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PubMed1995

Internucleosomal DNA fragmentation in ovine luteal tissue associated with luteolysis: in vivo and in vitro analyses.

Internucleosomal DNA fragmentation, a characteristic of apoptosis, can be visualized with agarose gel electrophoresis as discrete low-molecular-weight DNA fragments (laddering), in multiples of approximately 185 bp. CL were collected from superovulated ewes (control) or at 12 h after injection of prostaglandin F2 alpha (PGF2 alpha) on various days after hCG injection. The ability of PGF2 alpha on Days 8, 10, 12, and 14 (n > or = 3 per day per treatment) to induce luteal cell DNA fragmentation was evaluated. DNA was isolated and visualized on agarose gels. No DNA fragmentation was observed in CL from control ewes on Days 8, 10, or 12. Internucleosomal fragmentation of DNA (indicative of apoptosis) as well as nonspecific DNA fragmentation (indicative of non-apoptotic cell death) in CL from Day 14 controls was observed in two of four animals. Additionally, this pattern of DNA fragmentation was observed in CL from ewes treated with PGF2 alpha on all days. Evidence of DNA fragmentation was observed in luteal tissue after dissociation, yet no fragmentation was observed in unsliced, non-dissociated CL collected from Day 10 control ewes (incubated 4 h), or in sliced, non-incubated CL. Slicing and incubation alone were sufficient to initiate DNA fragmentation. A variety of approaches were utilized to inhibit DNA fragmentation. Only the addition of zinc acetate (1 mM) in the incubation medium throughout the 4-h incubation period prevented DNA fragmentation that was initiated by slicing (p < 0.05). There appear therefore, to be one or more intraluteal factors that directly initiate DNA fragmentation associated with cell death in luteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Mapping of neutralizing epitopes to fragments of the bovine coronavirus E2 protein by proteolysis of antigen-antibody complexes.

Neutralizing antigenic domains on bovine coronavirus gp100/E2 were mapped to fragments of this protein by proteolytic cleavage and fragment analysis. The procedure involved analysis of fragments generated after incubation of E2-monoclonal antibody complexes with various proteases. The smallest antibody-bound fragments obtained were a 50K fragment following Staphylococcus aureus V8 protease and submaxillary protease digestion, and a 37K fragment following trypsin digestion. Trypsin also produced a transient antibody-bound 50K fragment. A 40K fragment which was not bound by antibody was observed following digestions with all three proteases. The 50K fragments generated by V8, submaxillary protease and trypsin comigrated on gels and displayed the same altered mobility under non-reducing conditions, suggesting identity of these fragments and indicating the presence of disulphide linkages in these fragments. The 40K fragments generated by these three enzymes also comigrated and displayed the same altered mobility under non-reducing conditions. The 37K trypsin fragment contained both neutralizing domains, A and B.

Animals↗
PubMed1991

Fibrinogen degradation product fragment D increases endothelial monolayer permeability.

We assessed the effects of the two primary high-molecular-weight fibrinogen degradation products (FDP), fragments D and E, on the pulmonary vascular endothelial barrier function. Fragments D and E were purified to homogeneity by QAE Sephadex chromatography followed by gel filtration. Incubation of bovine pulmonary artery endothelial monolayers with 0.5-2.0 microM fragment D for 2 h caused a doubling of transendothelial 125I-albumin clearance rate (a measure of 125I-albumin permeability). Fragment E only produced a 0.6-fold increase in 125I-albumin clearance rate at concentration of 4.0 microM. Both FDP remained active in incubating media with serum. The permeability-increasing effect of fragment D was reversible and was not due to cell detachment or lysis. The fragment-D effect was time dependent and was associated with redistribution of endothelial F-actin microfilaments. The effect was independent of the carboxy-terminal sequence on gamma-chain of fragment D. Fragments D and E binding to pulmonary artery endothelial cells was specific and reversible, but fragment D binding was three-fold greater than fragment E, which may account for the greater permeability increase mediated by fragment D. The results indicate that FDP, especially fragment D, increase endothelial permeability to albumin. The response involves specific binding of fragment D to endothelial cells and redistribution of intracellular actin.

