Localization and characterization of a specific linear epitope of the Brucella DnaK protein

We have previously produced and characterized four monoclonal antibodies to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction. By use of a recombinant DNA technique, we have localized a linear epitope, recognized by two of these monoclonal antibodies (V78/07B01/G11 and V78/09D04/D08), in the last 21 amino acids of the C-terminal region of the Brucella DnaK protein. The C-terminal region has been reported to be the most variable region among DnaK proteins. The two other monoclonal antibodies (A53/09G03/D02 and A53/01C10/A10) failed to react with the recombinant clones and might recognize discontinuous epitopes of the Brucella DnaK protein. The four monoclonal antibodies reacted with all recognized Brucella species and biovars in immunoblotting after SDS-PAGE. Monoclonal antibodies V78/07B01/G11 and V78/09D04/D08 did not react with reported cross-reacting bacteria nor with bacteria of the α-2 subdivision of the class Proteobacteria for which a close genetic relationship with Brucella spp. has been reported. However, monoclonal antibodies A53/09G03/D02 and A53/01C10/A10 reacted with Phyllobacterium rubiacearum and/or Ochrobactrum anthropi, both bacteria of the α-2 subdivision of the class Proteobacteria. The Brucella genus DnaK specific epitopes could be of importance for diagnostic purposes.     

Characterization of monoclonal antibodies against human protein C specific for the calcium ion-induced conformation or for the activation peptide region

Three monoclonal antibodies have been produced that are specific for the activation peptide region in human protein C. These antibodies inhibited the activation of protein C by thrombin and by the thrombin-thrombomodulin complex. A fourth monoclonal antibody specifically recognized the Ca2+-stabilized conformation in protein C. This antibody bound both intact protein C and protein C from which the gamma-carboxyglutamic acid-containing region had been removed by limited proteolysis. These results indicate that this antibody recognizes the conformation in protein C stabilized by Ca2+ bound to the single binding site that is independent of gamma-carboxyglutamic acid.     

Monoclonal antibodies for human thrombomodulin which recognize binding sites for thrombin and protein C

Monoclonal antibodies for human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C, were prepared and their epitopes characterized. All six antibodies (MFTM-1-MFTM-6) bound to an elastase-digested active fragment of thrombomodulin, which contains six consecutive EGF domains. Binding of thrombomodulin to these antibodies did not depend on Ca2+ concentration. MFTM-4, MFTM-5, and MFTM-6 strongly inhibited protein C activation by thrombin and thrombomodulin. MFTM-4 and MFTM-5 inhibited thrombin binding to fixed thrombomodulin and bound to a recombinant mutant EGF456 protein, which contained the fourth, fifth, and sixth EGF domains of thrombomodulin. However, MFTM-6 did not inhibit thrombin binding to thrombomodulin and did not bind to EGF456 protein. Binding of thrombomodulin to fixed MFTM-4 or MFTM-5 was competitively inhibited by a recombinant mutant EGF45 protein which contained the fifth and sixth EGF-domains. These results suggest that epitopes of MFTM-4 and MFTM-5 are located in the fifth EGF domain of thrombomodulin. Thus, the binding site for thrombin is located in the fifth EGF domain. These results also suggest that an epitope for MFTM-6 is located at a region near the binding site for gamma-carboxyglutamic acid residues of protein C via Ca2+ on thrombomodulin.     

Heterogeneity of tubulin epitopes in mouse fetal tissues

A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves. Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

Heterogeneity of tubulin epitopes in mouse fetal tissues

Summary A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves.Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

Monoclonal antibodies to human protein C: effects on the biological activity of activated protein C and the thrombin-catalyzed activation of protein C1

Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the gamma-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzyme activity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibody-binding sites on the protein C molecule.     

Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design

Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8⁺ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8⁺ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97–99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including ²⁰⁰NS5²¹⁰ (94.84%, A*11:01), ⁵¹⁵NS5⁵²⁵ (98.71%, A*24:02), ²²⁵NS5²³² (99.35%, A*33:03), ⁵¹⁶NS5⁵²³ (98.71%, A*33:03), and ²⁸⁴NS5²⁹¹ (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including ²³⁴NS5²⁴¹ (96.77%, B*13:01), ⁹²NS5⁹⁹ (98.06%, B*15:01, B*15:02, and B*46:01), ²⁶²NS5²⁶⁹ (92.90%, B*38:02), and ⁵³⁸NS5⁵⁴⁷ (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including ⁵¹⁴NS5⁵²² (98.71%, C*01:02), ⁵¹⁴NS5⁵²⁴ (98.71%, C*01:02 and C*14:02), ⁹²NS5⁹⁹ (98.06%, C*03:02 and C*15:02), ³⁶²NS5³⁶⁹ (44.84%, C*03:04 and C*08:01), ²²⁵NS5²³² (99.35%, C*04:01), ²³⁴NS5²⁴¹(96.77%, C*04:01), ³⁶¹NS5³⁶⁹ (94.84%, C*04:01), ⁵¹⁵NS5⁵²² (98.71%, C*14:02), ⁵¹⁵NS5⁵²⁴ (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of ⁹²NS5⁹⁹ (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), ²⁰⁰NS5²¹⁰ (A*11:01), ³⁶²NS5³⁶⁹ (C*03:04, C*08:01), and ⁵¹⁴NS5⁵²⁴ (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.     

