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Biomedical subjects

I Theodor

Publications and source records attributed to I Theodor.

At least 19 recordsLinked to original sources
PubMed2001

Immunization with the Chlamydia trachomatis mouse pneumonitis major outer membrane protein can elicit a protective immune response against a genital challenge.

Infertility, ectopic pregnancy, and chronic abdominal pain are frequent complications of genital infections with Chlamydia trachomatis. In an attempt to produce a vaccine to protect against this pathogen we purified and refolded the C. trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP). This preparation, mixed with Freund's adjuvant using vortexing or sonication, was used to immunize BALB/c mice that were subsequently challenged in the upper genital tract. Vaginal cultures were taken on a weekly basis, and mice were mated 6 weeks after the challenge. Gels of the vortexed MOMP showed a predominant band with a molecular size of 62 kDa and weaker bands at 42 and 132 kDa, while the sonicated MOMP had a single band with a molecular size of 42 kDa. Following immunization with these two preparations, strong humoral and cell-mediated immune responses were detected in the mice inoculated with the vortexed MOMP. On the other hand, mice immunized with the sonicated MOMP had a strong humoral immune response but a relatively weak cell-mediated immune response, as determined by a T-cell lymphoproliferative assay and level of cytokine production by splenocytes. Vaginal cultures showed that the mice immunized with the vortexed MOMP were significantly protected, as determined by a decrease in the number of animals with positive cultures, the length of time the mice shed viable organisms, and the number of inclusion-forming units recovered per mouse. Animals immunized with the sonicated MOMP, on the other hand, showed a weaker level of protection based on the same three parameters. After mating, the number of fertile animals and number of embryos per mouse were significantly higher for the mice immunized with vortexed MOMP, but not for the mice immunized with sonicated MOMP, compared to those of the control groups. In conclusion, immunization with a purified and refolded preparation of the C. trachomatis MoPn MOMP confers a significant level of protection in mice against a genital challenge.

Animals↗
PubMed1999

Crotalase, a fibrinogen-clotting snake venom enzyme: primary structure and evidence for a fibrinogen recognition exosite different from thrombin.

Crotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence [see text] characteristic of a serine proteinase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin's fibrinogen-recognition exosite (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibitor, had no effect on crotalase. Spatial modeling of crotalase yielded a possible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85, Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also lacks thrombin's YPPW loop, as well as its functionally important ETW 146-148, and its heparin-binding site. The enzyme contains a single asparagine-linked glycosylation site, NFT, bearing neutral and amino sugars that account for 8.3% of the enzyme's total molecular weight of 29,027. The calculated absorbance of crotalase at 280 nm, 1%, cm(-1) is 15.2.

Amino Acid Sequence↗
PubMed1997

Monoclonal immunoglobulin A antibody to the major outer membrane protein of the Chlamydia trachomatis mouse pneumonitis biovar protects mice against a chlamydial genital challenge.

In order to analyze the protective role that IgA may play in a chlamydial infection two IgA monoclonal antibodies (mAb), MoPn 4-2 and MoPn 13-2, were raised against the major outer membrane protein (MOMP) of the Chlamydia trachomatis mouse pneumonitis (MoPn) biovar. mAb MoPn 4-2 was found to be serovar specific while mAb MoPn 13-2 was species specific. mAb MoPn 4-2 recognized a surface exposed conformational epitope as shown by its ability to bind to native EBs and nonreduced MOMP while failing to bind to heat and trypsin treated EBs, to reduced MOMP and to synthetic MOMP peptides. In contrast, mAb MoPn 13-2 recognized a nonconformational epitope since it was able to bind treated EBs, to reduced MOMP and to the synthetic peptide MTTWNPTISGSGI located in variable domain 4 of the MOMP. Both mAbs agglutinated intact EBs and had in vitro neutralizing activity. However, mAb MoPn 4-2 had a 20-fold higher in vitro neutralizing ability when compared to mAb MoPn 13-2 (50% neutralization at 5 micrograms ml-1 vs 100 micrograms ml-1). In an in vitro in vivo infectivity assay, mAb MoPn 4-2 protected mice against infertility when C. trachomatis MoPn elementary bodies were preincubated with the mAb before inoculation. In addition, passive transfer of mAb MoPn 4-2 resulted in significant protection as measured by a decrease in the number of mice infected, and in the intensity and duration of vaginal shedding. These results support previous findings suggesting that IgA antibodies can play a role in protection against a chlamydial infection, and further encourage work to develop vaccination strategies that elicit mucosal immunity.