Actins↗
PubMed1993

Different modes of fragmenting gallstones in extracorporeal shockwave lithotripsy.

Forty radiolucent gallbladder stones from eight patients were fragmented in vitro by extracorporeal shockwave lithotripsy, using the electromagnetic lithotripter Lithostar Plus (Siemens) at five different energy levels. The stones were characterized by size, computed tomography (CT) density, and cholesterol content. The largest residual fragment was measured after every 20 to 100 shock waves. As expected, fewer shock waves were required to achieve fragmentation at higher energy levels. When stones of the same size were compared, there were remarkable differences in the number of shock waves required for fragmentation. These differences must originate in other properties of the stones than size and number. Two different modes of fragmentation were observed: in one group of stones small, flat fragments were chipped off at the beginning of fragmentation ('chipping mode'). These stones initially lost about 25% of their weight as small fragments (< 1 mm) before breaking centrally into some large fragments. In the other group stones initially lost only about 10% of their weight as small fragments (< 1 mm) at the beginning of fragmentation and early broke centrally into some large fragments ('breaking mode'). Stones showing the chipping mode were almost pure cholesterol stones (> 97%) and required significantly less shock waves than stones of the same size showing the breaking mode (cholesterol content, 64-94%). This mode of fragmentation could not be predicted by CT density.

Cholelithiasis↗
PubMed1979

Metabolism of Okazaki fragments during simian virus 40 DNA replication.

Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one of three classes. Class I Okazaki fragments (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and were quantitatively identified by treating purified replicating DNA with Escherichia coli DNA ligase and then measuring the fraction of Okazaki fragments joined to longer nascent DNA chains. Similarly, class II Okazaki fragments (about 30%) were separated by a region of single-stranded DNA template (gap) that could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase, and class III fragments (about 50%) were separated by RNA primers that could be removed with E. coli DNA olymerase I, allowing the fragments to be joined with E. coli DNA ligase. These results were obtained with replicating SV40 DNA that had been briefly labeled with radioactive precursors in either intact cells or isolated nuclei. When isolated nuclei were further incubated in the presence of cytosol, all of the Okazaki fragments were converted into longer DNA strands as expected for intermediates in DNA synthesis. However, when washed nuclei were incubated in the abscence of cytosol, both class I and class II Okazaki fragments accumulated despite the excision of RNA primers: class III Okazaki fragments and RNA-DNA covalent linkages both disappeared at similar rates. These data demonstrate the existence of RNA primers in whole cells as well as in isolated nuclei, and identify a unique gap-filling step that is not simply an extension of the DNA chain elongation process concomitant with the excision of RNA primers. One or more factos found in cytosol, in addition to DNA polymerase alpha, are specifically involved in the gap-filling and ligation steps. The sizes of mature Okazaki fragments (class I) and Okazaki fragments whose synthesis was completed by T4 DNA polymerase were measured by gel electrophoresis and found to be broadly distributed between 40 and 290 nucleotides with an average length of 135 nucleotides. Since 80% and 90% of the Okazaments does not occur at uniformly spaced intervals along the DNA template. During the excision of RNA primers, nascent DNA chains with a single ribonucleotide covalently attached to the 5' terminus were identified as transient intermediates. These intermediates accumulated during excision of RNA primers in the presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki fragments blocked by RNA primers (class III) were found to have originated the farthest from the 5' ends of long nascent DNA strands. Thus, RNA primers appear to be excised in two steps with the second step, removal of the final ribonucleotide, being stimulated by concomitant DNA synthesis. These and other data were used to construct a comprehensive metabolic pathway for the initiation, elongation, and maturation of Okazaki fragments at mammalian DNA replication forks.

DNA Ligases↗
PubMed1979

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III.

