expression of the Ae and Ac3h laminin chains is associated with expression of MyoD and the mammalian myogenic differentiation program.

Development and characterization of an isolated and perfused tumor and skin preparation for evaluation of drug disposition.

The purpose of this study was to develop a model in which the regional pharmacokinetics of a drug in tumor and nontumorous tissue could be evaluated under a variety of physiological conditions. To this effect, the growth of a human choriocarcinoma cell line (JAR) was evaluated in pigs immunosuppressed with 25 mg cyclosporine/kg every 24 h. During an initial study, we demonstrated that suspensions containing approximately 3 million JAR cells with and without 1 million normal human fibroblasts injected s.c. into the inguinal region of pigs resulted in the growth of tumors consisting primarily of polygonal neoplastic cells. Multinucleate tumor cells, inflammatory cells, necrotic debris, and vascular endothelial cells were also present. Maximal tumor size was noted on day 12, after which time tumor regression occurred. The coinoculation of fibroblasts resulted in significantly larger tumors. Two single pedicle, axial pattern tubed flaps were created in the inguinal area of 4 pigs. JAR cells and fibroblasts were transplanted to one flap to allow for tumor formation. The other flap served as a nontumorous control. Both flaps were removed for perfusion with a physiological solution 11 days later. Glucose utilization, lactate concentrations, lactate dehydrogenase activities, and microscopic evaluation of skin samples were used to assess flap viability. All flaps remained viable for 8 h of perfusion. The only differences detected between nontumorous and tumor flaps was the initial perfusion pressure which was significantly lower in tumor flaps (P < 0.05). The isolated perfused tumor and skin flap is unique in that it consists of a tumor surrounded by normal tissue with an intact microvascular system and can be utilized to design regional pharmacokinetic studies describing drug distribution in tumor tissue.

O-glycosylation of the alpha-subunit does not limit the assembly of chorionic gonadotropin alpha beta dimer in human malignant and nonmalignant trophoblast cells.

Biosynthetic experiments were carried out in cultures of human malignant trophoblast cells (the JAR cell line) and in explants of normal first trimester human placental tissue to test the hypothesis that the O-glycosylation of the glycoprotein hormone alpha-subunit at Thr-39 regulates the assembly of the CG alpha beta dimer. This modification of alpha has been shown to ablate its ability to combine in vitro with the beta-subunit of bovine LH and might explain why JAR cells and placental explants secrete uncombined alpha- and beta-subunits in addition to the hCG alpha beta dimer. We have previously detected an O-linked carbohydrate chain at Thr-39 in preparations of secreted free alpha-subunit, but not dimer CG alpha from JAR culture medium. We report here evidence that the O-glycosylation of alpha does not regulate the biosynthetic assembly of the hCG dimer in cultures of JAR choriocarcinoma cells or first trimester placental explants. The intracellular precursor forms of alpha and beta that accumulate in the endoplasmic reticulum and combine in that compartment are not yet modified with O-linked carbohydrate, as determined by measurements of their [3H]galactosamine content after biosynthetic labeling of amino sugars with [3H]glucosamine. Furthermore, only half of the free alpha-subunit secreted by JAR cells and less than 10% of free alpha secreted by 10-week-old placental explants received the O-linked chain. This was shown by determining the ratio of the unglycosylated and glycosylated forms of the tryptic peptide from free alpha that contains the O-glycosylation site (residues 36-42). Based on these findings, we make the following conclusions. 1) O-Glycosylation of alpha-subunit is a late event in the secretory pathway of trophoblasts compared to the rapid combination in the rough endoplasmic reticulum of hCG subunit precursors to form alpha beta dimer. 2) Association of alpha with beta precludes the subsequent addition of the glycan to alpha at Thr-39. 3) The alpha molecules that fail to combine with beta in the endoplasmic reticulum are substrates for the later addition of O-linked carbohydrate, presumably in the Golgi complex, but only a fraction of the free alpha molecules are modified with O-linked carbohydrate.

Identification and characterization of subpopulations of the free alpha-subunit that vary in their ability to combine with chorionic gonadotropin-beta.

