Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175.

INTRODUCTION: Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery. METHODS: By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC). RESULTS: Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h. DISCUSSION: Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.

Parasitoid wasp venom elevates sorbitol and alters expression of metabolic genes in human kidney cells.

Venom from the parasitoid wasp Nasonia vitripennis dramatically elevates sorbitol levels in its natural fly hosts. In humans, sorbitol elevation is associated with complications of diabetes. Here we demonstrate that venom also induces this disease-relevant phenotype in human cells, and investigate possible pathways involved. Key findings are that (a) low doses of Nasonia venom elevate sorbitol levels in human renal mesangial cells (HRMCs) without changing glucose or fructose levels; (b) venom is a much more potent inducer of sorbitol elevation than glucose; (c) low venom doses significantly alter expression of genes involved in sterol and alcohol metabolism, transcriptional regulation, and chemical/stimulus response; (d) although venom treatment does not alter expression of the key sorbitol pathway gene aldose reductase (AR); (e) venom elevates expression of a related gene implicated in diabetes complications (AKR1C3) as well as the fructose metabolic gene (GFPT2). Although elevated sorbitol is accepted as a major contributor to secondary complications of diabetes, the molecular mechanism of sorbitol regulation and its contribution to diabetes complications are not fully understood. Our findings suggest that genes other than AR could contribute to sorbitol regulation, and more broadly illustrate the potential of parasitoid venoms for medical application.

Fibrinogen synthesized by cancer cells augments the proliferative effect of fibroblast growth factor-2 (FGF-2).

BACKGROUND: Fibroblast growth factor (FGF)-2 is a critical growth factor in normal and malignant cell proliferation and tumor-associated angiogenesis. Fibrinogen and fibrin bind to FGF-2 and modulate FGF-2 functions. Furthermore, we have shown that extrahepatic epithelial cells are capable of endogenous production of fibrinogen. OBJECTIVE: Herein we examined the role of fibrinogen and FGF-2 interactions on prostate and lung adenocarcinoma cell growth in vitro. METHODS: Cell proliferation was measured by (3)H-thymidine uptake and the specificity of FGF-2-fibrinogen interactions was measured using wild-type and mutant FGF-2s, fibrinogen gamma-chain (FGG) RNAi and co-immunoprecipitation. Metabolic labeling, immunopurification and fluorography demonstrated de novo fibrinogen production. RESULTS: FGF-2 stimulated DU-145 cell proliferation, whereas neither FGF-2 nor fibrinogen affected the growth of PC-3 or A549 cells. Fibrinogen augmented the proliferative effect of FGF-2 on DU-145 cells. The role of fibrinogen in FGF-2-enhanced DNA synthesis was confirmed using an FGF-2 mutant that exhibits no binding affinity for fibrinogen. FGG transcripts were present in PC-3, A549 and DU-145 cells, but only PC-3 and A549 cells produced detectable levels of intact protein. RNAi-mediated knockdown of FGG expression resulted in decreased production of fibrinogen protein and inhibited (3)H-thymidine uptake in A549 and PC-3 cells by 60%, which was restored by exogenously added fibrinogen. FGF-2 and fibrinogen secreted by the cells were present in the medium as a soluble complex, as determined by coimmunoprecipitation studies. CONCLUSIONS: These data indicate that endogenously synthesized fibrinogen promotes the growth of lung and prostate cancer cells through interaction with FGF-2.

Plasma fibrinogen gamma' chain content in the thrombotic microangiopathy syndrome.

BACKGROUND: Human fibrinogen gamma chain variants, termed gamma' chains, contain a unique 20-residue sequence after gamma chain residue 407 that ends at gamma'427, and is designated gamma'(427L). Full-length (FL) gamma'(427L) chains are constituents of a fibrin-dependent thrombin inhibitory system known as antithrombin I, whereas a gamma' chain processed in vivo, termed gamma'(423P), lacks the C-terminal tetrapeptide EDDL, and does not bind thrombin. Together, the gamma'(423P) and gamma'(427L) chains comprise the total plasma fibrinogen gamma' chain content. OBJECTIVES: Lowered plasma gamma' chain content (i.e. gamma' chain-containing fibrinogen/total fibrinogen ratio) has been shown to correlate with susceptibility to venous thrombosis, thus prompting this study on the total and FL gamma' chain content in 45 subjects with thrombotic microangiopathy (TMA), a disorder characterized by microvascular thrombosis. METHODS: We measured by enzyme-linked immunosorbent assay the total gamma' chain-containing fibrinogen/total fibrinogen (Total gamma'-fgn/Total fgn) ratio and the FL gamma' chain-containing fibrinogen/total fibrinogen (FL gamma'-fgn/Total fgn) ratio in these plasmas and in healthy subjects (n = 87). RESULTS: In healthy subjects, the mean Total gamma'-fgn/Total fgn ratio was 0.127, whereas the FL gamma'-fgn/Total fgn ratio was somewhat lower at 0.099 (P < 0.0001), a difference reflecting the presence of gamma'(423P) chains. In TMA plasmas, both the Total gamma'-fgn and FL gamma'-fgn/Total fgn ratios (0.099 and 0.084, respectively) were lower than those of their healthy subject counterparts (P < 0.0001). CONCLUSIONS: These findings in TMA suggest that reductions in the gamma' chain content indicate reduced antithrombin I activity that may contribute to microvascular thrombosis in TMA.

Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

Fibrinogen assembly, secretion, and deposition into extracellular matrix by MCF-7 human breast carcinoma cells.

A hallmark of breast carcinoma is the deposition of fibrinogen (FBG) without subsequent conversion to fibrin in the tumor stroma. In this study, the ability of the MCF-7 human breast cancer epithelial cell line to synthesize, secrete, and deposit FBG into the extracellular matrix (ECM) was examined. Whereas MCF-7 cells produced low levels of intact FBG, abundant levels of FBG intermediate complexes or degraded Aalpha, Bbeta, and gamma chain polypeptides were observed. Most of the Bbeta chain was degraded and missing an NH2-terminal peptide fragment. Reverse transcription-PCR analysis indicated that only gamma chain mRNA was present in detectable steady-state levels, although Southern hybridization revealed that the FBG Aalpha, Bbeta, and gamma chain genes were intact in MCF-7 cells. Immunostaining showed that extracellular FBG was bound to the surface of MCF-7 cells in a punctate pattern, reminiscent of receptor binding, rather than a fibrillar pattern characteristic of mature ECM. A similar punctate pattern of staining was observed when MCF-7 FBG was added to fibroblasts that normally assemble exogenous FBG into an extensive, fibrillar ECM, suggesting that MCF-7 cells are defective in assembly of a fibrillar ECM. The loss of FBG Bbeta chain NH2-terminal peptides may contribute to the lack of intact FBG assembly in MCF-7 cells, which may further affect its ability to assemble FBG into a fibrillar ECM. Taken together, the data suggest that endogenous synthesis and secretion of FBG is, at least in part, the source of FBG deposition in the ECM of breast cell carcinomas.

Calcium modulates plasmin cleavage of the fibrinogen D fragment gamma chain N-terminus: mapping of monoclonal antibody J88B to a plasmin sensitive domain of the gamma chain.

Plasmin sensitive sites are found on the A alpha, B beta and gamma chains of fibrinogen at regions joining the two C-terminal D fragments with the central E fragment. We have developed a monoclonal antibody (MoAb) reactive with this plasmin sensitive region on the human fibrinogen gamma chain and mapped its epitope. MoAb J88B reacts with gamma chains of both native as well as with reduced and denatured fibrinogen and fibrin, the CNBr fragment of the fibrinogen central domain, plasmin cleaved fragments D, gamma-gamma dimers, but not with plasmic fragments E. These data indicate that J88B maps to the plasmin sensitive domain localized to gamma 63-78. MoAb J88B failed to react with synthetic peptide gamma 70-78, which suggests that the epitope includes the newly exposed N-terminal residues gamma 63-70 of the early plasmic fragment D1A. As calcium has a marked influence on plasmin cleavage of C-terminal sites on the gamma chain, the effects of calcium on modulating plasmin cleavage of D1A to D1 were assessed in the absence or presence of J88B. The results indicated that calcium delays and J88B (+/- calcium) protects the gamma chain from plasmin cleavage at the N-terminus of D1A, suggesting that this enzymatically labile site is calcium-sensitive. Thus, MoAb J88B should prove useful in studies examining the structure of plasmin cleaved fibrinogen and fibrin.

Mechanisms of cytopenia in human immunodeficiency virus infection.