The effect of prothrombin fragment 2 on the inhibition of thrombin by antithrombin III has been studied. Fragment 2 was found to slow the rate of inhibition of thrombin by antithrombin III about 3-fold. The effect of prothrombin fragment 2 on antithrombin III inhibition was examined by comparing its action in the presence of either thrombin or meizothrombin (des fragment 1). The second order rate constants for antithrombin III inhibition of thrombin with saturating fragment 2 and antithrombin III inhibition of meizothrombin (des fragment 1) were the same. Prothrombin fragment 2 had no effect on either antithrombin III inhibition of meizothrombin (des fragment 1) or Factor Xa. The effect of the fragment on the reaction mechanism of thrombin inhibition was evaluated to see if the fragment altered binding of antithrombin III to thrombin or inhibited the formation of the covalent complex. The fragment was found to have no inhibitory effect on the rate of covalent complex formation, indicating that the protective effect of the fragment is by inhibiting binding of antithrombin III to thrombin. These data suggest that prothrombin fragment 2 may be an important factor in controlling the localization of clot formation by regulating the interaction between thrombin and antithrombin III.

Animals↗
PubMed2005

The Fc fragment mediates the uptake of immunoglobulin C from the midgut to hemolymph in the ixodid tick Amblyomma americanum (Acari: Ixodidae).

The phenomenon of immunoglobulin uptake from the midgut into the hemolymph has been observed in both argasid and ixodid ticks, but the mechanism of the uptake is not known. Using capillary feeding of adult Amblyomma americanum (L.) female ticks, we investigated the role of immunoglobulin G Fc and Fab fragments in the uptake process. Intact IgG and fragments labeled with 125I were introduced alone or as a mixture with a 10-fold molar excess of unlabeled compounds into ticks, and then the concentration of the compounds after 3 and 6 h of continuous feeding was assessed in the hemolymph. Our results showed that the uptake of Fc fragments was approximately 4 times higher than Fab fragments at 6 h of the feeding and that Fc but not Fab fragments competed with uptake of intact IgG. Glycosylation of the immunoglobulins did not seem to be required for uptake. There was not a statistically significant difference between the uptake of untreated IgG and IgG treated either with periodate or N-glycanase, and untreated Fc fragments or deglycosylated Fc fragments had equal activity in competition experiments with IgG. The uptake into hemolymph of IgG and Fc fragments, but not Fab fragments, was dependent on the pH of the feeding solution and showed a maximum at approximately pH 7.0. In addition, tick midgut cells bound Fc fragments with high affinity, estimated at 21 nM; the interaction with Fab fragments seemed nonspecific. Our results suggest that Fc fragments are the major determinant for the specific uptake of immunoglobulin G into tick hemolymph.

Animals↗
PubMed1998

Stimulation of inducible nitric oxide synthase in rat liver by hyaluronan fragments.

Hepatic injury and chronic wounding are characterized by increased synthesis of extracellular matrix proteins including hyaluronan (HA). Recently, it has been recognized that low-molecular-weight fragments of HA, but not native HA (e.g., high-molecular-weight HA), induce inflammatory gene expression, and activate the transcriptional regulator, nuclear factor kappaB (NF-kappaB). The inducible isoform of nitric oxide synthase (iNOS) is induced by cytokines and/or lipopolysaccharide (LPS) through the NF-kappaB signal transduction pathway. Because of this association, we hypothesized that HA fragments might also stimulate iNOS gene transcription. The aims of this study were therefore to determine whether HA or HA fragments induced iNOS in hepatic cells, and to characterize the signaling pathway. HA fragments (100 microg/mL) markedly stimulated iNOS messenger RNA (mRNA) in endothelial and Kupffer cells, but minimally induced this mRNA in hepatocytes and stellate cells. High-molecular-weight HA (200 microg/mL) had no effect on iNOS mRNA in any cell type. The addition of interferon gamma (IFN-gamma) to HA fragments resulted in stimulation of iNOS mRNA 2-, 3-, 4-, and 10-fold above that for HA fragments alone in hepatocytes, endothelial, Kupffer, and stellate cells, respectively. The combination of HA fragments and LPS did not result in an incremental increase in iNOS mRNA induction. iNOS protein and nitrite levels (used as a measure of NO production and NOS enzymatic activity) paralleled closely iNOS mRNA expression and increased proportionally to HA fragment concentration in a dose-dependent fashion. At 1 hour following stimulation, NF-kappaB DNA binding activity was detected in extracts from Kupffer cells stimulated with HA fragments, but not in those exposed to media alone or to high-molecular-weight HA. Finally, inhibitors of NF-kappaB blocked HA fragment-dependent iNOS mRNA induction in Kupffer and sinusoidal endothelial cells. The data indicate that HA fragments, but not high-molecular-weight HA, induce iNOS in liver, having the greatest effects on endothelial and Kupffer cells. We speculate that HA fragments may be an important stimulus for NO production in various forms of liver disease, particularly as a cofactor with inflammatory cytokines.

