Action of fibrin-stabilizing factor on cold-insoluble globulin and alpha2-macroglobulin in clotting plasma.

Experiments were performed to investigate whether proteins other than fibrin are substrates for activated fibrin-stabilizing factor (FSF, blood coagulation Factor XIII, plasma transglutaminase) in clotting whole plasma. Three fluorescently labeled polypeptides were identified in serum prepared by clotting normal, but not FSF-deficient, plasma in the presence of the fluorescent amine, N-(5-aminopentyl)-5-dimethyl-aminonaphthalene-1-sulfonamide (dansylcadaverine). The major labeled polypeptide had a Mr (estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate) of 1.6 times 10(5) and was found in the protein fraction precipitated by 33 to 50% saturated ammonium sulfate. The second had a Mr of 2.0 times 10(5), was found in the protein fraction insoluble in 33% saturated ammonium sulfate, and was precipitated by gamma-globulin directed against cold-insoluble globulin. The third had a Mr of 1.1 times 10(5) and was precipitated by 33 to 50% saturated ammonium sulfate. All three polypeptides were found in the first protein peak when labeled serum was chromatographed on Sephadex G-200. The immunoprecipitin arc containing alpha2-macroglobulin was fluorescent when labeled serum was analyzed by immunoelectrophoresis. These results indicate that alpha2-macroglubulin, cold-insoluble globulin, and an unidentified third protein with a subunit of Mr = 1.1 times 10(5) are transamidated by FSF in clotting plasma. The concentration of cold-insoluble globulin was decreased in serum formed at 37 degrees from normal, but not from FSF-deficient, plasma. The depletion of cold-insoluble globulin in normal serum was partially blocked by clotting in the presence of dansylcadaverine and completely blocked by clotting in the absence of calcium ions. Sera formed at 2 degrees from both normal and FSF-deficient plasma contained less cold-insoluble globulin than plasma. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of clots formed at 2 degrees demonstrated cross-linking of cold-insoluble globulin to fibrin in the normal, but not the FSF-deficient, sample. The serum concentration of alpha2-macroglobulin was the same as the plasma concentration irrespective of the conditions of clotting. Thus, the experiments suggest that FSF catalyzes the cross-linking of cold-insoluble globulin (but not alpha2-macroglobulin) to fibrin in clotting plasma.     

The cold-insoluble globulin of human plasma: studies of its essential structural features.

These investigations were directed at furnishing information on the essential structural features of the cold-insoluble globulin of human plasma. Amino acid and carbohydrate analyses showed that it is a glycoprotein (1.2% sialic acid, 1.8% hexose, 2.1% hexosamine) containing all of the amino acids usually found in proteins. Circular dichroic spectral analysis suggested that cold-insoluble globulin contained a very high proportion of beta-structure; no evidence for the presence of alpha-helix was found. Sedimentation velocity experiments at pH 7.0, in the presence or absence of dithiothreitol, plus related gel electrophoretic experiments at pH 8.4, indicated that the integrity of certain disulfide bridges was necessary for its solubility under "physiologic" conditions. In experiments in urea-containing solution, two sedimenting peaks were observed. The major one, amounting to more than 95% of the total, had an s20,w of 5.6 S, the minor peak had an s20,w of 7.3 S. Following disulfide bridge reduction a single symmetrical peak of 3.9 S was formed. Such behavior suggested that cold-insoluble globulin is a multichain molecule whose subunit chains are linked by disulfide bridging. Strong support for this conclusion was obtained from electrophoretic analyses in gels containing dodecylsulfate, in that cold-insoluble globulin manifested an increased rate of migration after reduction of disulfide bridges. The reduced cold-insoluble globulin band could be resolved into a closely spaced doublet, the components of which had molecular weights of 220 000 and 215 000, respectively. Since in sedimentation equilibrium experiments the molecular weight of the unreduced molecule was estimated to be 450 000, values in this range for the size of the subunit chain suggested that each cold-insoluble globulin molecule is composed of two covalently linked chains. The nature of the size heterogeneity of the reduced subunit chains is uncertain. However, the finding of a single type of NH2-terminal sequence ([Glu-Ala) in cold-insoluble globulin preparations, is consistent with the speculation that the smaller subunit may be a catabolic intermediate arising via release of peptide material containing the COOH-terminus of a parent chain.     