Human immunodeficiency virus (HIV) infection often has effects on the hematopoietic system which can be distinguished from the concurrent effects of medications or opportunistic infections. Exactly how the virus mediates these effects remains uncertain, but both in vivo and in vitro studies have pointed up possible direct and indirect modes of hematopoietic suppression. Whether a significant fraction of CD34+ cells in vivo are infected with HIV remains controversial, but most studies using in situ polymerase chain reaction techniques would suggest not. Other more indirect modes of hematopoietic cell suppression such as production of autoantibodies, production of other humoral inhibitory factors, T-cell mediated suppression of hematopoiesis, or production of inhibitory or stimulatory cytokines may also be contributory. It is probable that several of these mechanisms may occur simultaneously, and an increased understanding of their role may lead to improved strategies to correct the cytopenias which often accompany HIV disease.

Molecular characterization of mouse Pneumocystis carinii surface glycoprotein A.

Since the mouse offers an easily manipulated experimental animal model for the study of the immunopathogenesis of pneumonia caused by the opportunist Pneumocystis carinii, we cloned and characterized cDNAs encoding an abundant, immunogenic surface antigen termed glycoprotein A (gpA) from mouse P. carinii. A cDNA library was constructed in bacteriophage lambda gt11 from P. carinii-infected mouse lung poly(A+) RNA. Using a nucleic acid probe derived from a conserved region of the mouse P. carinii gpA structural gene, cDNAs encoding gpA were identified. A composite full-length gpA coding sequence was assembled from two overlapping cDNA clones. A DNA element homologous to the rat P. carinii upstream conserved sequence (UCS) was identified at the 5' end of several of the mouse P. carinii gpA cDNA clones, just upstream of the sequences encoding gpA structural gene isoforms. Using primer extension analysis, two neighboring putative transcriptional start sites were located on UCS-gpA mRNAs approximately 25 and 30 nt, respectively, upstream of the most 5' gpA cDNA clone isolated, suggesting a 5' UCS of 489 or 494 nucleotides in mouse P. carinii gpA. A comparative alignment of the composite mouse P. carinii gpA deduced amino acid sequence with gpA homologs from rat, human and ferret P. carinii demonstrated 156 identical residues, including 46 cysteines, further supporting the hypothesis for conserved secondary structure, as well as function, for gpA from all P. carinii.

Cloning and characterization of a conserved region of human and rhesus macaque Pneumocystis carinii gpA.

Although the genes encoding Pneumocystis carinii (Pc) glycoprotein A (gpA) display a high degree of host species-specific genotypic diversity, the Pc gpA derived from different host species share defined regions of significant homology in their primary amino acid (aa) structure. Using two degenerate oligodeoxyribonucleotide (oligo) primers corresponding to a conserved Cys region (Cys-primers) of the ferret (F), rat (R) and mouse (M) PcgpA, a 306-bp portion of the human (H) PcgpA was amplified from only one of three known HPc-infected lung samples using PCR. The deduced aa sequence of the HPc PCR product was 72% similar to the corresponding region of a published HPc gpA aa sequence. Because the conserved Cys-primers amplified only one of three samples of HPcgpA, a primer-pair was designed from sequences internal to the Cys-primer sequences of the HPcgpA PCR product (hPc). The hPc primers amplified the expected 254-bp product from each of the three HPc-infected lung DNA samples, suggesting that the Cys-primers may have either amplified a HPcgpA present in fewer copies in the genome of HPc or, alternatively, amplified a gene from an uncommon strain of Pc encoding an isoform variant of gpA not present in the other human isolates analyzed in this report. Restriction analysis of the amplified products demonstrated heterogeneity in the internal sequence, confirming that more than one gpA exists in HPc as well. To determine the relationship of HPcgpA to the gpA of Pc from another primate, the hPc primers were used successfully to amplify a 261-bp product from Pc-infected Rhesus macaque (Rm) lung genomic DNA. These results are consistent with our earlier findings that closely related host species are infected with Pc organisms encoding similar gpA, suggesting that the evolutionary divergence of Pc followed that of the mammalian host species.

Cloning and characterization of a lung-specific cDNA corresponding to the gamma chain of hepatic fibrinogen.