Animals↗
PubMed1993

Botulinum type A neurotoxin digested with pepsin yields 132, 97, 72, 45, 42, and 18 kD fragments.

Botulinum neurotoxin (NT) serotype A is a dichain protein made of a light and a heavy chain linked by at least one interchain disulfide; based on SDS-polyacrylamide gel electrophoresis their molecular masses appear as 147, 52, and 93 kD, respectively. Digestion of the NT with pepsin under controlled pH (4.3 and 6.0), time (1 and 24 hr), and temperature (25 and 30 degrees C) produced 132, 97, 42, and 18 kD fragments. The three larger fragments were isolated by ion-exchange chromatography. The 132 and 97 kD fragments are composed of 52 kD light chain and 72 and 45 kD fragments of the heavy chain, respectively. The sequences of amino terminal residues of these fragments were determined to identify the pepsin cleavage sites in the NT, which based on nucleotide sequence has 1295 amino acid residues (Binz et al., J. Biol. Chem. 265, 9153, 1990). The 42 kD fragment, beginning with residue 866, is the C-terminal half of the heavy chain. The 18 kD fragment, of which the first 72 residues were identified beginning with residue 1147, represents the C-terminal segment of the heavy chain. The 132 kD fragment (residue 1 to approximately 1146) is thus a truncated version of the NT without its 18 kD C-terminal segment. The 97 kD fragment (residue 1 to approximately 865) is also a truncated NT with its 42 kD C-terminal segment excised. These peptic fragments contain one or two of the three functional domains of the NT (binds receptors, forms channels, and intracellularly inhibits exocytosis of the neurotransmitter) that can be used for structure-function studies of the NT. This report also demonstrates for the first time that of the six Cys residues 453, 790, 966, 1059, 1234, and 1279 located in the heavy chain the later four do not form interchain disulfide links with the light chain; however, Cys 1234 and 1279 contained within the 18 kD fragment form intrachain disulfide. The electrophoretic behaviors of type A NT and its fragments in native gels and their comparison with botulinum NT serotypes B and E as well as tetanus NT suggest that each NT forms dimers or other aggregates and the aggregation does not occur when the 42 kD C-terminal half of the heavy chain is excised. Thus, the C-terminal half of the heavy chain appears important in the self-association to form dimers.

Amino Acid Sequence↗
PubMed1988

Uptake of polypeptide fragments of proteins by rat intestine in vitro and in vivo.

Minute amounts of intact proteins were previously shown to be taken up from the intestine into the systemic circulation of mature animals; fragments were not detected. In this study, we sought evidence for uptake of fragments in both in vitro and in vivo experiments. Polypeptide fragments produced by pepsin digestion of bovine serum albumin (ranging in molecular weight from approximately 6000 to 25,000) were labeled with 125I. Everted jejunal gut sacs prepared from rat intestine were incubated with labeled fragments. After incubation, fluid exposed to the serosal surface was applied to a Sephadex G-50 gel permeation column. Radioactivity was detected in fractions corresponding to the elution position of the fragments. Transfer of fragments from the mucosal to the serosal surface was temperature-dependent. In in vivo studies, labeled fragments were infused into the jejunum of rats. Blood samples obtained from a mesenteric vein or the portal vein contained labeled fragments. After infusion of unlabeled fragments, nanogram amounts of immunoreactive fragments were detected by radioimmunoassay of mesenteric and portal venous blood. Thus, polypeptide fragments of a potential food protein were capable of being transferred across the mucosa in vitro and in vivo. Failure to detect fragments in the systemic circulation most likely results from their rapid clearance.