The free (uncombined) alpha-subunit of hCG is secreted in excess over alpha beta dimer from both malignant and nonmalignant trophoblast cells and is secreted ectopically from a variety of other malignant cell types. The free alpha-subunits from various sources are distinguishable from those that combine because they migrate more heterogeneously and more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than dimer alpha. We have previously identified three posttranslational modifications that may contribute to the altered mobility of the free alpha-subunit and to its inability to combine with the beta-subunit: 1) preferential phosphorylation of the free alpha-subunit, 2) O-glycosylation of free alpha, and 3) differences in the processing of the asparagine-linked oligosaccharides between the free and combinable forms. We have purified three populations of the alpha-subunit from the JAR choriocarcinoma cell line and from ChaGo, a bronchogenic carcinoma cell line that ectopically synthesizes only the alpha-subunit, in order to identify the posttranslational modifications that contribute to the altered mobility on SDS-PAGE. Fractionation of the oligosaccharides released from the alpha forms with peptide N-glycosidase has shown that the faster migrating alpha forms on SDS-PAGE have less completely processed oligosaccharide chains. Twenty-two to 25% of the JAR free alpha and 35-41% of the ChaGo alpha forms that migrate the fastest on SDS-PAGE recombine with beta in an in vitro recombination assay under conditions where 62% of the dimer alpha form recombines. In contrast, only 5-12% and 16-21% of the JAR free alpha and ChaGo alpha forms, respectively, that migrate the slowest on SDS-PAGE recombine with beta. The form of JAR free alpha least capable of combining with beta contains on O-linked glycan on Thr-39. This same site is a substrate for phosphorylation by JAR cells. However, most of ChaGo alpha fails to recombine with beta even though ChaGo alpha contains little O-linked carbohydrate. These results suggest that the larger asparagine-linked complex glycans on the slower migrating alpha forms are the major limiting factor for subunit combination. Although these modifications may not be rate limiting for combination in the rough endoplasmic reticulum, they may prevent dimerization of the free subunits later in the secretory pathway.

Conformational intermediates in the production of the combinable form of the beta-subunit of chorionic gonadotropin.

Human trophoblastic cells synthesize and secrete hCG as well as uncombined forms of the alpha- and beta-subunits of hCG. We have previously reported that the rate-limiting step in alpha beta-dimer assembly in cultured JAR choriocarcinoma cells is a conformational change in beta-subunit accompanied by the formation of intramolecular disulfide bonds. We now report on the intermediate steps in the acquisition of this combinable conformation by the beta-subunit. The earliest biosynthetically labeled form of beta detected in JAR cells is a precursor termed p beta 1 that lacks at least one of the intramolecular disulfide bonds found in mature beta-subunit, that does not combine with alpha-subunit, and that does not react with a monoclonal antibody specific for free beta. The p beta 1 precursor rapidly assumes (within 5 min) a new conformation termed p beta 2 that, in contrast to p beta 1, migrates more slowly on nonreduced sodium dodecyl sulfate-polyacrylamide gels, combines with alpha to form the hCG dimer, and reacts with the monoclonal anti-free beta antibody. Pulse-chase kinetic experiments support the following sequence of events: p beta 1----uncombined p beta 2----combined p beta 2. The transition of p beta 1 to uncombined p beta 2 involves the formation of at least one intramolecular disulfide bond coincident with the conformational shift of the p beta molecule. Furthermore, treatment of the nonreduced subunits with trypsin releases a [35S]cysteine-labeled peptide from p beta 1, but not from either form of p beta 2. This peptide presumably contains one of the two crucial cysteine residues that participate in forming the disulfide bond that distinguishes p beta 1 from the p beta 2 forms. Dimer p beta 2 differs from both p beta 1 and uncombined p beta 2 in that it contains an O-linked N-acetylgalactosamine, which represents the first step in the formation of the O-linked glycans of beta-subunit. Dimer p beta 2 is, therefore, the most fully processed and kinetically the latest of the three p beta forms that appear in JAR cell lysates. We conclude that formation of an appropriate array of intramolecular S-S bonds accompanies the acquisition of a combinable conformation of beta-subunit, and we have identified intermediate steps in the pathway leading to this conformational change. The data suggest that it is the achievement of this conformation by beta-subunit that limits the alpha beta combination reaction rather than the amount or conformation of alpha-subunit.