The gene (gamma FBG) encoding the fibrinogen gamma chain (gamma FBG) has been shown to exhibit both tissue-specific and ubiquitous expression. To confirm the identity of the gamma FBG transcripts expressed in extrahepatic tissue, lung tissue was chosen as a model of extrahepatic gamma FBG gene expression. A ferret lung cDNA clone bank was constructed in lambda gt11 and several positive plaques were isolated using cross-species hybridization with the rat gamma FBG cDNA. Sequence data of the longest clone, designated pFLG gamma 3, was compared at the nucleotide and deduced amino acid (aa) levels with sequences of gamma FBG from other species. The results indicated that the identity of the ferret lung-specific gamma FBG cDNA to pig, rat, bovine and human gamma FBG cDNAs ranged from 78-88%; the similarity of the ferret lung-specific gamma FBG deduced aa sequence ranged from 84-88% across species. Cysteine aa involved in intra- and inter-chain disulfide-bonded secondary and tertiary structure are absolutely conserved in ferret gamma FBG. The putative cell-cell adhesion sites for both platelet alpha IIb beta 3 and intercellular adhesion molecule-1 receptor binding to ferret gamma FBG are > 90% similar to the corresponding sites in the human gamma FBG. The results of Northern hybridization indicated that the ferret lung gamma FBG mRNA was equivalent in size to the liver gamma FBG mRNA; Southern hybridization suggested that ferret gamma FBG is a single-copy gene, as is the gamma FBG of other species. Lung-specific gamma FBG expression was localized to epithelial cells of the large and small airways and chondrocytes by in situ RNA:RNA hybridization. The functional significance of gamma FBG expression in lung is not presently known. Since expression of FBG is up-regulated 2-10-fold in the liver during an inflammatory event, it is possible that lung-specific gamma FBG expression occurs predominantly during lung disease or injury.

Molecular characterization of KEX1, a kexin-like protease in mouse Pneumocystis carinii.

Expression screening of a Pneumocystis carinii-infected mouse lung cDNA library with specific monoclonal antibodies (mAbs) led to the identification of a P. carinii cDNA with extensive homology to subtilisin-like proteases, particularly fungal kexins and mammalian prohormone convertases. The 3.1 kb cDNA contains a single open reading frame encoding 1011 amino acids. Structural similarities to fungal kexins in the deduced primary amino acid sequence include a putative proenzyme domain delineated by a consensus autocatalytic cleavage site (Arg-Glu-Lys-Arg), conserved Asp, His, Asn and Ser residues in the putative catalytic domain, a hydrophobic transmembrane spanning domain, and a carboxy-terminal cytoplasmic domain with a conserved tyrosine motif thought to be important for localization of the protease in the endoplasmic reticulum and/or Golgi apparatus. Based on these structural similarities and the classification of P. carinii as a fungus, the protease was named KEX1. Southern blotting of mouse P. carinii chromosomes localized kex1 to a single chromosome of approximately 610 kb. Southern blotting of restriction enzyme digests of genomic DNA from P. carinii-infected mouse lung demonstrated that kex1 is a single copy gene. The function of kexins in other fungi suggests that KEX1 may be involved in the post-translational processing and maturation of other P. carinii proteins.

Fibrinogen binding potentiates FGF-2 but not VEGF induced expression of u-PA, u-PAR, and PAI-1 in endothelial cells.

Endothelial cell responses at sites of injury occur in a fibrin matrix and are regulated by growth factors including those of the FGF and VEGF families. The pericellular proteolytic balance is important in these responses, and FGF-2 and VEGF up-regulate endothelial cell u-PA, u-PAR and PAI-1. Because both VEGF and FGF-2 bind to fibrinogen, we have examined the capacity of fibrinogen to modulate the up-regulation of these proteins by FGF-2 and VEGF. Confluent cultures of endothelial cells were exposed to FGF-2, VEGF, and fibrinogen or to combinations of growth factors with fibrinogen. Changes in mRNA levels of u-PA, u-PAR and PAI-1 were measured by Northern blot. FGF-2 increased u-PA, u-PAR, and PAI-1 mRNA, but there was a significantly greater induction when fibrinogen was added to FGF-2 at all concentrations. The potentiation by fibrinogen was particularly evident at an FGF-2 concentration of 0.1 ng mL(-1), which resulted in non-significant change in transcript levels by itself, but significantly increased up to 2.6-fold with fibrinogen. VEGF also increased endothelial cell expression of u-PA, u-PAR and PAI-1, but this effect was not potentiated by fibrinogen. Addition of LM609, a monoclonal antibody to alphaVbeta3, significantly inhibited induction of u-PA mRNA and activity by fibrinogen-bound FGF-2 compared to FGF-2. A monoclonal antibody to FGFR1 also inhibited u-PA mRNA expression induced by fibrinogen-bound FGF-2. We conclude that fibrinogen increases the capacity of FGF-2, but not of VEGF, to up-regulate u-PA, u-PAR, and PAI-1 in endothelial cells and that fibrinogen-bound FGF-2 requires alphaVbeta3 binding to up-regulate endothelial cell u-PA.