Alveolitis, Extrinsic Allergic↗
PubMed1997

Immunization with an acellular vaccine consisting of the outer membrane complex of Chlamydia trachomatis induces protection against a genital challenge.

The ability to induce protection against a genital challenge was studied in BALB/c female mice with three Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) preparations as well as an acellular vaccine consisting of the chlamydial outer membrane complex (COMC). The MOMP preparations were extracted with three different types of detergents, sodium dodecyl sulfate (SDS), n-octyl-beta-D-glucopyranoside (OGP), and Zwittergent 3-14 (Z3-14). A positive immunization control consisted of mice inoculated intranasally with 10(4) C. trachomatis MoPn inclusion-forming units (IFU). Mice inoculated with ovalbumin served as a negative control. Furthermore, a sham-immunized, nonchallenged group was included as a fertility control. Two weeks after the last immunization, the mice were challenged in the left ovarian bursa with 10(5) C. trachomatis MoPn IFU. Vaginal swabs were collected for culture, vaginal and serum samples were assayed for chlamydial-specific antibodies, and splenocytes were collected to determine the lymphoproliferative response. At 42 days after the challenge, the mice were mated with proven male breeder mice. Animals that were considered to be pregnant (as determined by weight) were killed, and the embryos were counted. A significant humoral and cell-mediated immune response was observed in all the groups of mice inoculated with chlamydial antigens. Antibodies to variable domain (VD)1 of the MOMP were detected in serum samples from all the immunized groups. However, antibodies to VD3 and VD4 were detected only in the groups immunized with the Z3-14-MOMP and the COMC. Mice immunized with COMC developed significant immunoglobulin A chlamydia-specific antibodies in the vagina, while mice immunized with the detergent-extracted MOMPs had low antibody titers. Following the intrabursal challenge, a significant decrease in the intensity and duration of vaginal shedding was noted in the mice immunized with COMC and a moderate decrease was noted in the group immunized with OGP-MOMP. No protection against the infection was noted in the groups of animals immunized with SDS- and Z3-14-MOMP. Furthermore, of the mice immunized with the COMC preparation, only 25% (4 of 20) shed C. trachomatis, as determined by vaginal culture, while 83% (40 of 48) of the control mice inoculated with ovalbumin were culture positive (P < 0.05). In addition, after mating, the mice inoculated with COMC were found to have fertility rates comparable to those of the control sham-immunized, nonchallenged animals (70% [14 of 20] versus 81% [17 of 21], respectively [P > 0.05]), and there were no significant differences between the average number of embryos per mouse in the two groups (5.1 versus 5.9, respectively [P > 0.05]). In contrast, mice immunized with the purified MOMP preparations were not protected against infertility. In summary, a preparation of the COMC protected mice against infection and infertility, supporting the feasibility of the development of an acellular vaccine against C. trachomatis infections.

Animals↗
PubMed1996

Role of neutrophils in controlling early stages of a Chlamydia trachomatis infection.

Inoculation of mice with the granulocyte-depleting monoclonal antibody RB6-8C5 showed that neutrophils are critical protective effector cells during a Chlamydia trachomatis infection. In addition, administration of monoclonal antibody 2E6 demonstrated that extravasation of neutrophils into the peritoneal cavity in response to inoculation with the C. trachomatis MoPn biovar is dependent on the surface beta-2 integrin molecule CD18.

Animals↗
PubMed1990

Isoforms of a highly specific B beta-chain fibrinogenase from the venom of Crotalus atrox: preliminary observations.

