Search PubMed⌕ Search

Biomedical subjects

J C Moore

Publications and source records attributed to J C Moore.

At least 37 records · Page 2Linked to original sources
PubMed1999

Calpain activity in bone marrow transplant-associated thrombotic thrombocytopenic purpura.

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) is not well understood. Recent studies have described a platelet aggregating factor which has been characterized as a calcium-dependent cysteine protease (calpain) in patients with TTP. A type of TTP, sometimes called secondary TTP, has been associated with bone marrow transplantation (BMT). However, unlike primary adult TTP, BMT-TTP has important differences and often does not respond well to plasma exchange. We describe the measurement of calpain activity in a group of BMT patients (with and without the clinical syndrome of transplant-associated TTP). Calpain was measured using a functional assay (14C-serotonin platelet release with inhibition by the cysteine protease inhibitor, leupeptin) in the sera of patients following autologous (auto) or allogeneic (allo) BMT. We also independently diagnosed and graded the BMT-TTP on the day of blood sampling using a scale that related to the percentage schistocytes and lactic dehydrogenase level. Calpain activity was detected in 1/8 (13%) grade 0-1 (6 auto, 2 allo); 6/16 (38%) grade 2 (3 auto, 13 allo) 9/16 (56%) grade 3 (2 auto, 14 allo) and 8/8 (100%) grade 4 BMT-TTP. Pre-BMT samples were tested in 10 allo-BMT patients who had positive calpain results post-BMT. One patient gave positive results before the transplant. This patient developed grade 4 BMT-TTP (day 24 post-BMT) and died despite apheresis. Positive calpain results were highly associated with neurologic symptoms, P < 0.001. Nineteen of 24 (79%) patients with positive results had neurologic symptoms compared to three of 21 (14%) patients with negative results. In conclusion, calpain was detected in half of the BMT patients with mild to moderate BMT-TTP (grades 2-3) and was uniformly found in those with severe (grade 4) BMT-TTP. Typically the calpain activity develops as TTP complicates the transplant process. It is unknown whether calpain contributes to the pathogenesis of this disorder, or is a secondary event.

Adult↗
PubMed1999

A prospective study of protein-specific assays used to investigate idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is a disorder in which platelets, sensitized by autoantibodies, are destroyed by the reticuloendothelial system. The diagnosis of ITP has been a clinical one because assays measuring platelet-associated IgG (PAIgG) have low specificity. The recently introduced assays that measure antibodies against specific platelet glycoproteins (GP) offer the possibility of improved specificity. In this report we describe two prospective studies. In the first study we compared two protein-specific assays (AC and MAIPA) looking for the presence of autoantibodies against GP IIb/IIIa in 81 patient samples. These results were compared with an immunoradiometric assay for PAIgG. The second study investigated the enhanced sensitivity of measuring anti-GP Ib/IX autoantibodies in 76 patient samples. The protein-specific assays were able to differentiate immune from non-immune thrombocytopenia (specificity 91%, sensitivity 39%), whereas the PAIgG assay could not (specificity 19%, sensitivity 78%). The addition of the Ib/IX AC assay maintained a specificity of 92% while increasing the diagnostic sensitivity to 66%. In contrast to the PAIgG assay, there was no correlation between the platelet count and the likelihood or degree of positivity within the control samples using the glycoprotein assays. These studies confirm that glycoprotein assays can be used as diagnostic tests for ITP.

Antibodies↗
PubMed1998

A europium fluoroimmunoassay for measuring peptide binding to MHC class I molecules.

A fluoroimmunoassay employing europium-streptavidin and time-resolved fluorimetry was used to measure binding of biotin-labeled peptides to H-2Dk molecules. A fluorescein-labeled octameric peptide from the middle T (MT) protein of mouse polyoma virus was identified that specifically binds to purified Dk using a previously-established assay based on size exclusion chromatography. A europium immunoassay was adapted to measure binding of a biotin-derivative of this peptide to purified Dk. The assay was found to be sensitive and highly specific. Binding was optimal at pH 5.0 and 24-27 degrees C, and it was not enhanced in the presence of additional beta2-microglobulin (beta2m). Excellent results were also obtained in experiments with fixed cells that express Dk. This assay is expected to be useful for high volume, routine analysis of peptide binding to MHC class I molecules.