Characterization of a monoclonal antibody, D73H, that maps to a highly conserved region on fibrinogen Bbeta chain.

The primary structure of fibrinogen is highly conserved across species, yet often times monoclonal antibodies produced against the fibrinogen of one species will not crossreact with the fibrinogen of another. Herein, we describe the production and characterization of murine MAb, D73H, raised against human fibrinogen. D73H crossreacts with a highly conserved epitope on the Bbeta chain of fibrinogen from human, rat, bovine, guinea pig, and mouse. Western blotting revealed that D73H reacted with the Bbeta chain of plasmin fragment D, localizing its epitope to Bbeta134-461. A 7 kDa band was identified by D73H in Western blots of reduced fibrinogen CNBr-fragments. N-terminal sequencing mapped this fragment to Bbeta243-253, further localizing the epitope to Bbeta243-305. In silico analysis indicated that Bbeta243-305 is predominantly hydrophilic, and surface probability prediction indicated three potential antigenic determinants corresponding to Bbeta252-258, Bbeta262-269, and Bbeta279-286. Further in silico analysis of the crystal structure of fibrinogen fragment D-D indicated that Bbeta262-269 (FGRKWDPY) is predominantly alpha-helical and located on the surface of the molecule adjacent to a bend imposed in the beta chain at residue 260, which is near the junction between the rigid coiled-coil domain and the globular C-terminus. A synthetic peptide corresponding to Bbeta261-272 competitively inhibited the binding of D73H to the Bbeta chain of denatured intact fibrinogen and reduced and denatured Bbeta chain in Western blots, experimentally proving the validity of these predictive algorithms. Together these data indicate that, although plasmin resistant, Bbeta chain residues Bbeta261-272 comprising the D73H epitope are highly conserved across species, surface exposed, and immunogenic.

Increased expression of plasminogen activator inhibitor-1 in R. rickettsii-infected endothelial cells.

Changes in PAI-1 expression in human umbilical vein endothelial cells (HUVEC) were studied following in vitro infection with Rickettsia rickettsii. A 1.8-fold increase in secreted PAI-1 activity occurred in infected versus control cultures (p = 0.03) at 24 h but not at earlier timepoints. A similar increase (1.4-fold) in secreted PAI-1 antigen (p < 0.005) was measured by ELISA. To determine whether this increase was due to increased synthesis of PAI-1, HUVEC were metabolically labeled with 35S-methionine concurrent with R. rickettsii infection. Such infection resulted in a 1.9-fold increase in labeled PAI-1 in the medium at 24 h (p = 0.036). Increase steady-state levels of PAI-1 mRNA were detected as early as 18 h by Northern blot analysis, peaking (5.5-fold) at approximately 24 h. These results indicate that PAI-1 production is increased in RR-infected endothelial cells, an effect that may contribute to the vascular occlusions noted in Rocky Mountain spotted fever.

Tumors and fibrinogen. The role of fibrinogen as an extracellular matrix protein.

The progression of a tumor from benign and localized to invasive and metastatic growth is the major cause of poor clinical outcome in cancer patients. Much like in a healing wound, the deposition of fibrin(ogen), along with other adhesive glycoproteins, into the extracellular matrix (ECM) serves as a scaffold to support binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during angiogenesis and tumor cell growth. Inappropriate synthesis and deposition of ECM constituents is linked to altered regulation of cell proliferation, leading to tumor cell growth and malignant transformation. Fibrin deposition occurs within the stroma of a majority of tumor types. In contrast, abundant FBG, not fibrin, is present within the stroma of breast cancers. It is thought to originate from exudation of plasma FBG and subsequent deposition into the tumor stroma and not endogenous synthesis and secretion of FBG by breast tumor cells. However, we show that MCF-7 human breast cancer cells synthesize and secrete FBG polypeptides, suggesting that the origin of FBG in the stroma of breast carcinoma may be due to endogenous synthesis and deposition. Moreover, FBG assembles into ECM as conformationally altered FBG, not as fibrin. Studies in our laboratory demonstrate that FBG alters the ability of breast cancer cells to migrate. Together, the results of studies from our laboratory, as well as the laboratories of others, indicate that the presence of fibrin(ogen) within the tumor stroma likely affects the progression of tumor cell growth and metastasis. This review focuses on FBG within tumors and its relationship with other tumor constituents, ultimately focusing on the role of FBG in breast cancer.