Adhesion of rat hepatocytes to collagen

Rat hepatocytes, freshly isolated with a collagenase perfusion technique, were found to attach within 1 h on collagen substrates and on culture dishes coated with cold insoluble globulin (CIG) or asialoceruloplasmin (AC). Spreading was observed on collagen and CIG but not on AC. Both attachment and spreading occurred in a simple balanced salt solution in the absence of serum. In the absence of serum no attachment was observed on plain plastic dishes or on dishes coated with serum albumin or other plasma proteins, unless divalent manganese ions were present. In the presence of manganese the hepatocytes attached to all surfaces tested, but no spreading occurred. Attachment to collagen occurred equally well to collagens type I or type III both in the native, fibrillar state and in the denatured state. Collagen attachment required magnesium ions but did not appear to involve the collagen-linked carbohydrates. Different mechanisms were found to operate in hepatocyte attachment to collagen and to AC; the latter is most likely mediated by the hepatocyte surface receptor involved in recognition and uptake of asialoglycoproteins. The role of CIG in hepatocyte attachment to collagen was investigated. Data are presented suggesting that this glycoprotein, which mediates the adhesion of fibroblasts to collagen, is not required for hepatocyte attachment to collagen.     

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.     

Comparison of the structures of human fibronectin and plasma cold-insoluble globulin

Human amniotic fluid fibronectin and plasma fibronectin (cold-incoluble globulin) are indistinguishable both immunologically and by amino acid composition. Cyanogen bromide and tryptic peptides also suggest substantial structural homology. However, carbohydrate analysis has demonstrated additional saccharides in fibronectin and an overall increase in carbohydrate content relative to coldinsoluble globulin. Furthermore, limited proteolytic cleavage of the two proteins indicates differences in primary structure or in conformation. Using affinity-purified antibodies to cold-insoluble globulin, a glucosamine-labeled pronaseresistant component, probably proteoglycan, was found to coprecipitate with fibronectin, suggesting an association between these two macromolecules in the connective tissue matrix.     

Attachment of phenoloxidase to fungal cell walls in arthropod immunity

In crayfish, phenoloxidase was located in the hemocytes. The plasma had infinitesimal enzyme activity. A phenoloxidase preparation from hemocytes precipitated spontaneously after approximately 1.5 hr at 22°C, which became attached spontaneously to glass, Plexiglas, and polystyrene plastic. The enzyme preparation could also become attached to Saccharomyces cerevisiae cell walls. Attachment was mediated by a proteinaceous substance, since trypsin significantly decreased the degree of attachment. Calcium ions were also necessary for attachment. A β-1,3-glucan, laminaran, partially prevented attachment to the fungal cell walls. Heparin caused precipitation of the phenoloxidase preparation from hemocytes. In crayfish cuticle, proteins with associated phenoloxidase activity were attached to cell walls of Aphanomyces astaci as well as to those of S. cerevisiae.     

Adhesion of murine cells to glass microbeads: substrate-adsorbed serum proteins.

Small (1 mm diameter) glass beads treated with acid and alkali provided a satisfactory substratum for the attachment and growth of normal and SV-40 transformed Balb/c 3T3 murine cells. Cells attached to the beads with similar, though slower kinetics as to flat glass surfaces, and spread and grew with their usual morphology; when detached by EGTA treatment, they left behind cellular substrate-attached material (SAM) which was similar electrophoretically to that obtained from tissue culture plastic. Because of their small size and rough etched surfaces, the beads have a large surface area and high adsorptive capacity, and so are a useful tool to isolate specific serum proteins adsorbed from the culture medium that may be important for cell attachment and spreading. The adsorbed serum proteins were solubilized with SDS and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. They included all reduced species adsorbed to tissue culture plastic, and only small amounts of one other major protein extracted from a “bacteriological” polystyrene surface on which cells could not grow. Profiles of unreduced samples differed considerably. The profile of adsorbed proteins varied little with tiem (5 minutes–4 days), temperature (4°–37°C), pH (5–9), presence of the protease inhibitor PMSF, or serum concentration (0.1–10%). Much of the adsorbed protein, qualitatively similar to the SDS-extracted material, could be eluted with H₂O or phosphate-buffered saline. Purified albumin and fibrinogen bound avidly to the beads; the material adsorbed from serum contained a large amount of albumin, however, little fibrinogen and no cold-insoluble globulin (as a 220 K protein) could be detected by Coomassie blue stained SDS-PAGE gels.     