HLA genetic profile of Mapuche (Araucanian) Amerindians from Chile

Amerindian Mapuche (Araucanians) are now living in Chile and Argentina at both sides of Andean Mountains. They are anthropologically and genetically different from southernmost South America Patagonian Amerindians. Most of the HLA alleles found in our Mapuche sample are frequent or very frequent in North and South America Amerindians: (1) Class I: A*02:01, A*03:01, A*68:01, B*39:09, B*51:01, (2) Class II: DRB1*03:01, DRB1*04:03, DRB1*07:01, DRB1*08:02, DRB1*14:02, DRB1*16:02. One of the nine most frequent extended haplotypes seems to be from European origin, suggesting the existence of a degree of admixture with Europeans in our Mapuche sample. It has been calculated of about 11 % admixture. Three of the extended haplotypes are also found in other Amerindians and five of them are newly found in Mapuche Amerindians: A*68:01-B*39:09-DRB1*08:02-DQB1*04:02; A*68:01-B*51:01-DRB1*04:03-DQB1*03:02; A*29:01-B*08:01-DRB1*03:01-DQB1*02:01; A*02:01-B*15:01-DRB1*04:03-DQB1*03:02; A*33:01-B*14:02-DRB1*07:01-DQB1*03:03. The medical importance of calculating HLA profile is discussed on the diagnostic (HLA and disease) and therapeutical bases of HLA pharmacogenomics and on the construction of a virtual transplantation HLA list profile. Also, anthropological conclusions are drawn.     

Amino acid residues in the P6-P'3 region of thrombin-activable fibrinolysis inhibitor (TAFI) do not determine the thrombomodulin dependence of TAFI activation.

Thrombin bound to thrombomodulin activates thrombin-activable fibrinolysis inhibitor (TAFI) and protein C much more efficiently than thrombin alone. Although thrombomodulin has been proposed to alter the thrombin active site, the recently determined structure of the thrombin-thrombomodulin complex does not support this proposal. In this study, the contribution of amino acids near the activation site of TAFI toward thrombomodulin dependence was determined, utilizing four variants of TAFI with specific substitutions in the P6-P'3 region surrounding the Arg-92 cleavage site. Two point mutants had either the Ser-90 or Asp-87 of TAFI replaced with Ala, a third mutant had the thrombin activation site of the fibrinogen Bbeta-chain substituted into positions 91-95 of TAFI, and a fourth mutant had the thrombin activation site of protein C substituted into positions 90-95 of TAFI. Each of these mutants was expressed, purified, and characterized with respect to activation kinetics and functional properties of the enzyme. Even though fibrinogen is poorly cleaved by thrombin-thrombomodulin, the fibrinogen activation site does not significantly alter the thrombomodulin dependence of TAFI activation. The TAFI variant with the protein C activation sequence is only slowly activated by thrombin-thrombomodulin, and not at all by free thrombin. Mutating Asp-87 to Ala increases the catalytic efficiency of activation 3-fold both in the presence and absence of thrombomodulin, whereas mutating Ser-90 to Ala effects only minor kinetic differences compared with wild type TAFI. The thermal stabilities and antifibrinolytic properties of the enzymes were not substantially altered by any of the mutations that allowed for efficient activation of the enzyme. We conclude that residues in the P6-P'3 region of TAFI do not determine the thrombomodulin dependence of activation, which lends support to the argument that the role of thrombomodulin is to optimally orient thrombin and its substrate, rather than to allosterically alter the specificity of the thrombin active site.     

Identification of the protein C/activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor

The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two alpha-helices that sit upon an 8-stranded beta-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the active site with fluorescein) interactions, whereas two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mouse EPCR. We found that 5 of the 10 candidate residues for protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope for one of the blocking mAbs. Three-dimensional molecular modeling of EPCR indicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two alpha-helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility.     

Calcium binding to the epidermal growth factor homology region of bovine protein C

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.     

The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody

Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.     

Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Activation of the anticoagulant human plasma serine protease zymogen, protein C, by a complex of thrombin and the membrane protein, thrombomodulin, generates activated protein C, a physiologic anti-thrombotic, anti-inflammatory and anti-apoptotic agent. Alanine-scanning site-directed mutagenesis of residues in five surface loops of an extensive basic surface on protein C was used to identify residues that play essential roles in its activation by the thrombin-thrombomodulin complex. Twenty-three residues in the protein C protease domain were mutated to alanine, singly, in pairs or in triple mutation combinations, and mutants were characterized for their effectiveness as substrates of the thrombin-thrombomodulin complex. Three protein C residues, K192, R229, and R230, in two loops, were identified that provided major contributions to interactions with thrombin-thrombomodulin, while six residues, S190, K191, K217, K218, W231, and R312, in four loops, appeared to provide minor contributions. These protein C residues delineated a positively charged area on the molecule's surface that largely overlapped the previously characterized factor Va binding site on activated protein C. Thus, the extensive basic surface of protein C and activated protein C provides distinctly different, though significantly overlapping, binding sites for recognition by thrombin-thrombomodulin and factor Va.     