Animals↗
PubMed1995

Biodistribution and tumor localization of 111In-labeled unmodified and modified F(ab')2 fragments of human monoclonal IgM (16.88) in a nude mouse model.

Unmodified F(ab')2 and modified Fab'-BMH-Fab' fragments of human monoclonal IgM (16.88) were compared for biodistribution and tumor localization in nude mice bearing LS-174T human colon carcinoma xenografts. Although both unmodified and modified fragments of IgM cleared rapidly from the blood, the radioactivity retentions for each fragment in liver and kidney were significantly different. Kidney uptake of the modified fragment was about 4-fold lower than kidney uptake of the unmodified fragment. Radioactivity uptake in liver was 2-4-fold higher for the modified fragment. Lower liver and higher kidney uptake of unmodified fragments reflected the labile disulfide linkage of F(ab')2 in their hinge region and the subsequent behavior of the Fab' fragments resulting from the reduction of the disulfide linkage. Higher liver and lower kidney retention of modified fragments, on the other hand, resulted from the different cleavage mechanism of the stable thioether linkage. Tumor targeting was similar for unmodified and modified fragments at approx. 4% of injected dose per gram. These results indicate that the changes in fragment linkage chemistry may provide different pharmacokinetic patterns in vivo and improve the therapeutic application of radiolabeled fragments in human patients.

Animals↗
PubMed1990

Identification and characterization of a 43-kilodalton laminin fragment from the "A" chain (long arm) with high-affinity heparin binding and mammary epithelial cell adhesion-spreading activities.

A recently described procedure of reduction and carboxymethylation followed by heparin-Sepharose chromatography [Arumugham et al. (1988) Connect. Tissue Res. 18, 135-147] was used to characterize high-affinity heparin binding fragments of the laminin "A" chain. Two laminin fragments of Mr 53K and 43K selectively bound to the heparin-Sepharose column from the chymotrypsin digest of laminin, indicating that these fragments originate from the "A" chain. Without reduction and carboxymethylation but in the presence of 2.0 M urea, the heparin-Sepharose-bound material from the chymotrypsin laminin digest contains all the attachment-promoting activity for normal mouse mammary epithelial cells. The reduced 200-kDa intact three short arm fragment, fragments of Mr 70K-160K obtained either from laminin or from the reduced 200-kDa three short arm fragment, and the 53-kDa heparin binding fragment were all inactive in promoting the adhesion of mouse mammary epithelial cells. The mammary epithelial cell adhesion and spreading properties of laminin are associated with the high-affinity heparin binding 43-kDa fragment. The mammary epithelial cells attach to the 43-kDa fragment substrate and synthesize laminin, collagen type IV, and desmoplankins I and II as are the cells attached to laminin substrate and to the cells grown on tissue culture dishes. The biologically active 43-kDa fragment is generated from laminin, but not from the three short arm fragment. These results suggest that normal mouse mammary epithelial cells interact with laminin through a single site which is present in the 43-kDa heparin binding fragment located on the long arm of the "A" chain.

Animals↗
PubMed1995

Intracellular A beta 1-42 aggregates stimulate the accumulation of stable, insoluble amyloidogenic fragments of the amyloid precursor protein in transfected cells.