Laboratory studies on adhesion of microalgae to hard substrates

Adhesion of Chlorella vulgaris(chlorophyceae), Nitzschia amphibia(bacillariophceae) and Chroococcus minutus(cyanobacteria) to hydrophobic (perspex, titanium and stainless steel 316-L), hydrophilic (glass) and toxic (copper, aluminium brass and admiralty brass) substrata were studied in the laboratory. The influence of surface wettability, surface roughness, pH of the medium, culture age, culture density, cell viability and presence of organic and bacterial films on the adhesion of Nitzschia amphibia was also studied using titanium, stainless steel and glass surfaces. All three organisms attached more on titanium and stainless steel and less on copper and its alloys. The attachment varied significantly with respect to exposure time and different materials. The attachment was higher on rough surfaces when compared to smooth surfaces. Attachment was higher on pH 7 and above. The presence of organic film increased the attachment significantly when compared to control. The number of attached cells was found to be directly proportional to the culture density. Attachment by log phase cells was significantly higher when compared to stationary phase cells. Live cells attached more when compared to heat killed and formalin killed cells. Bacterial films of Pseudomonas putida increased the algal attachment significantly. %     

Locomotion and adhesion of neutrophil granulocytes : Effects of albumin, fibrinogen and gamma globulins studied by reflection contrast microscopy

Proteins as well as materials of low molecular weight have marked effects on the rate of locomotion, adhesion and cell shape of human neutrophil granulocytes in vitro. Plasma protein preparations differ qualitatively with respect to their chemokinetic activity. Human serum albumin (HSA), fibrinogen and acid-treated gamma globulin without polymers have a positive chemokinetic effect on neutrophils suspended in Gey's solution. Standard gamma globulin (SGG) or acid-treated gamma globulin with polymers have marked negative chemokinetic activity. Three different mechanisms are presumably responsible for the low rate of locomotion observed in Gey's solution alone, Gey's solution containing acid-treated gamma globulin with polymers or SGG, respectively: (a) too firm adhesion to the substratum; (b) lack of adhesion to the substratum; and (c) impaired capacity to perform shape changes. The relationship between attachment of cells to the substratum and the rate of neutrophil locomotion has been investigated. It appears that the pattern of adhesion rather than cell attachment as measured by the proportion of neutrophils adhering to the substratum is a meaningful correlate to locomotion. Two different patterns of adhesion can be distinguished by means of reflection-contrast microscopy: (a) the pattern characterized by uniform grey areas is compatible with efficient locomotion; (b) a pattern characterized by large black areas at the cell periphery. It is associated with neutrophils in Gey's solution which fail to displace themselves efficiently. This suggests that reflection-contrast microscopy may be helpful in distinguishing contacts allowing locomotion to occur from contacts impeding neutrophil locomotion.     

A cell spreading factor in human serum that is not cold-insoluble globulin

Gel filtration of human serum shows that there are two separate peaks of activity that will cause fibroblasts in culture to adopt their characteristic bipolar morphology (i.e., cell spreading). One of these activities has a molecular weight just below 250 000 D while the other is in the range 65 000–85 000 D. The lower molecular weight activity is unlikely to be related to cold-insoluble globulin, yet is quantitatively the more significant.     

Heterogeneity of the cold-insoluble globulin of human plasma (CIg), a circulating cell surface protein.

