Expression and intracellular localization of the human N-acetylmuramyl-L-alanine amidase, a bacterial cell wall-degrading enzyme

N-acetylmuramyl-L-alanine amidase (NAMLAA) specifically degrades peptidoglycan, which is a major component of bacterial cell walls with strong inflammatory properties. For instance, peptidoglycan is capable of stimulating peripheral blood cells to release pro-inflammatory cytokines and is capable of inducing chronic arthritis in an animal model. In a previous study we found that degradation of peptidoglycan by purified NAMLAA reduced its inflammatory effects. To determine where NAMLAA is located in tissues, monoclonal antibodies against purified NAMLAA were produced for use in immunohistochemistry, immunoelectron microscopy, flow cytometric analysis, and Western blotting. The immunohistochemical studies showed NAMLAA-positive cells in human spleen, liver, arthritic synovial tissues, and lymph nodes. In flow cytometric studies of blood and bone marrow, neutrophilic and eosinophilic granulocytes proved to be positive. Monocytes were negative, although they do contain lysozyme, the other important peptidoglycan-degrading enzyme. However, mature macrophages obtained by bronchoalveolar lavage and subsequent selection based on autofluorescence did possess NAMLAA. In immunocytochemical staining of blood smears, thrombocytes were also positive for NAMLAA. Western blot analysis and immunoelectron microscopy of neutrophils and eosinophils showed that NAMLAA is located in azurophilic granules of neutrophils and in secretory vesicles and crystalloid-containing granules of eosinophils. Flow cytometric analysis of blood and bone marrow from different French-American-British-classified acute myeloid leukemia (AML) patients showed that AML-M2 myeloblasts were the first in the granulocyte maturation lineage that were positive for NAMLAA. The more immature AML, such as AML-M0 and AML-M1, did not express NAMLAA. CD15- and CD13-negative megakaryoblasts, corresponding to AML-M7, were also positive for NAMLAA. The expression pattern of NAMLAA in the myeloid lineage suggests that the monoclonal antibody AAA4, recognizing NAMLAA, is useful for discrimination between AML in the monocyte lineage and in the granulocyte lineage.     

Effects of no precursor and donor on neuronal activity of rat hippocampal slices

Human N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) degrades peptidoglycan, a major component of bacterial cell walls with potent pro-inflammatory cytokine-inducing properties. We postulate that degradation of peptidoglycan by N-acetylmuramyl-L-alanine amidase is important for the inactivation of inflammatory peptidoglycan products in human tissues. The inflammatory activities of peptidoglycan digested by lysozyme and/or amidase were investigated using two properties of peptidoglycan: its capacity to induce the release of the inflammatory cytokines IL-1, IL-6 and TNF-alpha in vivo and in vitro and its capacity to induce arthritis in Lewis rats. The results show that after subsequent treatment with both lysozyme and amidase, the peptidoglycan products were unable to induce arthritis in Lewis rats. The production of pro-inflammatory cytokines in mice after intravenous injection of cell wall fragments was lower after in vitro degradation of the cell wall fragments by amidase. These in vivo results were confirmed with whole blood assays in which the production of pro-inflammatory cytokines was measured after stimulation with lysozyme- and amidase-treated peptidoglycan. The results show that human N-acetylmuramyl-L-alanine amidase possesses an enzymatic activity capable of inactivating inflammatory peptidoglycan by lowering its cytokine-inducing properties.     

Induction of tumour-suppressor phosphoprotein p53 in the apoptosis of cultured rat cerebellar neurones triggered by excitatory amino acids