We have analyzed the effect of internalized amyloid beta-protein (A beta) 1-42 aggregates on the metabolism of the amyloid precursor protein (APP) in stably transfected 293 cells. The amount of potentially amyloidogenic fragments of APP immunoprecipitated by anti-carboxyl-terminal APP and anti-A beta antibodies is dramatically enhanced by the treatment of the cells with A beta 1-42, which is resistant to degradation, but not A beta 1-28, which does not accumulate in cells. This accumulation of amyloidogenic carboxyl-terminal fragments is specific, since there is relatively little effect of A beta 1-42 on the amount of the nonamyloidogenic alpha-secretase carboxyl-terminal fragment. The amyloidogenic fragments accumulate in the same nonionic detergent-insoluble fraction of the cell that contains the internalized A beta 1-42. Western analysis indicates that a subset of the amyloidogenic fragments react with antibodies that recognize a conformation of A beta that is specifically associated with aggregated forms of A beta, suggesting that the adoption of this aggregation-related conformation may be an early event which precedes the final processing that produces A beta. Pulse-chase analysis of the [35S]Met-labeled 16-kDa amyloidogenic fragment indicates that it is relatively stable in A beta 1-42-treated cells, with a half-life of approximately 50 h. This fragment is degraded with a half-life of 30 min in control cells treated with A beta 1-28. In contrast, the turnover of the nonamyloidogenic alpha-secretase product is not significantly altered by the presence of A beta 1-42. The continuous uptake of A beta 1-42 from the medium is not required for the stimulation of amyloidogenic fragment accumulation, suggesting that the presence of intracellular A beta 1-42 aggregates establishes a new pathway for APP catabolism in cells which leads to the long term stability of the fragments. If these amyloidogenic fragments of APP ultimately give rise to A beta, then the production of A beta may be an autocatalytic, "runaway" process in cells containing A beta 1-42 nuclei. It is conceivable that the accumulation of insoluble APP and amyloidogenic fragments of APP in response to A beta 1-42 aggregates may mimic the pathophysiology of dystrophic neurites, where the accumulation of intracellular APP and APP fragments has been documented by immunohistochemistry.

Amyloid↗
PubMed1976

Limited hydrolysis of the polypeptide chain elongation factor Tu by trypsin. Isolation and characterization of the polypeptide fragments.

The digestion of EF-Tu-GDP (or EF-Tu-GTP) by trypsin [EC 3.4.21.4] under native conditions has been shown to proceed through two different and characteristic stages. 1. In the first phase, the protein is transformed into a fragment (Fragment A) with a molecular weight of 39,000 by exposure to trypsin for a relatively short period of time. Fragment A is unable to catalyze the binding of aminoacyl-tRNA to ribosomes. The ability to promote two partial steps of the binding reaction, i.e., formation of the aminoacyl-tRNA-EF-Tu-GTP ternary complex as well as the methanol-stimulated, ribosome dependent GTPase reaction, was rapidly destroyed. On the other hand, the ability to interact with guanine nucleotides as well as EF-Ts survived well during prolonged digestion. 2. In the second phase of digestion, a nick is introduced in Fragment A to yield two subfragments (Fragments B and C). These two fragments exist as a hybrid molecule which migrates as a single peak on a Sephadex G-75 column, and which dissociates into Fragments B and C only in the presence of 6 M guanidine hydrochloride or 5% sodium dodecyl sulfate. The molecular weights of Fragments B and C, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, were 22,000 and 12,000 respectively. The hybrid molecule still retained one mole of bound guanine nucleotide and was resistant to further tryptic digestion. 3. Three sulfhydryl groups of EF-Tu were found to be present in Fragment B, both by amino acid analysis of the purified fragments and also by electrophoresis of tryptic digests labeled with N-ethyl[14C]maleimide. 4. The tryptic digestion of EF-Tu-GDP (or EF-Tu-GTP) labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) at SH2 (the second SH), caused a 30% decrease in the fluorescence emission during the first rapid phase of digestion. This indicates that destruction of the hydrophobic environment near SH2 of EF-Tu occurred in the early phase of tryptic digestion. 5. The kinetic studies on the reaction of ANM with EF-Tu before and after tryptic digestion indicated that both Fragment A and the hybrid molecule reacted with ANM in the presence of GTP three to four times more rapidly than in the presence of GDP. Thus, it appears that the ability to induce conformational transition near SH2 by a change of nucleotide ligands is still retained in the hybrid molecule consisting of Fragments B and C.