Fibrinogen modulates gene expression in wounded fibroblasts.

Fibrinogen (FBG) has long been regarded as serving essentially a hemostatic role by its conversion from a soluble, plasma protein to an insoluble fibrin gel. However, several extrahepatic sites of FBG biosynthesis have been identified. Indeed, we have demonstrated that both lung epithelial cell derived and plasma FBG assemble into the extracellular matrix (ECM) of epithelial cells and fibroblasts. In this report, we determined that FBG assembly into the ECM is a cell dependent step that occurs in the absence of de novo protein synthesis. Using an in vitro model of wound repair, we examined the role of FBG in modulating gene expression. Data collected from cDNA array analysis indicated that FBG downregulates steady state levels of fibronectin mRNA, whereas cyclin D1 mRNA levels were upregulated in fibroblasts. Taken together, these data suggest that FBG may function independently of hemostasis in cellular adhesive interactions to modulate cellular signaling processes during wound repair.

Antigenic properties of recombinant glycosylated and nonglycosylated Pneumocystis carinii glycoprotein A polypeptides expressed in baculovirus-infected insect cells.

Since a continuous culture system is not yet available for the opportunistic fungal pathogen Pneumocystis carinii, obtaining suitable amounts of purified P. carinii antigens free of mammalian-host lung contaminants is difficult. Hence, production of recombinant antigen possessing epitopes found in native P. carinii antigens is critical for immunological studies. We utilized the baculovirus expression vector system (BEVS) in insect cells to determine whether B-cell epitopes present in the protein core of a native P. carinii surface glycoprotein were conserved in the recombinant polypeptide, and to investigate its glycosylation by insect cells. B-cell epitopes were retained, but the insect cells appeared to hyperglycosylate the recombinant protein.

The charge-heterogeneity of human fibrinogen as investigated by 2D electrophoresis.

The charge-heterogeneity of human plasma fibrinogen subunit chains was characterized by two-dimensional electrophoresis (2DE). Western blotting with antibodies specific for the gamma-chain demonstrated that the gamma-chains focus at varying isoelectric points (pI). This microheterogeneity was also observed in fibrinogen secreted from hepatocytic cells and in recombinant fibrinogen expressed in Chinese hamster ovary (CHO) cells. Further, covalent gammagamma-dimerization by FXIIIa was not influenced by the charge-heterogeneity, and removal of the carbohydrate did not reduce the number of gamma-chain pI variants. These observations suggest that the microheterogeneity of the gamma-chain is a multifactorial phenomenon that is not due to physiologic modification of the glycoprotein in circulation.

Fibrinogen naples I (B beta A68T) nonsubstrate thrombin-binding capacities.

Fibrinogen Naples I (Bbeta A68T) is characterized by defective thrombin binding and fibrinopeptide cleavage at the fibrinogen substrate site in the E domain. We evaluated the fibrinogen of three homozygotic members of this kindred (II.1, II.2, II.3) who have displayed thrombophilic phenotypes and two heterozygotic subjects (I.1, I.2) who were asymptomatic. Electron microscopy of Naples I fibrin networks showed relatively wide fiber bundles, probably due to slowed fibrin assembly secondary to delayed fibrinopeptide release. We evaluated 125I-thrombin binding to the fibrin from subjects I.1, I.2, II.1, and II.2 by Scatchard analysis with emphasis on the high-affinity site in the D domain of fibrin(ogen) molecules containing a gamma chain variant termed gamma'. Homozygotic subjects II.1 and II.2 showed virtually absent low-affinity binding, consistent with the Bbeta A68T mutation, whereas heterozygotes I.1 and I.2 showed only moderately reduced low-affinity binding. The homozygotes also showed impaired high-affinity thrombin binding, whereas that of the heterozygotes was nearly the same as normal. Genomic sequencing of the gamma' coding sequence (I.2, II.2), ELISA measurements of two gamma' chain epitopes (L2B, gamma'409-412, and IF10, gamma'417-427) (I.2, II.1, II.2, II.3), and mass spectrometry of Naples I fibrinogen (II.2) showed no differences from normal, thus indicating that there were no abnormal structural modifications of the gamma' chain residues in Naples I fibrinogen. However, thrombin reportedly utilizes both of its available exosites for binding to high- and low-affinity sites on normal fibrin, suggesting that binding is cooperative. Thus, reduced high-affinity thrombin binding to homozygotic Naples I fibrin may be related to the absence of low-affinity binding sites.