We have isolated the collagenase/gelatinase activity of fibronectin from a bovine lens capsule hydrolysate, using heparin-agarose, gelatin-agarose, immunopurification with polyclonal antibodies directed against bovine plasma fibronectin, and immunopurification with a monoclonal antibody directed against the extra-domain A of cellular fibronectin. The expression of collagenase/gelatinase activity by the purified fibronectin fragment was dependent on the incubation time at 37 degrees C and the addition of gelatin to the purified sample. Under these conditions, the purified fibronectin fragment exhibited collagenase/gelatinase activity, as measured by means of gelatin zymography and the intramolecularly quenched fluorogenic substrate of collagenases (7-methoxycoumarin-4-yl)-acetylprolylleucylglycylleucyl-[3-(2,4-di nitrophenyl)-L-2,3-diaminopropionyl]-alanylarginylamide. This activity was due to proteins of 47 kDa and 37 kDa, as indicated by the gelatin-zymography pattern. When the processing was analyzed, by means of SDS/PAGE under reducing conditions, purified starting material of 66 kDa and 55 kDa was observed, and molecular masses of 45, 30 and 27 kDa were found for the processed samples. Under these conditions, the processing was more significant when a substrate, i.e the fluorogenic peptide or gelatin, was added to the processing mixture. An inhibition-profile study showed a zinc-dependent collagenase activity. Using the 45-kDa chymotryptic fragment from human plasma fibronectin, which contains the collagen-binding site, the same results were obtained. These results allow us to define a thiol-dependent zinc metalloproteinase expressed after limited proteolysis of both basement membrane and plasma fibronectins. This proteinase contains a collagen-binding domain, a zinc-binding sequence, and a cysteine involved in catalysis. This enzyme is a member of the thimet family of zinc metalloproteinases.     

Structural characterization of the cyanelle peptidoglycan of Cyanophora paradoxa by 252Cf plasma desorption mass spectrometry and fast atom bombardment/tandem mass spectrometry

A strategy for the structural characterization of the four major NaBH4-reduced peptidoglycan monomers derived from muramidase-digested peptidoglycan from the cyanelles of the flagellate Cyanophora paradoxa Korschikoff is described. Initial molecular weight determination of these glycopeptides was performed by positive and negative ion plasma desorption mass spectrometry. Due to the presence of two pairs of disaccharide tripeptide and disaccharide tetrapeptide monomers differing in mass by 112 units, respectively, an as yet unknown peptidoglycan modification either at the carbohydrate or at the peptide moiety was assumed. beta-Elimination of the disaccharide unit from the unreduced peptidoglycan monomers yielded the corresponding (modified) N1-lactyltripeptides and -tetrapeptides, respectively. These peptides, N-terminally blocked with lactic acid, unambiguously showed the modification to be located on the peptide moiety. By positive ion fast atom bombardment/hybrid tandem mass spectrometry of the reduced peptidoglycan monomers as well as of the corresponding deglycosylated monomers (= N1-lactylpeptides) the modification was determined to be linked to the glutamic acid moiety. Based on combined data from plasma desorption mass spectrometry, tandem mass spectrometry, accurate mass measurement and amino acid analysis of the acid hydrolysate after derivatization with o-phthaldialdehyde by high-performance liquid chromatography we could establish the structure of the modification as N-acetylputrescine. Finally, the confirmation of the linkage of the glutamic acid to diaminopimelic acid via the gamma-COOH was based on the presence of a-type peptide backbone fragment ions in the positive ion plasma desorption mass spectra of the modified N1-lactylpeptides.     

Familial Creutzfeldt-Jakob disease. Codon 200 prion disease in Libyan Jews

alpha 2-antiplasmin, a plasma glycoprotein of the serpin superfamily, is the primary physiological inhibitor of plasmin, the key enzyme in fibrin degradation. Previous purification methods utilize lengthy multistep protocols with low yields or use monoclonal antibodies that are expensive or difficult to make. With a relatively small investment, a chicken was immunized with keyhole limpet hemocyanin-conjugated to alpha 2-antiplasmin C-terminal 26 residue synthetic peptide and the peptide-specific antibody (IgY) was isolated from the egg yolks of hens using the peptide affinity column. Based on the interaction between this IgY and alpha 2-antiplasmin, pure alpha 2-antiplasmin was isolated from human plasma in two steps: (a) citrated plasma was precipitated with 15% PEG-8000 to remove the bulk of plasma proteins while retaining the majority of alpha 2-antiplasmin activity; and (b) the alpha 2-antiplasmin was affinity-purified from the supernatant using the IgY column. Yields were typically 48% and the purity and authenticity of the alpha 2-antiplasmin were verified by gel electrophoresis, Western Blot analysis, N-terminal sequence, and amino acid analysis.     

Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

Five monoclonal antibodies (mAbs) that recognize human glutamate dehydrogenase (GDH) have been selected and designated as monoclonal antibodies hGDH60-6, hGDH60-8, hGDH63-10, hGDH63-11, and hGDH91-14. A total of five mAbs recognizing different epitopes of the enzyme were obtained, two of which inhibited human GDH activity. When total proteins of human homogenate separated by SDS- PAGE, were probed with mAbs, a single reactive protein band of 55 kDa, which co-migrated with purified recombinant human GDH was detected. When the purified GDH was incubated with each of the mAbs, its enzyme activity was inhibited by up to 58%. Epitope mapping analysis identified, two subgroups of mAbs recognizing different peptide fragments. Using the individual anti-GDH antibodies as probes, the cross reactivities of brain GDH obtained from human and other animal brain tissues were investigated. For the human and animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 55 kDa when hGHD60-6, hGHD60-8, or hGHD91-14 mAbs were used as probes. However, the anti-human GDH mAbs immunoreactive to bands on Western blots reacted differently on the immunoblots of the other animal brains tested, i.e., the two monoclonal antibodies hGDH63-10 and hGDH63-11 only produced positive results for human. These results suggest that human brain GDH is immunologically distinct from those of other mammalian brains. Thorough characterization of these anti-human GDH mAbs could provide potentially valuable tool as immunodiagnostic reagents for the detection, identification and characterization of the various neurological diseases related to the GDH enzyme.     

Evaluation of sperm damage and techniques for sperm clean-up

In this study we describe the partial purification and characterization of the HeLa cell oligopeptidase M or endopeptidase 3.4.24.16. The HeLa enzyme was isolated initially by its ability to hydrolyse a nonapeptide substrate (P9) which was cognate to the N-terminal cleavage site of preproTGF alpha. The enzyme was shown to be a metalloprotease as it was inhibited by Zn(2+)-chelating agents and DTT, and had an approximate molecular weight of 55-63 kD determined by gel filtration. Neurotensin, dynorphin A1-17 and GnRH1-9 were rapidly degraded by the enzyme while GnRH1-10 and somatostatin were not. Neurotensin was cleaved at the Pro10-Tyr11 bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). The K(m) for neurotensin cleavage was 7 microM and the Ki for the specific 24.16 dipeptide inhibitor (Pro-ile) was 140 microM which were similar to those observed from the human brain enzyme [Vincent et al. (1996): Brain Res 709:51-58]. Through the use of specific antibodies, the purified HeLa enzyme was shown to be oligopeptidase M. This enzyme and its closely related family member thimet oligopeptidase were shown to co-elute during the isolation procedure but were finally separated using a MonoQ column. Oligopeptidase M is located mainly in mitochondria though it was detected on the plasma membrane in an inactive form. The results obtained demonstrate the first recorded instance of this enzyme in human tissue cultured cells, and raise the issue of its function therein.     

Significance of IgG for the activity of factor XII measured in human plasma

The plasma level of factor XII (FXII) was measured in samples from healthy young men. The activated contact factor was assayed as prekallikrein activator (PKA), as S-2222 amidase, and in radial immunodiffusion tests. By removing the bulk of IgG on protein G columns before the activation procedure, the functional activities increased to about 135%. In such test preparations, PAGE immunoblot experiments with polyclonal antibodies against FXII showed, in addition to FXIIa (80 kD), a double band with a molecular weight of about 46 kD. This protein could also be detected with a light-chain-specific monoclonal antibody to FXII, but not with such an antibody directed against its heavy chain. The 46-kD band was also observed in plasma deficient in FXII. The amidase assays indicated that the minor part of FXIIa was present in some kind of association with another protease. To obtain a correct estimation of total FXIIa in the amidase assays a sufficiently high level of FXI was required compared to that of FXII. The PKA assays were generally carried out with a prekallikrein (PK) substrate containing IgG. By replacing this substrate by PK free from IgG additional PKA activity was observed, the activity appearing also in plasma deficient in FXII.     