Identification of epitopes associated with hCG and the beta hCG carboxyl terminus by monoclonal antibodies produced against a synthetic peptide

We have produced a library of monoclonal antibodies directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl terminus of hCG. Five antibodies, designated FB01, FB02, FB03, FB04, and FB00, were developed and analyzed with respect to affinity and specificity for epitopes on human chorionic gonadotropin (hCG) and beta hCG by enzyme-linked immunoabsorbent and radioimmunoassays (RIA). All monoclonal antibodies demonstrated low affinity constants (approximately 10(-7) liters/mol) compared with those obtained by immunization with native beta hCG. One antibody, namely FB00, bound only to the synthetic peptide, whereas all other monoclonal antibodies recognized either free native beta hCG or both beta hCG and HCG. Antibodies produced against the synthetic peptide did not cross-react with other glycoprotein hormones such as LH, TSH, and FSH. Characterization of the monoclonal antibody-binding sites revealed the presence of at least four separate and distinct epitopes on the last 35 amino acids of beta hCG. Indeed, one epitope recognized by FB01 is located between residues 109 and 118, whereas another antigenic region recognized by FB04 appears to be present on the 109-121 portion of the molecule near or at position 118. One additional antigenic site was localized between residues 118 and 136. Finally, FB00 recognized an epitope located on the last 10 amino acids (136-145) of beta hCG. With the use of such antibodies, two- and three-site monoclonal RIA were developed and employed to detect free beta hCG and hCG in sera of patients with choriocarcinoma. These assays may be useful in the detection of beta hCG- and hCG-producing tumors and subsequent monitoring of patients in response to surgery and/or chemotherapy.     

HLA Class I and II Blocks Are Associated to Susceptibility,Clinical Subtypes and Autoantibodies in Mexican Systemic Sclerosis (SSc) Patients

IntroductionHuman leukocyte antigen (HLA) polymorphism studies in Systemic Sclerosis (SSc) have yielded variable results. These studies need to consider the genetic admixture of the studied population. Here we used our previously reported definition of genetic admixture of Mexicans using HLA class I and II DNA blocks to map genetic susceptibility to develop SSc and its complications.MethodsWe included 159 patients from a cohort of Mexican Mestizo SSc patients. We performed clinical evaluation, obtained SSc-associated antibodies, and determined HLA class I and class II alleles using sequence-based, high-resolution techniques to evaluate the contribution of these genes to SSc susceptibility, their correlation with the clinical and autoantibody profile and the prevalence of Amerindian, Caucasian and African alleles, blocks and haplotypes in this population.ResultsOur study revealed that class I block HLA-C*12:03-B*18:01 was important to map susceptibility to diffuse cutaneous (dc) SSc, HLA-C*07:01-B*08:01 block to map the susceptibility role of HLA-B*08:01 to develop SSc, and the C*07:02-B*39:05 and C*07:02-B*39:06 blocks to map the protective role of C*07:02 in SSc. We also confirmed previous associations of HLA-DRB1*11:04 and –DRB1*01 to susceptibility to develop SSc. Importantly, we mapped the protective role of DQB1*03:01 using three Amerindian blocks. We also found a significant association for the presence of anti-Topoisomerase I antibody with HLA-DQB1*04:02, present in an Amerindian block (DRB1*08:02-DQB1*04:02), and we found several alleles associated to internal organ damage. The admixture estimations revealed a lower proportion of the Amerindian genetic component among SSc patients.ConclusionThis is the first report of the diversity of HLA class I and II alleles and haplotypes Mexican patients with SSc. Our findings suggest that HLA class I and class II genes contribute to the protection and susceptibility to develop SSc and its different clinical presentations as well as different autoantibody profiles in Mexicans.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Summary Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

共基质对10株细菌降解芘的作用

从石油污染的污泥中分离出10株细菌(SB01—SB10),研究了有(或无)共基质(葡萄糖Glu,或菲PHE)对细菌降解芘(PYR)的影响.结果表明：当以PYR为唯一碳源和能源时(MS₁),SB01的PYR降解率最高,5 d可降解30.4%;以Glu为共代谢基质时(MS₂),SB09的PYR降解率最高,可达37.7%;以PHE为共代谢基质时(MS₃),SB10的PYR降解率为50.2%.Glu抑制SB01、SB03对PYR的降解,对SB01抑制作用最明显,使SB01的PYR降解率降低7.9%;Glu对SB02、SB07、SB08、SB10降解率无明显促进或抑制作用.PHE对细菌降解PYR均有促进作用,对SB10的促进作用最明显,使其降解率提高298%.Glu与PHE对SB04和SB09降解PYR的促进作用无显著差异,而对其它各菌株而言,PHE对PYR降解的促进作用大于Glu.     

The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives.

Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.     

Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases: crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 A resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl(1-) instead of SO(4)(2-).