Three variants of a Crotalus atrox venom enzyme that cleaves primarily the Arg42 - Ala bond of the B beta-chain of human fibrinogen were separated by high-performance liquid chromatography and were shown by N-terminal sequence analysis to be amino acid isoforms. All are single-chain glycoproteins whose approximate molecular weights lie between 26,000 and 28,000. The isoenzymes also cleaved the A alpha-chain at Arg491 - His, though much more weakly, with strengths for both bonds that varied among the isoforms.

Amino Acid Sequence↗
PubMed1989

Catroxobin, a weakly thrombin-like enzyme from the venom of Crotalus atrox. NH2-terminal and active site amino acid sequences.

Two thrombin-like isoenzymes, termed catroxobins, were purified by gel filtration and ion exchange chromatography to electrophoretic homogeneity from the venom of the Western diamondback rattlesnake, Crotalus atrox. By SDS-polyacrylamide gel electrophoresis their molecular weights were estimated to be 25,000 and 26,200. A 43-residue NH2-terminal sequence, containing the active histidine residue, was the same for the two isoenzymes. In addition, a 33-residue internal peptide from catroxobin I contained a normal active serine sequence. These sequences were highly homologous to other thrombin-like venom enzymes, and to pancreatic kallikrein and trypsin, but less so to the B chain of thrombin. Catroxobin, possessing 89 TAME esterase units/mg of protein, clotted human fibrinogen very slowly, releasing fibrinopeptide A and a small amount of fibrinopeptide B. No other evidence of cleavage of the fibrinogen molecule was revealed by polyacrylamide gel electrophoresis or HPLC.

Amino Acid Sequence↗
PubMed1989

On the location in the thrombin B chain of substrate recognition sites for fibrinopeptide release and factor XIII activation.

Thrombin, a serine proteinase comprised of two disulfide-linked subunits (A chain and B chain), induces clotting by releasing fibrinopeptide A from fibrinogen and then influences the character of the resulting fibrin by releasing fibrinopeptide B and by activating factor XIII. While the active center of thrombin is known to reside in its B chain, the subunit location of the structural determinants that govern the specific release of fibrinopeptides A and B and the activation of factor XIII have not been established. We have investigated the subunit location within the thrombin molecule of the determinants of substrate specificity for these actions using an isolated, immobilized B-chain preparation. Isolated B chain was prepared by covalently linking the intact thrombin molecule to Sepharose beads via the carbohydrate chain attached to asparagine 53 of its B chain, then reducing the single interchain disulfide bond to release the A chain, and finally reoxidizing the intrachain disulfide bonds of the immobilized B chain, allowing it to refold. The isolated, immobilized B chain of thrombin induced clotting of purified fibrinogen, releasing both fibrinopeptides A and B as demonstrated by HPLC and by electrophoresis of reduced fibrin chains. In addition, the B-chain preparation activated added factor XIII, yielding electrophoretically characteristic cross-linked fibrin chains.

Animals↗
PubMed1986

Thrombin-like enzyme from the venom of Bitis gabonica. Purification, properties, and coagulant actions.

Gabonase, an enzyme which acts on fibrinogen and factor XIII in uniquely thrombin-like ways, was purified to electrophoretic homogeneity from the venom of Bitis gabonica. On sodium dodecyl sulfate-polyacrylamide electrophoresis, the reduced protein behaved as a single chain with Mr = 30,600. The enzyme contains 20.6% carbohydrate, no free sulfhydryl groups and hence, from amino acid analysis, five disulfide bonds. Its extinction coefficient (E1%1cm) at 280 nm is 9.6. Its pI is 5.3. Gabonase has an active serine residue, is inactivated by phenylmethanesulfonyl fluoride, and has an active histidine which reacts with the chloromethyl ketone of tosyl-L-lysine. Its NH2-terminal amino acid sequence (Val-Val-Gly-Gly-Ala-Glu-Cys-Lys-Ile-Asp-Gly-His-Arg-Cys-Leu-Ala-Leu-Leu -Tyr-) is homologous to the B chain of thrombin. The activity of the enzyme is stabilized by calcium ion. It exhibits strong N alpha-p-tosyl-L-arginine methyl esterase activity, hydrolyzes tripeptide nitroanilide derivatives weakly or not at all, and cleaves no peptide bonds in insulin, glucagon, or the S peptide of ribonuclease. Gabonase clots fibrinogen with a specific activity of 45 NIH thrombin-equivalent units/mg, releasing both fibrinopeptides A and B and showing substrate inhibition at fibrinogen concentrations of 3 mg/ml or greater. The enzyme also activates factor XIII. It is not inactivated by either heparin or hirudin.