Animals↗
PubMed1997

Inefficient peptide binding by cell-surface class II MHC molecules.

The efficiency of peptide loading onto surface class II MHC molecules in intact APC was investigated, using a previously defined europium immunoassay as well as a simplified Western blot procedure. Conditions normally employed for peptide loading in T cell stimulation assays were suboptimal for peptide binding, which is enhanced at low pH, in the presence of protease inhibitors, and the absence of competing serum proteins. In contrast to some earlier reports, our results indicate that the rate of peptide loading by class II molecules is not enhanced in the environment of the plasma membrane. Peptide association rates were similar for purified and cell-surface class II molecules. As previously reported, rapid peptide binding can be achieved by reconstituting class II molecules into total cellular membranes. We report that this activity is due solely to HLA-DM (which is not present at the cell surface), since it can be specifically removed by immunodepletion with an anti-DM mAb. Thus, we find no evidence for additional cellular cofactors capable of catalyzing peptide binding to class II molecules.

Amino Acid Sequence↗
PubMed1997

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences.

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.

Carboxylic Ester Hydrolases↗
PubMed1997

Optimizing industrial enzymes by directed evolution.

Enzymes can be tailored for optimal performance in industrial applications by directing their evolution in vitro. This approach is particularly attractive for engineering industrial enzymes. We have created an efficient para-nitrobenzyl esterase over six generations of random point mutagenesis and recombination coupled with screening for improved variants. The best clones identified after four generations of sequential random mutagenesis and two generations of random recombination display more than 150 times the p-nitrobenzyl esterase activity of wild type towards loracarbef-p-nitrobenzyl ester in 15% dimethylformamide. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (< 2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.

Carboxylic Ester Hydrolases↗
PubMed1997

Acute and chronic toxicity of azinphos-methyl to two estuarine species, Mysidopsis bahia and Cyprinodon variegatus.

The acute and chronic toxicity of azinphos-methyl (Guthion) was evaluated for two estuarine species in the laboratory. Mysids (Mysidopsis bahia) and sheepshead minnows (Cyprinodon variegatus) were selected as the representative invertebrate and vertebrate estuarine test species, respectively. The toxicological endpoints determined for each species included the 96-h LC50, the no-observed-effect concentration (NOEC), the maximum acceptable toxicant concentration (MATC), and the acute-to-chronic ratio. The 96-h LC50 value derived for sheepshead minnows (2.0 microg/L) was seven times higher than the 96-h LC50 value (0.29 microg/L) derived for mysids. The MATCs were 0.024 microg/L and 0.24 microg/L for the mysid and the sheepshead minnow, respectively. The estimated acute-to-chronic ratios were 12 for mysids and 8.3 for sheepshead minnows.

Animals↗
PubMed1997

Peptide binding to mixed isotype Abeta(d)Ealpha(d) class II histocompatibility molecules.

Previous studies have demonstrated that mixed isotype A beta(d) E alpha(d) molecules are expressed in transfected cell lines and that the level of expression is very low in normal B cells from H-2(d) mice. T-cell responses restricted by A beta(d) E alpha(d) are induced in H-2(d) mice immunized with the synthetic peptides YL2 and FL2 or with sperm whale myoglobin, despite the low concentration of mixed isotype molecules expressed on antigen-presenting cells. In the present study, the peptide binding behavior of A beta(d) E alpha(d) was investigated. A peptide from the cytoplasmic domain of invariant chain, I(1-18), was observed to bind with high affinity to purified A beta(d) E alpha(d). Binding was optimal at pH 5, indicating that these molecules prefer to bind peptide in the acidic environment of endosomal compartments similar to other murine class II proteins. YL2 and FL2 bind to A beta(d) E alpha(d) with slightly lower affinity. The selective restriction of YL2- and FL2-specific T cells to mixed isotype molecules was accounted for by the observation that these peptides do not bind to either I-E(d) or I-A(d). By contrast, myoglobin peptides bind to both parental and mixed isotype molecules. None of the A beta(d) E alpha(d)-restricted peptide determinants bind to A beta(d) E alpha(d) with extremely high affinity. Thus it is unlikely that these peptides occupy an unusually high fraction of mixed isotype molecules during antigen presentation in vivo. It is more likely that the presence of a subpopulation of high-affinity T cells capable of being stimulated by very low concentrations of A beta(d) E alpha(d)/peptide complexes is responsible for the unusual A beta(d) E alpha(d)-restricted response observed with some antigens.

