Albert E. Chung

Properties of a basement membrane-related glycoprotein synthesized in culture by a mouse embryonal carcinoma-derived cell line.

Two glycoproteins, GP-1 and GP-2, have been isolated from an extracellular membrane synthesized in cell culture by an embryonal carcinoma-derived cell line. The amino acid and carbohydrate compositions have been determined. Both proteins are rich in half-cystine residues and contain approximately 12-15% carbohydrate. Antibodies have been obtained against one of the glycoproteins, GP-2, in rabbits. The antibody reacts with basement membranes from adult mouse and human kidney glomeruli and tubules, and all basement membranes tested from mouse embryonic tissues. The molecular properties of GP-2 are superficially similar to LETS protein; however, immunological and other criteria show that they are distinct proteins. The presence of LETS protein and GP-2 in basement membranes suggests that there are subtle interactions which are important in adhesion of epithelial cells to basement membranes.

cAMP Regulates Ca2+-dependent Exocytosis of Lysosomes and Lysosome-mediated Cell Invasion by Trypanosomes

Ca2+-regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca2+ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca2+-dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins Gs and Gi, consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a β-adrenergic agonist that activates adenylyl cyclase, enhances Ca2+-dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca2+] i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca2+-induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca2+ and a parasite/host cell interaction known to involve Ca2+-dependent lysosomal fusion.

Immunohistochemical localization of entactin and laminin in mouse embryos and fetuses

The temporal appearance and the anatomic distribution of entactin in the developing mouse embryo was studied by light and electron microscopic immunohistochemistry and compared with the appearance and the distribution of laminin. Immunohistochemically detectable entactin first appeared in the hatched blastocysts, in contrast to laminin which became apparent as early as the 8-cell stage of development. However, beginning with the appearance of entactin in the hatched blastocyst, antibodies to these two basement membrane glycoproteins co-localized in all extraembryonic matrices. These findings indicate that the synthesis of entactin and laminin is asynchronous in the early stages of mouse embryonic development but becomes synchronized in the postimplantational stages of development.

Neurologic Defects and Selective Disruption of Basement Membranes in Mice Lacking Entactin-1/Nidogen-1

Entactin-1 (nidogen-1) is an ubiquitous component of basement membranes. From in vitro experiments, entactin-1 was assigned a role in maintaining the structural integrity of the basement membrane because of its binding affinity to other components, such as type IV collagen and laminin. Entactin-1 also interacts with integrin receptors on the cell surface to mediate cell adhesion, spreading, and motility. Targeted disruption of the entactin-1 gene in the mouse presented in this study revealed a duplication of the entacin-1 locus. Homozygous mutants for the functional locus lacked entactin-1 mRNA and protein and often displayed seizure-like symptoms and loss of muscle control in the hind legs. The behavior patterns suggested the presence of neurologic deficits in the central nervous system, thus providing genetic evidence linking entactin-1 to proper functions of the neuromuscular system. In homozygous mutants, structural alterations in the basement membranes were found only in selected locations including brain capillaries and the lens capsule. The morphology of the basement membranes in other tissues examined superficially appeared to be normal. These observations suggest that the lost functions of entactin-1 result in pathologic changes that are highly tissue specific.

The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues.

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.

A novel extracellular membrane elaborated by a mouse embryonal carcinoma-derived cell line

Abstract An extracellular membranous structure is synthesized by an embryonal carcinoma-derived cell line, M1536-B3, in suspension cultures. Analysis of the solubilized membranous structure on polyacrylamide gels in sodium dodecyl sulfate yielded two major classes of glycoproteins with molecular weights of approximately 230,000 and 320,000 respectively. The amino acid composition of the purified membranous structures revealed the absence of both hydroxyproline and hydroxylysine. Carbohydrate analysis demonstrated the presence of fucose, xylose, mannose, galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. These carbohydrates represented approximately 9% of the weight of the membrane. A comparison of the electrophoretic patterns of cells grown on monolayers and in suspension revealed a marked accumulation of the glycoproteins under the latter growth conditions. D -[3H]-glucosamine was incorporated into these two and a third major glycoprotein by cells in suspension culture.

Cationic lipid-mediated co-transfection of insect cells.