Cocaine and butyrylcholinesterase (BChE): determination of enzymatic parameters

In humans, the plasma enzyme, butyrylcholinesterase (E.C. 3.1.1.8), metabolizes cocaine to the water-soluble, pharmacologically inactive compounds, ecgonine methylester and benzoic acid. Homogeneous enzyme was purified from human plasma and used to determine the enzyme kinetic parameters of Km and Vmax with cocaine as the substrate. The KM (11.9 microM) indicates that cocaine is tightly bound to the four active sites of the native tetramer. The Vmax (1.17 microM/min) is 50-fold greater than cocaine catalytic antibodies. Administration of purified human butyrylcholinesterase to a cocaine-intoxicated patient would be expected to shift the metabolism to the inactive metabolites and reduce the toxicity.     

Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.     

Tissue fibronectin is an endogenous ligand for galectin-1

A 14K beta-galactoside-binding lectin (galectin-1) is present in many animal tissues. In a search for endogenous ligands, we surveyed galectin-1-binding proteins in human placenta. Extract of human placenta with 2 M urea was applied to a Sepharose 4B column conjugated with galectin-1 purified from frog (Rana catesbeiana) eggs. Two major proteins eluted with 100 mM lactose from the column-bound fraction showed apparent molecular masses of 220 and 180 kDa on SDS-PAGE under reducing conditions. Western blotting analysis using monoclonal antibodies indicated that these proteins were fibronectin and laminin, respectively. Most placental and amniotic fibronectins bound strongly to the column, whereas almost all plasma fibronectin passed through the column. The galectin-1, fibronectin and laminin were immunohistochemically shown to be co-localized in the extracellular matrix of placental tissue. In a cell attachment assay, rhabdosarcoma cells adhered to a plate coated with placental fibronectin, even in the presence of GRGDS peptide, if galectin-1 were also present. This adhesive effect of galectin-1 was inhibited by lactose. These results indicate that tissue fibronectin, as well as laminin, serve as endogenous ligands for galectin-1, suggesting that galectin-1 may play a role in assembly of the extracellular matrix, or in the control of cell adhesion based on lectin-extracellular matrix interaction.     

A new peptide-N4-(acetyl-beta-glucosaminyl)asparagine amidase from soybean (Glycine max) seeds: purification and substrate specificity

We report here the isolation and characterization of a peptide-N4-(acetyl-beta-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the beta-aspartylglycosylamine linkage (GlcNAc beta 1-->Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.     

Atherosclerotic disease in patients undergoing cataract extraction. A nationwide case-control study. The Cataract Patient Outcomes Research Team

The antitumor activity of cytostatic 5'-deoxy-5-fluorouridine (5'-dFUrd) depends on its being converted to 5-fluorouracil (5-FUra) by the enzyme thymidine phosphorylase (dThdPase, EC 2.42.4). We prepared mouse anti-human dThdPase monoclonal antibodies to serve as tools for clinical studies with this drug. Partially purified dThdPase obtained form HCT116 human colon cancer cells grown in athymic mice was used as and antigen for the immunization of mice. Six hybridomas were cloned which produced anti-human dThdPase antibodies, as detected by Western blot analysis with human dThdPase. With these antibodies, we developed an ELISA method sensitive enough to measure dThdPase levels, even in tumor tissue samples weighing as little as 10 mg. In addition, one monoclonal antibody was suitable for immunologically staining the enzyme in tumor tissues. Thus, these anti-human dThdPase monoclonal antibodies could be used to measure levels of the enzyme in tumor cells, which is essential for the activation of 5'-dFUrd.     

Purification and characterization of endogenous digoxin-like immunoreactive factors in chicken blood

Studies have been performed to determine whether an endogenous material capable of binding to digoxin antibodies is present in the chicken plasma. In the blood of 12 chickens without feed control, endogenous digoxin-like immunoreactive factors (DLIF) binding of digoxin antibodies in enzyme immunoassays amounted to 866 / 302 pg digoxin equivalents/mL of plasma (mean +/- SEM). Immunoreactivity of DLIF increased to 1848***331 pg/mL with a double value of control after boiling and acid pretreating the plasma. The major purification steps employed in this report were gel filtration column chromatography, high performance liquid chromatography (HPLC) and isoelectric focusing (IEF). Using HPLC for the separation, at least 10 chicken DLIFs with different molecular weight (MW) have been found. The MW of the smallest is 300 daltons (Da) while the largest is 100 kDa. The value of the isoelectric point of the most abundant type of DLIF from untreated chicken plasma is 6.3 as determined by IEF. The partially purified DLIF inhibits Na+, K(+)-ATPase from a porcine cerebral cortex as well as three human red blood cell membrane preparations in a dose-response fashion.     