Animals↗
PubMed1997

Intact proteins can bind to class II histocompatibility molecules with high affinity.

The ability of intact protein antigens to bind to purified class II histocompatibility molecules was investigated. Intact bovine ribonuclease (RNase) inhibited peptide binding to DR1 with a potency similar to that of a high affinity peptide or irreversibly denatured RNase. Similarly, horse myoglobin (Mb) was a potent inhibitor of peptide binding to I-E(k). I-E(k)-Mb complexes were directly visualized as a distinct band with reduced mobility on SDS PAGE. Direct binding experiments with biotin-labeled proteins demonstrated that Mb and RNase bind to class II molecules through the peptide-binding groove with high affinity, and that binding occurs in the absence of detergent. The possibility that HLA-DM can catalyse the binding of intact protein antigens was supported by the observation that DM enhances the binding of biotin-RNase to DR1. Our results provide further support for the hypothesis that intact, partially unfolded protein antigens can act as ligands for initial interaction with class II molecules.

Animals↗
PubMed1997

Proteolytic degradation of high molecular weight kininogen in acute thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized by thrombocytopenia and schistocytic haemolytic anaemia. The majority of patients have normal coagulation parameters including the activated partial thromboplastin time (APTT). An intracellular cysteine protease, calpain, has been found in the plasma of many patients with acute TTP, and we hypothesize that it participates in the pathogenesis of the disorder. However, certain aspects of the disorder remain unresolved. For example, high molecular weight kininogen (HK) is one of the primary plasma inhibitors of calpain, and it also can act as a substrate for calpain. Consequently, one might anticipate that the HK could be defective or altered in TTP. In this report we describe the analysis of HK in plasma from live patients with acute TTP and following recovery. The HK was studied immunogenically and functionally. We observed that the HK in plasma samples from patients with acute TTP was proteolysed. This degradation was not observed in remission samples from the same patients. However, both acute and remission TTP samples had normal HK coagulant activity (acute samples, x = 0.84 +/- 0.26 U/ml; remission samples, x = 0.89 +/- 0.21 U/ml; control samples, x = 0.87 +/- 0.05 U/ml). Although the TTP plasmas were able to inhibit calpain activity, less inhibition activity was found in the acute samples (acute: mean inhibition 71 +/- 2.4%; remission: mean 92 +/- 2.1%; control samples: mean 93 +/- 5.4%; P < 0.001). Normal HK treated with calpain also had reduced calpain inhibition capacity (mean 58%). This study suggests that although HK is proteolysed during acute TTP, the proteolysis occurs without a major loss in the coagulation function or depletion of the protease inhibitory activity of HK.

Acute Disease↗
PubMed1996

An autosomal dominant, qualitative platelet disorder associated with multimerin deficiency, abnormalities in platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen and an epinephrine aggregation defect.