Amino Acids↗
PubMed1995

Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388.

Fragments of human thrombomodulin (TM) have been expressed in large quantities in the Pichia pastoris yeast expression system and purified to homogeneity. Fermentation of P. pastoris resulted in yields of 170 mg/l TM. Purification to homogeneity resulted in an overall 10% yield, so that quantities of approximately 20 mg purified fragments can be readily obtained. Smaller fragments of TM, such as the individual fourth or fifth domains, were not active, nor were equimolar mixtures of the two domains. These results demonstrate that the fourth and fifth epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM. The fragment containing the fourth and fifth EGF-like domains [TMEGF(4-5)] had 10% the specific activity of rabbit TM. Comparison of the M388L mutant TMEGF(4-5) fragment with the same mutant TMEGF(4-5-6) fragment showed that the fragment with the sixth domain had a 10-fold better Km value for thrombin than the fragment that did not contain the sixth domain; this factor completely accounts for the higher specific activity of the fragments containing the sixth domain. Comparison of the wild-type and M388L mutants showed that the M388L mutation resulted in a 2-fold increase in kcat for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the 2-fold increase in specific activity of these mutant fragments.

Amino Acid Sequence↗
PubMed1989

Identification of functional antigenic segments of toxic shock syndrome toxin 1 by differential immunoreactivity and by differential mitogenic responses of human peripheral blood mononuclear cells, using active toxin fragments.

When toxic shock syndrome toxin 1 was subjected to papain hydrolysis, two serologically active fragments of 16.3 kilodaltons (16K fragment) and 12.4 kilodaltons (12K fragment) were generated, whereas a third fragment of 9.7 kilodaltons (10K fragment) was inactive. The biologic activities of the fragments were evaluated in vitro by determining their ability to promote nonspecific proliferation of human peripheral blood mononuclear cells. The 12K fragment was significantly (P less than or equal to 0.013) more stimulatory than the 16K fragment. When human peripheral blood mononuclear cells were preincubated for a period of 24 h with various concentrations of the 16K fragment, followed by incubation with a constant amount (2 x 10(-2) ng/ml) of whole toxin, the level of DNA synthesis induced by the holotoxin was reduced by approximately 60% when compared with that of controls exposed to whole toxin alone. The 12K fragment did not demonstrate a similar blocking effect. Immunoblots of the toxic shock syndrome toxin 1 digest, which were exposed to monoclonal antibodies (MAbs) developed against native toxin, depicted the presence of two different antigenic regions (epitopes). One MAb, 8-5-7, which has been shown previously to inhibit the biologic activity of the holotoxin in vitro and in vivo, reacted primarily with the 12K fragment. A second MAb, 10-6-1, that did not neutralize interleukin-1 production reacted primarily with the 16K fragment. On the basis of the differential mitogenic responses and the identification of heterologous epitopes, it was concluded that the functional region of the holotoxin can be partitioned into at least two functional segments encompassed between amino acid residues 53 and 87 and between amino acid residues 88 and 194 on the polypeptide chain.

Antibodies, Bacterial↗
PubMed1998

Oral vaccination against tetanus: comparison of the immunogenicities of Salmonella strains expressing fragment C from the nirB and htrA promoters.