Purification and characterization of a rat hepatic acetyltransferase that can metabolize aromatic amine derivatives

Rat liver cytosol is capable of N-acetylation of arylamines, O-acetylation of arylhydroxylamines and N,O-acyltransfer of arylhydroxamic acids. The objective of this study was to characterize the enzyme(s) responsible for these reactions. A partially purified acetyltransferase preparation from rat liver cytosol was used to produce five mouse monoclonal IgG1S that bound to acetyltransferase on Western blots and affected one or more of the acetylation reactions. Two immunoaffinity columns were prepared by covalently cross-linking monoclonal antibodies to protein A-Sepharose. The first column permitted recovery of a single, immunoreactive 32 kDa protein that was capable of catalyzing all three reactions, while the second removed all three acetylation activities from a partially purified enzyme preparation and yielded a single, immunoreactive 32 kDa protein on elution. The harsh conditions necessary for elution from the latter column precluded recovery of an active enzyme. Although Western blots from SDS-PAGE at all stages of purification showed a single 32 kDa protein, purification was associated with the production of multiple, immunochemically reactive peptides with higher pIs. Direct enzymatic assays of these immunochemically reactive components after isoelectric focusing on polyacrylamide gels demonstrated that a single 32 kDa, pI 4.5 protein is capable of all three cytosolic acetylation activities. A second 32 kDa protein, pI 4.8, was able to carry out N-acetylation but not N,O-acetyltransfer. Immunoreactive components with pIs > 4.8 that were formed during purification were catalytically inactive. However, isoelectric focusing in solution of cytosolic preparations that had been subjected only to gel filtration gave a single 32 kDa immunoreactive peptide that was capable of all three acetylation reactions. Buffer concentration differentially affected the enzymatic activities of the enzyme, i.e. as a pH 7.4 buffer was decreased from 50 mM sodium pyrophosphate to 2 mM, the ability to N-acetylate arylamines was lowered while the abilities for O-acetylation and N,O-acetyltransfer were unaffected. It has been shown that a single 32 kDa protein carries out all of the acetylation reactions in rat liver cytosol. Although it cannot be ruled out that other similarly sized and closely related enzymes that share antigenic sites are also capable of these acetylation reactions, these studies suggest that instabilities of the major peptide responsible for these activities, as reflected in changes in isoelectric point, may be responsible for changes in the enzymatic potentials of this peptide.     

Relationship between phase variation in colony morphology, intrastrain variation in cell wall physiology, and nasopharyngeal colonization by Streptococcus pneumoniae

Streptococcus pneumoniae undergoes phase variation in colony morphology, which has been implicated as a factor in the pathogenesis of pneumococcal disease. Phenotypic differences between opaque and transparent colony forms correlate with differences in rates of autolysis. This study examined whether differences in autolysis are caused by differences in expression of the major amidase, LytA, or the structure of its peptidoglycan substrate. No significant difference was detected by high-pressure liquid chromatography analysis of stem peptides released after treatment of purified peptidoglycan with amidase. Differences in the rate of digestion of purified cell walls, furthermore, did not correlate with susceptibility to autolysis. Lower levels of autolysis in opaque variants, however, was associated with decreased levels of immunodetectable LytA on colony immunoblots and Western blots (immunoblots). Diminished cell-surface-associated LytA in opaque variants was also demonstrated by whole-cell inhibition enzyme-linked immunosorbent assay. Since transparent variants have been shown both to colonize the nasopharynx more efficiently in an animal model and to express more surface-exposed LytA, it was determined whether LytA contributes to colonization in a neonatal rat model of pneumococcal carriage. Defined mutants in the lytA gene were used to show that there was no significant contribution by LytA to nasopharyngeal colonization in this model. Although the expression of LytA was shown to undergo phase variation in association with colony morphology, lytA mutants are still capable of phenotypic variation in colony morphology, which suggests that other factors are responsible for intrastrain differences which affect colonization.     