Multimerin is a massive soluble, multimeric protein found in platelets and endothelial cells. Recent studies identified multimerin as a specific coagulation factor V binding protein, complexed with platelet, but not plasma, factor V. These findings led us to investigate individuals with inherited factor V deficiencies for possible multimerin abnormalities. Platelet proteins were evaluated using immunoassays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunoprecipitation, and direct binding studies. Patients with factor V Quebec, a disorder with abnormal platelet factor V, had a quantitative deficiency in multimerin (n = 11 tested; mean, 12.5%; range, 5% to 27% of the normal pool; normal range, 45% to 214%) with a normal multimer pattern. Quantitative and qualitative abnormalities were detected in their platelet factor V. An unrelated patient who was deficient in platelet and plasma factor V had normal platelet multimerin. The levels of platelet beta-thromboglobulin, von Willebrand factor, thrombospondin, and fibrinogen antigen were normal in the factor V Quebec patients. However, proteins with abnormal mobility were detected in their platelet lysate and releasate, and their platelet thrombospondin, von Willebrand factor, and fibrinogen showed evidence of proteolytic degradation. Platelet counts of the factor V Quebec patients ranged from mildly thrombocytopenic to low normal (mean, 159 x 10(9)/L; range, 104 to 198 x 10(9)/L). In addition, their platelets failed to aggregate in response to 6 to 10 micromol/L epinephrine despite normal numbers of platelet alpha 2-adrenergic receptors. These data indicate that patients with factor V Quebec have an inherited bleeding disorder distinct from other platelet disorders and associated with multiple abnormalities, including multimerin deficiency, abnormal platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen, and an epinephrine aggregation defect.

Afibrinogenemia↗
PubMed1996

An insulin peptide that binds an alternative site in class II major histocompatibility complex.

We report that a peptide from the B chain of insulin, B(10-30), binds with high affinity to multiple class II proteins, including IAb,d,k, IEd,k, and DR1. The ability of B(10-30) to inhibit the binding of other peptide antigens to class II does not correlate with its affinity for class II. B(10-30) only weakly inhibits the binding of antigenic peptides. Conversely, peptides with high affinity for the peptide-binding groove of various class II proteins do not inhibit B(10-30) binding. The rate of association of B(10-30) with class II is unusually rapid, approaching saturation in 1-2 h compared with 1-2 d for classical peptide antigens in the same conditions. The dissociation rate is also relatively rapid. The B(10-30) peptide inhibits the binding of the super-antigen staphylococcal enterotoxin B (SEB) to IAk. It also inhibits SEB-mediated T cell activation. These observations support the conclusion that B(10-30) binds to a site outside the peptide-binding groove. Our findings indicate that short-lived peptide-class II complexes can be formed through interactions involving the SEB-binding site and raise the possibility that alternative complexes may serve as T cell receptor ligands.

Amino Acid Sequence↗
PubMed1996

A structural transition in class II major histocompatibility complex proteins at mildly acidic pH.

Peptide binding by class II major histocompatibility complex proteins is generally enhanced at low pH in the range of hydrogen ion concentrations found in the endosomal compartments of antigen-presenting cells. We and others have proposed that class II molecules undergo a reversible conformational change at low pH that is associated with enhanced peptide loading. However, no one has previously provided direct evidence for a structural change in class II proteins in the mildly acidic pH conditions in which enhanced peptide binding is observed. In this study, susceptibility to denaturation induced by sodium dodecyl sulfate (SDS) detergent or heat was used to probe the conformation of class II at different hydrogen ion concentrations. Class II molecules became sensitive to denaturation at pH 5.5-6.5 depending on the allele and experimental conditions. The observed structural transition was fully reversible if acidic pH was neutralized before exposure to SDS or heat. Experiments with the environment-sensitive fluorescent probe ANS (8-anilino-1-naphthalene-sulfonic acid) provided further evidence for a reversible structural transition at mildly acidic pH associated with an increase in exposed hydrophobicity in class II molecules. IAd conformation was found to change at a higher pH than IEd, IEk, or IAk, which correlates with the different pH optimal for peptide binding by these molecules. We conclude that pH regulates peptide binding by influencing the structure of class II molecules.

Acids↗
PubMed1996

Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents.

Through sequential generations of random mutagenesis and screening, we have directed the evolution of an esterase for deprotection of an antibiotic p-nitrobenzyl ester in aqueous-organic solvents. Because rapid screening directly on the desired antibiotic (loracarbef) nucleus p-nitrobenzyl ester was not feasible, the p-nitrophenyl ester was employed. Catalytic performance on the screening substrate was shown to reasonably mimic enzyme activity toward the desired ester. One p-nitrobenzyl esterase variant performs as well in 30% dimethylformamide as the wildtype enzyme in water, reflecting a 16-fold increase in esterase activity. Random pairwise gene recombination of two positive variants led to a further two-fold improvement in activity. Considering also the increased expression level achieved during these experiments, the net result of four sequential generations of random mutagenesis and the one recombination step is a 50-60-fold increase in total activity. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (< 2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model developed based on its homology to acetylcholinesterase and triacylglycerol lipase. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.