Biosynthesis and deposition of a noncovalent laminin-heparan sulfate proteoglycan complex and other basal lamina components by a human malignant cell line.

The basal lamina components laminin, heparan sulfate proteoglycan (HSPG), and type IV collagen were synthesized and codeposited in the extracellular matrix (ECM) by a cultured human cell line from gestational choriocarcinoma (JAR). Laminin and HSPG formed a noncovalent complex detected by the coimmunoprecipitation of HSPG with laminin from cell lysates and culture media. The complex was stable in the cell lysis buffer that contained detergents (1% Triton X-100, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate) and sodium chloride (from 0.15 to 1.0 M), but was dissociated by adding 8 M urea to the detergent lysates. Even though JAR cells produced roughly equal amounts of HSPG and chondroitin sulfate proteoglycan, only HSPG complexed with laminin, suggesting a specific interaction between these basal lamina components. The laminin-HSPG complex was deposited and retained in the ECM. This was shown biochemically by isolating an enriched fraction of ECM from JAR cells cultured on native type I collagen gels. At steady state, more than half (52%) of the laminin-HSPG in the culture was recovered in the ECM fraction, in contrast to 16% of the total laminin and 29% of the total type IV collagen, which were secreted to a greater extent than laminin-HSPG into the culture medium. The retention of the laminin-HSPG complex in the ECM suggests that it may participate in the assembly of the basal lamina-like extracellular matrix deposited by JAR cultures. Omission of ascorbate from the culture medium abolished the ECM deposition of type IV collagen but had little effect on the deposition of laminin or laminin-HSPG. This demonstrates that the stable deposition of laminin-HSPG and laminin in the collagen-based choriocarcinoma cultures is not dependent on an assembled network of type IV collagen.

Biosynthesis and secretion of laminin and laminin-associated glycoproteins by nonmalignant and malignant human keratinocytes: comparison of cell lines from primary and secondary tumors in the same patient.

Laminin biosynthesis was compared in four pairs of human squamous cell carcinoma cultures derived from primary and recurrent or metastatic tumors in four patients with cancer of the larynx and hypopharynx to determine if changes in laminin production accompany tumor progression. Laminin profiles of the malignant cells were compared with laminin biosynthesized by nonmalignant human keratinocytes. Pulse-chase biosynthetic labeling of the cultures with [35S]methionine established that all of the squamous carcinoma cell lines synthesize immunoreactive A (Mr 400,000), B1 (Mr 205,000), and B2 (Mr 200,000) laminin subunits; assemble them to form the intact laminin molecule (Mr 950,000); and secrete a portion of the laminin they produce into the culture media. One aspect of laminin expression unique to keratinocytes, both malignant and nonmalignant, was the occurrence of three additional glycoprotein forms (Mr 195,000, 170,000, and 160,000) in the laminin immunoprecipitates. In contrast to the laminin subunits, these glycoproteins were not immunoreactive with the anti-laminin antiserum on Western blots. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis without and with reduction of disulfide bonds revealed that the laminin immunoprecipitates contained a family of oligomeric molecules. These ranged in apparent molecular weight from 370,000 to 950,000 and were composed of laminin subunits and the glycoprotein forms linked by interchain disulfide bonds. The malignant keratinocyte cell lines from different patients were distinguishable in terms of the array of laminin and glycoprotein forms displayed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the rate of [35S]methionine incorporation into laminin during the pulse-labeling, the fraction of [35S]methionine-labeled laminin secreted into the medium during the chase incubation, and the absolute amount of laminin secreted into the culture medium as determined by enzyme-linked immunosorbent assay. However, cell lines established from primary and metastatic or recurrent cancer in the same patient were indistinguishable in their profile of laminin biosynthesis and secretion. In comparison to primary cultures of nonmalignant foreskin basal keratinocytes, the malignant cells secreted into the culture medium a larger fraction of the laminin that they produce. This is an indication that the malignant keratinocytes in culture deposited a less stable basal lamina-like extracellular matrix than their malignant counterparts. The diminished integrity of the basal lamina matrix may be an important factor in the invasive growth of human epithelial cancer.