The cold-insoluble globulin of human plasma (CIg), a circulating cell surface protein, exists in multiple molecular forms. Most molecules are found as two chain (MR approximately 220 000 per chain) disulfide-bridged dimeric units but several minor components of smaller size have also been identified; based upon their migration rates in dodecyl sulfate gel electrophoretic experiments, the smaller molecules characterized in this study range in molecular size from 235 000 to 146 000. The component of molecular weight 235 000 apparently represents a two chain disulfide-bridged derivative of larger parent molecules (one chain of 220 000 plus a smaller remnant), whereas smaller CIg components appear to be single chain proteins. These observations plus electrophoretic analyses of samples of plasmic digests of CIg indicate that the interchain disulfide bridging in the two chain molecule is located in a segment within approx. 175 residues of the NH2- or COOH-terminus.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Pectin and Xyloglucan Influence the Attachment of Salmonella enterica and Listeria monocytogenes to Bacterial Cellulose-Derived Plant Cell Wall Models

Minimally processed fresh produce has been implicated as a major source of foodborne microbial pathogens globally. These pathogens must attach to the produce in order to be transmitted. Cut surfaces of produce that expose cell walls are particularly vulnerable. Little is known about the roles that different structural components (cellulose, pectin, and xyloglucan) of plant cell walls play in the attachment of foodborne bacterial pathogens. Using bacterial cellulose-derived plant cell wall models, we showed that the presence of pectin alone or xyloglucan alone affected the attachment of three Salmonella enterica strains (Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, and Salmonella enterica subsp. indica M4) and Listeria monocytogenes ATCC 7644. In addition, we showed that this effect was modulated in the presence of both polysaccharides. Assays using pairwise combinations of S. Typhimurium ATCC 14028 and L. monocytogenes ATCC 7644 showed that bacterial attachment to all plant cell wall models was dependent on the characteristics of the individual bacterial strains and was not directly proportional to the initial concentration of the bacterial inoculum. This work showed that bacterial attachment was not determined directly by the plant cell wall model or bacterial physicochemical properties. We suggest that attachment of the Salmonella strains may be influenced by the effects of these polysaccharides on physical and structural properties of the plant cell wall model. Our findings improve the understanding of how Salmonella enterica and Listeria monocytogenes attach to plant cell walls, which may facilitate the development of better ways to prevent the attachment of these pathogens to such surfaces.     

A Seed Storage Protein with Possible Self-Affinity through Lectin-Like Binding

The primary storage protein of oat (Avena sativa L.) seeds, globulin, was shown to have a specific carbohydrate-binding activity. The globulin was capable of hemagglutinating rabbit red blood cells and this hemagglutination was inhibited by the β-glucan, laminarin, as well as by carbohydrate which had been cleaved from the native globulin. Globulin with carbohydrate-binding activity was isolated from cell wall preparations and from defatted flour. The lectin activity apparently resides in the α-subunit of the globulin and has affinity for the carbohydrate which is O-glycosidically linked to the globulin. A portion of this carbohydrate is attached to the β-subunit. Two affinity columns were synthesized utilizing laminarin and the carbohydrate from the native globulin as ligands. The hemagglutinating activity bound to both of these columns. The activity was specifically eluted from the globulin-carbohydrate affinity column with carbohydrate cleaved from native globulin by an alkali-catalyzed β-elimination. The possible roles of this unique self-binding capacity are discussed.     

Collagen synthesis by bovine aortic endothelial cells in culture.

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.     