Amino Acid Substitution↗
PubMed1996

Isolation and characterization of cysteine proteinase in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon disorder characterized by thrombocytopenia, schistocytic haemolytic anaemia, fever, neurologic lesions, and renal failure. A platelet aggregating factor has been postulated to be responsible for this disorder, but its precise identity remains debated. Two different groups of investigators have provided evidence that the platelet aggregating factor is a cysteine proteinase. We have suggested that it was calpain, whereas others have suggested that it was calpain, whereas others have suggested that it was cathepsin L. To help resolve this issue, we have studied the platelet activating activity found in the acute serum samples from 10 different TTP patients as well as purified calpain and cathepsin L. The TTP activity was measured functionally (platelet serotonin release assay and casein lysis assay) and antigenically (immunodepletion using anti-calpain and anti-cathepsin L antibodies and antigenic analysis using SDS PAGE). The TTP serum paralleled the activity of the purified calpain and had optimal pH activity of 7.5. The purified cathepsin L activity had optimal activity at low pH (5.5) and activity was no longer measurable at pH 7.5. Similarly, a specific cathepsin L inhibitor (Z-Phe-Phe-CHN2) had no effect on the activity of the TTP samples nor on purified calpain, but it did abolish the activity activity of purified cathepsin L. The platelet activating of the TTP samples was detectable in the microparticle pellet following ultracentrifugation of TTP serum, and could be immunodepleted using antibodies to calpain but not to cathepsin L. These studies indicate that the microparticle-associated platelet activating factor in TTP corresponds to calpain.

Blood Platelets↗
PubMed1995

Oral administration of dihydroartemisinin and ferrous sulfate retarded implanted fibrosarcoma growth in the rat.

In the presence of iron, dihydroartemisinin forms free radicals and causes cell death. Since most cancer cells have high rates of iron intake, dihydroartemisinin would have selective cytotoxic effect on cancer cells. The present experiment was designed to study the effect of dihydroartemisinin and ferrous sulfate on the growth of implanted fibrosarcoma in the rat. We found that the growth rate of the tumor was significantly retarded by daily oral administration of ferrous sulfate followed by dihydroartemisinin. No significant tumor growth retardation effect was observed in rats treated with either dihydroartemisinin or ferrous sulfate alone. The drug treatment did not significantly affect body weight compared with untreated tumor-implanted animals and no apparent toxic effect was observed after drug treatment. An artemisinin analog-ferrous salt combination may provide a novel approach for cancer therapy.

Administration, Oral↗
PubMed1995

Factor V is complexed with multimerin in resting platelet lysates and colocalizes with multimerin in platelet alpha-granules.

Factor V stored in platelets is an important source of factor Va for the prothrombinase complex. Investigations of potential platelet factor Va-binding proteins, using factor Va light chain affinity chromatography, identified a disulfide-linked multimeric protein with a reduced mobility of 155 kDa in the column eluate. Immunodepletion and immunoblotting indicated that this protein was multimerin. Multimerin specifically bound factors V and Va and the isolated factor Va light chain, but not the heavy chain of factor Va. Factor V stored in platelets, but not plasma factor V, was found to be complexed with multimerin. Multimerin immunodepletion of resting platelet lysates was associated with the removal of factor V and the loss of factor V coagulant activity. Immunoelectron microscopic studies colocalized factor V with multimerin in the alpha-granules of resting platelets. With thrombin-induced platelet activation, we observed dissociation of factor Va-multimerin complexes, multimerin-independent membrane binding of factor Va, and prothrombinase activity that was not inhibitable by multimerin antibodies. This study indicates that platelet factor V is stored as a complex with multimerin and suggests a possible role for multimerin as a carrier protein for factor V stored in platelets.

Animals↗