Dipeptidyl peptidase IV from porcine seminal plasma: purification, characterization, and N-terminal amino acid sequence

Dipeptidyl peptidase IV (DPP IV) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approximately 290,000 on PAGE in the absence of sodium dodecyl sulfate (SDS) and 310,000 on Sephacryl S-300 HR column chromatography, and to be 115,000 and 105,000 on SDS-PAGE in the absence and presence of beta-mercaptoethanol. The enzyme is suggested to be composed of three identical subunits. The enzyme rapidly hydrolyzed the substrate Gly-Pro-MCA, and weakly the substrate Lys-Ala-MCA. It was strongly inhibited by diisopropylphosphofluoridate (DFP), and moderately by both phenylmethyl-sulfonyl fluoride (PMSF) and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). It was also strongly inhibited by zinc ion. The amino acid sequence of the first 18 residues of the enzyme was Asn-Lys-Gly-Thr-Asp-Asp-Ala-Ala-Ala-Asp-Ser-Arg-Arg- Thr-Tyr-Thr-Leu-Thr-. This sequence was highly homologous to the sequences in the rear of the transmembrane site of human and rat liver DPP IVs and mouse thymus DPP IV. The native DPP IV is suggested to be released into the seminal plasma after the cleavage of the hydrophobic N-terminal domain by chymotrypsin-like or pepsin-like enzymes. Other properties of DPP IV including kinetic parameters, pH stability and heat stability were characterized.     

Both cellular and soluble forms of thrombomodulin inhibit fibrinolysis by potentiating the activation of thrombin-activable fibrinolysis inhibitor

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity. The enzyme, TAFIa, potently attentuates fibrinolysis. TAFI activation, like protein C activation, is augmented about 1250-fold by thrombomodulin (TM). In this work, the effects of both soluble and cellular forms of TM on TAFI activation-dependent suppression of fibrinolysis were investigated. Soluble TM included in clots formed from purified components, barium citrate-adsorbed plasma, or normal human plasma maximally increased the tissue plasminogen activator-induced lysis time 2-3-fold, with saturation occurring at 5, 10, and 1 nM TM in the three respective systems. Soluble TM did not effect lysis in the system of purified components lacking TAFI or in plasmas immunodepleted of TAFI. In addition, the antifibrinolytic effect of TM was negated by monoclonal antibodies against either TAFI or TM. The inhibition of fibrinolysis by cellular TM was assessed by forming clots in dialyzed, barium citrate-adsorbed, or normal plasma over cultured human umbilical vein endothelial cells (HUVECs). Tissue plasminogen activator-induced lysis time was increased 2-fold, with both plasmas, in the presence of HUVECs. The antifibrinolytic effect of HUVECs was abolished 66% by specific anti-TAFI or anti-TM monoclonal antibodies. A newly developed functional assay demonstrated that HUVECs potentiate the thrombin-catalyzed, TM-dependent formation of activated TAFI. Thus, endothelial cell TM, in vitro at least, appears to participate in the regulation of not only coagulation but also fibrinolysis.     

Production of monoclonal antibodies to prostromelysin (ProMMP-3) and establishment of a quantitative prostromelysin ELISA assay

Human prostromelysin (59 kDa) was purified from the conditioned medium of IL-1-stimulated human dermal fibroblasts and anti-prostromelysin monoclonal antibodies were produced and identified by ELISA assay. Using prostromelysin, a C-terminally truncated recombinant form of prostromelysin consisting of amino acids 1-255, and their respective activated enzymes, we have begun mapping the epitopes recognized by these monoclonal antibodies. Various patterns of reactivity against the proenzymes and activated enzymes were observed. In further attempts to map the epitopes, we employed synthetic peptides representing hydrophilic regions of the primary amino acid sequence of prostromelysin. Our monoclonal antibodies did not recognize these peptides, suggesting that the antibodies may be recognizing conformational epitopes composed of non-linear portions of prostromelysin. Using these monoclonal antibodies, we have developed a quantitative prostromelysin sandwich ELISA assay.     

Mapping and serodiagnostic application of a dominant epitope within the human herpesvirus 8 ORF 65-encoded protein

A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi's sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8.