Plasma clearance and liver metabolism of native and asialo-human transcortin in the rat

Plasma kinetics and liver metabolism of iodanated human corticosteroid-binding protein have been studied in ovariectomized female rats. ¹²⁵I-labeled human corticosteroid-binding globulin prepared by a modified chloramine T reaction was shown to be physically intact and biologically active. Intravenously injected ¹²⁵I-labeled human corticosteroid-binding globulin was shown to give a complex clearance pattern from the plasma, with half-lives of 7.5 and 51 min. Estrogen injections had no effect on plasma clearance rate. Direct involvement of liver plasma membrane receptors for asialoglycoproteins in ¹²⁵I-labeled human corticosteroid-binding globulin metabolism was demonstrated in vivo and in vitro using asialofetuin as a competitive inhibitor. ¹²⁵I-labeled human asialo-corticosteroid-binding globulin was cleared from the plasma with a half-life of less than 1 min, while the simultaneous injection of 5 mg asialofetuin maintained the circulating plasma levels. Asialofetuin also slowed the clearance of intact ¹²⁵I-labeled human corticosteroid-binding globulin from the plasma ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=90}\text{min}$). Binding of ¹²⁵I-labeled human asialo-corticosteroid-binding globulin to rat liver plasma membranes in vitro was inhibited in a dose-dependent manner by asialofetuin, but not by intact human corticosteroid-binding globulin or fetuin. ¹²⁵I-labeled human corticosteroid-binding globulin did not bind significantly to the membranes. It is concluded that human corticosteroid-binding globulin clearance from rat plasma is rapid and that the carbohydrate moiety of human corticosteroid-binding globulin is involved in its clearance and catabolism by the liver.     

Cellular prion protein in human plasma–derived extracellular vesicles promotes neurite outgrowth via the NMDA receptor–LRP1 receptor system

Exosomes and other extracellular vesicles (EVs) participate in cell–cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α₂-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α₂-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrP^C) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrP^C-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor–related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R–LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrP^C. Finally, EVs harvested from rat astrocytes carried PrP^C and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrP^C with the NMDA-R–LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.     

ENTRY OF A CHLORELLA-VIRUS INTO ITS HOST CELL1

The endosymbiotic Chlorella sp. (Chlorophyceae) of Paramecium bursaria (Ciliata) can be infected by a double-stranded DNA-containing virus (Chlorella-virus) that has a phagelike entry mechanism. Electron micrographs show that soon after attachment of the virus to the algal cell wall, a hole is formed through which the viral DNA enters the alga. Biochemical studies on a European Chlorella-virus system suggest that digestion of the algal cell wall is caused by glycolytic enzymes, one of which was identified as a β-d -glucosidase. Enzymes are bound to the virus capsid and are activated only after or by the attachment of the virus to its cognate alga or to preparations of the algal cell wall. Common features of viral cell wall-digesting enzymes and algal autolysins are discussed.     

Treponema pallidum fibronectin-binding proteins

Putative adhesins were predicted by computer analysis of the Treponema pallidum genome. Two treponemal proteins, Tp0155 and Tp0483, demonstrated specific attachment to fibronectin, blocked bacterial adherence to fibronectin-coated slides, and supported attachment of fibronectin-producing mammalian cells. These results suggest Tp0155 and Tp0483 are fibronectin-binding proteins mediating T. pallidum-host interactions.     

Prospore Membrane Formation Defines a Developmentally Regulated Branch of the Secretory Pathway in Yeast

Spore formation in yeast is an unusual form of cell division in which the daughter cells are formed within the mother cell cytoplasm. This division requires the de novo synthesis of a membrane compartment, termed the prospore membrane, which engulfs the daughter nuclei. The effect of mutations in late-acting genes on sporulation was investigated. Mutation of SEC1, SEC4, or SEC8 blocked spore formation, and electron microscopic analysis of the sec4-8 mutant indicated that this inability to produce spores was caused by a failure to form the prospore membrane. The soluble NSF attachment protein 25 (SNAP-25) homologue SEC9, by contrast, was not required for sporulation. The absence of a requirement for SEC9 was shown to be due to the sporulation-specific induction of a second, previously undescribed, SNAP-25 homologue, termed SPO20. These results define a developmentally regulated branch of the secretory pathway and suggest that spore morphogenesis in yeast proceeds by the targeting and fusion of secretory vesicles to form new plasma membranes in the interior of the mother cell. Consistent with this model, the extracellular proteins Gas1p and Cts1p were localized to an internal compartment in sporulating cells. Spore formation in yeast may be a useful model for understanding secretion-driven cell division events in a variety of plant and animal systems.