Amino Acid Sequence↗
PubMed1985

Action of crotalase, an enzyme with thrombin-like and kallikrein-like specificities, on tripeptide nitroanilide derivatives.

Since crotalase has thrombin-like and kallikrein-like functional and structural properties, we compared the actions of crotalase, thrombin and plasma kallikrein on 13 tripeptide nitroanilide substrates. Initial rates of hydrolysis were determined at 27 degrees C, pH 8.3, and used to construct Lineweaver-Burk plots from which Km and Vmax were determined. The ratio of kcat/Km was taken as a measure of enzymatic specificity. Crotalase yielded kcat/Km values for the following nitroanilide substrates in descending order of magnitude: H-D-NLeu-CHA-Arg, H-D-Pro-HHT-Arg, Tos-Gly-Pro-Arg, H-D-PhGly-Phe-Arg, Cbo-Glu(BuO)-Gly-Arg, H-D-But-CHA-Lys, H-D-CHG-But-Arg, H-D-NLeu-HHT-Lys, H-D-HHT-Ala-Arg, Bz-Pro-Phe-Arg, Tos-Gly-Pro-Lys, MeS-Leu-Gly-Arg, MeO-CO-CHG-Gly-Arg. This pattern of specificity correlated only roughly with those of thrombin and kallikrein.

Chromogenic Compounds↗
PubMed1983

Fibrinogen Seattle releases half the normal amount of fibrinopeptide B.

Fibrinogen Seattle, a clinically silent, slow-clotting dysfibrinogen, releases 50% of the normal amount of fibrinopeptide B as assessed by amino acid analysis. The reduced dysfibrin exhibited equal quantities of chains with B beta- and beta-charge mobility on polyacrylamide gel electrophoresis in 2 M urea at low pH. By these same techniques, the release of fibrinopeptide A was normal. Clots formed by repolymerizing the thrombin and batroxobin dysfibrin monomers showed a maximal turbidity that was lower than normal. Fibrinogen Seattle was indistinguishable from normal fibrinogen by radial immunodiffusion and immunoelectrophoresis. Degradation by plasmin and transamination by factor XIIIa were normal. The characteristics of fibrinopeptide release by fibrinogen Seattle distinguish it from other reported dysfibrinogens.

Amino Acids↗
PubMed1982

Kallikrein-like activity of crotalase, a snake venom enzyme that clots fibrinogen.

During the amino acid sequence determination of crotalase (EC 3.4.21.30), the thrombin-like enzyme from the venom of Crotalus adamanteus (eastern diamondback rattlesnake), we found that, in addition to the expected structural homology with bovine thrombin (EC 3.4.21.5), there was even greater homology with porcine pancreatic kallikrein (EC 3.4.21.8). In exploring further the similarities between crotalase and kallikrein, several striking observations were made. First, crotalase was rapidly and specifically inhibited by the tripeptide affinity labeling derivative prolylphenylalanylarginine chloromethyl ketone, which is known to be a specific inhibitor of kallikrein. Second, NaDodSO4/acrylamide gel electrophoresis revealed that crotalase cleaves the plasma kallikrein-susceptible bonds in human high molecular weight kininogen, producing an intermediate with procoagulant activity. Crotalase-catalyzed cleavage of high molecular weight kininogen also liberates kinin as evidenced by rat blood pressure bioassay. Finally, crotalase exhibits substrate specificity not only for the thrombin chromogenic substrate S-2238 but also for the kallikrein substrates S-2302 and S-2266. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen, was not one of the activities exhibited by crotalase.

Animals↗