We have found the in vivo-regulated nirB promoter (PnirB) to be effective for directing expression of a number of antigens in salmonella in vivo. We wished to determine if other in vivo-regulated promoters have utility for antigen expression in salmonella and to compare the effectiveness of these promoters with that of PnirB. To this end, we have devised a scheme that allows the promoter element of the PnirB-fragment C plasmid pTETnir15 to be swapped with other promoters of interest. We demonstrate the usefulness of this system by replacing PnirB with PhtrA to create plasmid pTEThtrA1. htrA is a stress response gene that is required for virulence of salmonella in mice and survival within macrophages. Expression of fragment C in Salmonella typhimurium BRD509 (aroA aroD) harboring pTEThtrA1 (strain BRD937) correlated with growth temperature in vitro. A comparison was made of the immune responses to fragment C elicited in mice immunized orally with BRD937 or BRD847 (BRD509/pTETnir15) or subcutaneously with purified fragment C plus alhydrogel. High levels of anti-fragment C antibodies that persisted for at least 12 weeks were present in all groups of mice. Vaccination with BRD937 was the most effective means of immunization: the serum immunoglobulin G (IgG), IgA, and IgM anti-fragment C titers were higher in the BRD937-immunized mice throughout the duration of the study than in mice in the other groups. The kinetics of the serum anti-fragment C responses were different in different groups. The response was most rapid in the BRD937 group, with the titers almost at peak levels at 2 weeks postimmunization. Only the mice immunized with BRD937 or BRD847 developed an intestinal IgA response to fragment C. Again, the response was superior in the BRD937 group. The peak of the intestinal response was delayed with respect to the serum response. Analysis of the IgG subtype response to fragment C revealed a dominant IgG2a response in the salmonella-immunized mice, indicating a type 1 helper T-cell response to fragment C, whereas the major subtype in the group parenterally immunized with fragment C plus alhydrogel was IgG1. The IgG1/IgG2a ratio was much higher in sera of BRD937-immunized mice than in sera of BRD847-immunized mice. At 15 to 20 weeks after immunization, the mice immunized with BRD937 or BRD847 were solidly immune to tetanus toxin and salmonella. The immune responses to fragment C seen in mice immunized with BRD937 are the strongest we have observed and indicate that the htrA promoter may be very useful for expressing foreign antigens in salmonella vaccine strains.

Administration, Oral↗
PubMed1975

Immune responses to the cleavage-associated neoantigens of fibrinogen in man. Identification and characterization of humoral antibodies specific for cleavage fragments.

Cleavage of human fibrinogen and fibrin by plasmin is associated with modification of native antigenic expression and the exposure of cleavage-associated neoantigenic sites on the derivative molecular fragments. In this study, the presence of humoral antibodies in man to cleavage-associated neoantigens has been demonstrated by primary antigen binding radioimmunochemical assays. Specific binding of radioiodinated human fibrinogen D fragment by serum immunoglobulins was demonstrated in 52 of 59 random normal human sera and was independent of immunoglobulin concentration. Binding was mediated by F (ab)2 fragments of IgG, and specificity for neoantigens was indicated by the capacity of the D fragment but not native fibrinogen to competitively inhibit the antibody. The population distribution of antibody to these cleavage-associated neoantigens indicated the presence of a major group of individuals (77%) with a mean antigen binding capacity of 11.8 pmol/ml serum. Two minor populations with : (a) low or undetectable binding capacities (less than 6.0 pmol/ml serum) and (b) exhibiting markedly elevated binding capacities (less 18.0 pmol/ml serum) were delineated. Independent of these features, sera could also be readily separated into two groups that differed with respect to relative antibody affinity. The antibodies in most sera exhibited marked heterogeneity of binding affinity, whereas a small group of sera contained antibodies exhibiting relative homogeneity of binding affinity. Specific antibody was rather equally distributed between the major immunoglobulin classes, and in no serum was the antibody restricted to a single immunoglobulin class. Antibodies capable of binding fibrinogen fragments X, Y, and D and fibrin D fragment were detected in most sera. The quantity of antibody differed for different fragments with X greater than Y congruent to D greater than fibrin D. The presence of antibody capable of binding any single fragment was statistically correlated with the presence of antibody capable of binding other cleavage fragments. No antibody to the E fragment was detected. Antibody to cleavage fragments was not demonstrable in sera containing fibrinogen or fibrin cleavage fragments. Demonstration of this humoral immune response to the products of the fibrinolytic systems provides a new interface between the coagulation and immune system.

Antibodies↗