Detection of a glycosylated, incompletely folded form of chorionic gonadotropin beta subunit that is a precursor of hormone assembly in trophoblastic cells.

The alpha and beta subunits of human chorionic gonadotropin are secreted both as a combined, noncovalently linked dimer form as well as uncombined, free forms by human trophoblastic cells. We have utilized the cultured choriocarcinoma cell line JAR to determine what regulates the combination of the two subunits. The human chorionic gonadotropin subunits produced by JAR cells were biosynthetically labeled with [35S] cysteine or [3H]mannose by a pulse-chase protocol, purified by immunoprecipitation with specific antisera that recognize free or combined subunits, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing or reducing conditions. Radioactively labeled bands were eluted from the gels and analyzed for total counts/minute incorporated, the ratio of free thiols to intramolecular cystine disulfides, and oligosaccharide composition. In some experiments, labeled gel bands were eluted with trypsin under nonreducing conditions, and the trypsin-released peptides were analyzed by high performance liquid chromatography. Using these procedures, the following results were obtained. The earliest, biosynthetically labeled form of the beta subunit detected in JAR cells contains high mannose N-linked oligosaccharides and has one-half of its incorporated cysteines present as free thiols. This form, termed pre-beta 1, has not yet combined with the alpha subunit even though the biosynthetically labeled alpha subunit is present in the cells at the same time. The pre-beta 1 form has a t1/2 of about 4 min and has a precursor-product relationship with a more completely disulfide-bonded form, termed pre-beta 2, which does combine with the alpha subunit to form a dimer. A subset of beta molecules produced in JAR cells does not attain the same disulfide bonding pattern as the pre-beta 2 form, does not combine with the alpha subunit, and is secreted as a free beta subunit into the culture medium. On the other hand, the earliest detectable form of the alpha subunit in JAR cells has all its thiols present as cystine disulfides, at a time when dimerization with the beta subunit has not yet taken place. These results strongly suggest that intramolecular disulfide bond formation in the beta subunit is the crucial and rate-limiting event in alpha beta dimer formation. The subset of beta molecules that remain free do not appear to form the appropriate intramolecular disulfides and thus do not achieve the correct conformation to combine with the alpha subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Laminin production by murine melanoma cells: possible involvement in cell motility.

Three lines of B16 melanoma cells (B16-F1, B16-F10 and B16-BL6) were examined for motility in the micropore filter assay and for synthesis in culture of the basal lamina glycoprotein laminin. All three lines synthesized laminin as judged by the incorporation of [35S]methionine into immunoreactive laminin and secreted (or shed) laminin into the culture medium as indicated by biosynthetic labeling studies and enzyme-linked immunosorbent assays. Immunoreactive laminin was also seen on the surface of the cells as indicated by immunofluorescence staining and by complement-mediated killing. Analysis of [35S]methionine-labeled laminin immunoprecipitates by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) both with and without reduction of intersubunit disulfide bonds revealed that all three cell lines produced a similar array of laminin forms, and that the Mr = 950 kD laminin molecule (but not the uncombined subunits) was secreted into the culture medium. Laminin biosynthesis appeared to be limited by the availability of the Mr = 400 kD A subunit as shown by the intracellular accumulation of excess B subunit in the form of uncombined B subunit (Mr = 200 kD) and as a disulfide-linked B dimer (Mr = 400 kD). The motility of all three cell lines was stimulated four- to five-fold by the addition of either exogenous laminin from the EHS sarcoma or culture medium from the B16 cells containing the secreted laminin. The stimulated motility was inhibited by antilaminin serum. These observations suggest that the laminin synthesized by the B16 melanoma cells themselves may facilitate their motility.

The effect of carbonyl cyanide trifluoromethoxyphenylhydrazone and methylamine on the processing and secretion of the glycoprotein hormone chorionic gonadotropin by human choriocarcinoma cells.

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells.

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.