Current co-transfection protocols use calcium phosphate precipitation for cultured insect (Sf-9) cells followed by multiple agarose overlays to purify recombinant baculoviruses (1). We report a more effective and convenient method that uses the cationic lipid N[1,1-(2,3-dioleyloxy)propoyl]-N,N,N,trimethylammonium chloride, or DOTMA (2, 3), for cotransfections of Sf-9 cells and uses 96-well microtiter plates to identify and purify recombinant baculoviruses. The cotransfection method is not pH sensitive and may be used with vector DNA from mini-lysate protocols. DOTMA is sold by Gibco/BRL under the trade name LipofectinTm. The cDNA genes for both the aand fl-chains of hemoglobin were cloned into the baculovirus transfer vector pAcYM1 (4) and verified by restriction enzyme digestion, partial DNA sequencing, and dot-blot hybridization (results not shown). One isg of CsCl-purified transfer vector for the a-chain of hemoglobin was mixed with 2Atg of AcMNPV DNA and 30 Ag of DOTMA in a polystyrene container. The solution was brought to a volume of 50 A1 with water, mixed, and allowed to sit at room temperature for 15 minutes. The DNA/DOTMA complex was mixed with 3 ml of serum-free Graces medium supplemented to 0.33% lactalbumin hydrolysate and 0.33% yeastolate and added to a 25-cm2 flask containing 3 x 106 Sf-9 cells. The cells were then incubated without serum at 27°C for 3-24 hours. After this incubation, the medium was supplemented to 10% fetal bovine serum in a total volume of 6 ml and the cells incubated for a week at 27°C. 10-5 to 10-7 dilutions of the co-transfection medium were prepared and each dilution used to infect Sf-9 cells seeded in a 96-well microtiter plate at 1 x 104 cells/well. The plates were incubated at 27°C for a week. Each well was scored for the presence of viral infection and the viral titer of the cotransfection medium calculated by end-point dilution (1). We find that the viral titer from the co-transfection of Sf-9 cells using DOTMA is at least 20-fold greater than the titer obtained from the calcium phosphate precipitation method. The cells from each plate were lysed and screened with a 32P-labeled probe to identify recombinant viral samples. Figure la shows an autoradiograph of a nitrocellulose filter from a 96-well dot-blot hybridization of a plate of cells infected with a 10-5 dilution of the co-transfection medium. The percentage of recombinant virus present in the co-transfection medium is estimated to be 5-10%, a 5-50-fold increase in recombinant virus over the calcium phosphate precipitation method. We find that an overnight incubation of Sf-9 cells in the presence of DOTMA provides optimal recombinant virus production. The microtiter plate screening procedure was done a second time to purify recombinant virus from wild-type virus. This process was also performed for the fl-chain of hemoglobin. Purified recombinant viruses for both chains (Ha and Hfl) were used to infect 25-cm2 culture flasks of Sf-9 cells (1). Cells were collected at 72 hours post-infection, washed, resuspended in sample buffer, and analyzed on a 15% SDS-polyacrylamide gel. Figure lb shows that proteins identical in size to each chain are expressed in the infected cells.

Domain-specific Interactions between Entactin and Neutrophil Integrins: G2 DOMAIN LIGATION OF INTEGRIN α3β1 AND E DOMAIN LIGATION OF THE LEUKOCYTE RESPONSE INTEGRIN SIGNAL FOR DIFFERENT RESPONSES

Extracellular matrix proteins activate neutrophils to up-regulate many physiologic functions that are necessary at sites of tissue injury. To elucidate the ligand-receptor interactions that mediate these functions, we examined neutrophil activation by the basement membrane protein, entactin. Entactin is structurally and functionally organized into distinct domains; therefore, we utilized glutathione S-transferase -fusion proteins encompassing its four major domains, G1, G2, E, and G3, to assess interactions between entactin and neutrophil integrin receptors. We show that the E domain, which contains the single RGD sequence of entactin, is sufficient for ligation of the β3-like integrin, leukocyte response integrin, and signaling for chemotaxis. Moreover, the G2 domain signals for stimulation of Fc receptor-mediated phagocytosis via ligation of α3β1. This receptor-ligand interaction was revealed only after stimulation of neutrophil by immune complexes or phorbol esters. Interestingly, the E domain does not enhance phagocytosis, and the G2 domain is not chemotactic. Furthermore, cleavage of entactin with the matrix metalloproteinase, matrilysin, liberates peptides that retain E domain-mediated chemotaxis and G2 domain-mediated enhancement of phagocytosis. These studies indicate that multiple domains of entactin have the ability to ligate individual integrins expressed by neutrophils and to activate distinct functions.

Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin.

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.

Oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase from Azotobacter vinelandii: III. The path of hydrogen in the enzymatic reaction

Abstract Isocitrate dehydrogenase from Azotobacter vinelandii transfers hydrogen from threo - d 8 -isocitrate to the A side of TPN + . The decarboxylation of threo d 8 isocitrate to 2-ketoglutarate occurs with retention of configuration. The enzyme catalyzes the labilization of tritium from [3-T]2-ketoglutarate in the presence of Mg 2+ and TPNH which suggests an ordered addition of substrates in the reductive carboxylation of 2-ketoglutarate to isocitrate.