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J M Conroy

Publications and source records attributed to J M Conroy.

54 records · Page 3Linked to original sources
PubMed1984

An enzyme-linked immunosorbent assay for zein and other proteins using unconventional solvents for antigen adsorption.

An enzyme-linked immunosorbent assay (ELISA) performed in polystyrene microtiter plates that can detect and quantitate the maize prolamin zein is described. The assay yields positive reactions with as little as 1 ng of antigen and uses solvents not ordinarily employed in ELISA methods. A systematic investigation of zein adsorption to polystyrene in various solvents supports the hypothesis that antigen binding occurs through nonpolar interactions. The method was also used to determine structural relationships among three zein polypeptides differing in size and charge. Additional experiments indicate that a number of soluble proteins are absorbed to polystyrene in the denaturing agent urea and retain immunological reactivity. The retention of antigen reactivity after solubilization in 6-8 M urea suggests that ELISA methods may be applicable to other proteins which are insoluble, or rendered insoluble, in aqueous buffers.

Cross Reactions↗
PubMed1984

Macrophage-derived prostaglandin E modulation of the mixed-lymphocyte reaction: an anomaly of increased production and decreased T-cell susceptibility during tumor growth.

One-way mixed-lymphocyte reactions (MLR) were used to assess macrophage (M phi)-derived factor-mediated modulation of normal and tumor-bearing host (TBH) T-cell immune responsiveness. Normal and TBH M psi culture supernatants contained the inhibitory substance prostaglandin E (PGE) in concentrations of 10(-8) to 10(-9) M, with TBH M phi supernatant containing approximately twice the amount of PGE as its normal counterpart. Normal and TBH MLR reactivity were both suppressed by the addition of normal host M phi supernatant. However, TBH T cells were less inhibited by TBH M phi supernatant (55%) as compared to normal host T cells (73%). Although dialyzed M phi supernatants were less inhibitory (17-19%) on normal host T-cell MLR reactivity, TBH T-cell responses were enhanced (20-46%). Indomethacin or eicosatetraynoic acid treatment of M phi reduced PGE levels in the supernatants and in general enhanced MLR reactivity. When PGE1 and PGE2 were titrated in the MLR, normal host T lymphocytes were more susceptible to inhibition than were TBH. Concentrations of PGE1 and PGE2 comparable to that found in normal host M phi supernatants caused approximately 38% inhibition whereas whole M phi supernatants decreased MLR reactivity by greater than 70%, suggesting that another factor(s) was necessary to account for the additional M phi-mediated suppression of lymphocyte function. Isoelectric focusing was used to fractionate normal host M phi supernatant. Two factors with isoelectric points in the pH ranges 7.0-8.5 and 4.5-5.0 were inhibitory in the MLR. An enhancing factor was also identified with an pI in the range of pH 6.0-7.0. These data suggest that TBH M phi-derived PGE production was increased over its normal counterpart, but that TBH T cells were less susceptible to its effect and an additional factor(s) was working in concert with PGE.

Animals↗
PubMed1983

A simple and rapid dot-immunobinding assay for zein and other prolamins.

A simple method for the immunochemical assay of zein and other prolamins is described. Alcohol-solubilized zein is spotted onto chromatography paper disks or strips, incubated with antiserum (30-60 min), washed with several changes of Tris-buffered saline (10 min), incubated with protein A-peroxidase (30 min), and washed with several changes of Tris-buffered saline (10 min). the binding of zein-specific antibody is visualized by peroxidase-catalyzed color production from 4-chloro-1-naphthol. The method is simple, inexpensive, and rapid (2 h total time) and has potential application to other insoluble antigens.

Densitometry↗
PubMed1983

Heterogeneity in anticatalytic activity of antibody specific for horseradish peroxidase.

Rabbit antibodies specific for horseradish peroxidase are heterogeneous in their ability to inhibit enzyme activity. Heterogeneity was demonstrated by fractionation of the total antiperoxidase pool by differential ammonium sulfate precipitation and differential elution of antibody from enzyme affinity columns. Both fractionation methods yielded antibody subpopulations that differed in anticatalytic activity. Some antibody subpopulations decreased enzyme activity almost completely at low molar ratios of antibody to peroxidase. Other subpopulations were not effective inhibitors even at great molar excess. Admixture experiments demonstrated that inefficient antibody pools decreased the anticatalytic effect of highly inhibitory antibody. The degree of inhibition observed with unfractionated antiserum is a reflection of the interaction of various antibody subpopulations with the enzyme. No correlation was found between the immunoglobulin class of antiperoxidase and anticatalytic efficiency in analyses of numerous antisera. The determinant specificity of an antiperoxidase molecule determines its anticatalytic ability. A peptide fragment (mol. wt 22,500) of peroxidase prepared by partial tryptic digestion bount 60 to 70% of the total antiperoxidase in a number of antisera. However, the peptide did not bind inhibitory antibodies.

Animals↗
PubMed1983

The effect of alternate substrates and substrate concentration on the antibody-mediated inhibition of horseradish peroxidase.

Inhibition of the catalytic activity of horseradish peroxidase by rabbit antisera was measured using alternate enzyme substrates. The same general inhibition curve patterns were obtained regardless of the electron donor or hydroperoxide used. In all cases typical biphasic inhibition patterns were found and complete inhibition of enzyme activity was never observed. Measurement of the degree of inhibition as a function of substrate concn revealed a dependence of anticatalytic activity on hydroperoxide concn. As the concn of hydrogen peroxide in the assay mixture increased, there was a corresponding increase in the inhibition observed. On the other hand, the degree of inhibition was not dependent on the concn of electron donor (dianisidine) in the assay mixture. Spectrophotometric experiments with an electron donor analogue demonstrated that antibodies do not inhibit peroxidase activity by excluding electron donor molecules from enzyme binding sites. The results have suggested possible mechanisms for the antibody-mediated inhibition of peroxidase activity.

2,6-Dichloroindophenol↗
PubMed1983

Immunoregulation of the mixed lymphocyte reaction by macrophage-derived factors: functional and biochemical separation of enhancing and inhibitory factors.

Positive and negative immunoregulation of the mixed lymphocyte reaction (MLR) occurred through release of macrophage(MO)-derived, soluble enhancing and inhibitory factors. Macrophages, sonicated or cultured in low concentrations, produced nondialyzable, soluble factor(s) capable of enhancing the MLR; but the culture supernatant had no biologically detectable levels of Interleukin 1, Interleukin 2, or interferon. Production of enhancing supernatants was not affected by pretreatment of MO with trypsin or anti-Thy 1 antibody plus complement. In contrast, MO cultured in high concentrations yielded an inhibitory supernatant factor(s) which suppressed MLR reactivity even when MO were cultured in the presence of indomethacin. Culturing MO with proteolytic enzyme inhibitors increased the yields of the inhibitory and enhancing factors. Both factors were precipitable with ammonium sulfate and could be separated into several biologically active fractions using anion exchange chromatography.

Animals↗
PubMed1982

Inhibition of horseradish peroxidase activity by specific antibody: determinant specificity of anticatalytic antibodies.

Rabbit antisera specific for horseradish peroxidase inhibit the catalytic activity of the enzyme. All antibodies prepared against the holoenzyme react with the peroxidase apoenzyme. However, only a minority (30-45%) of the total antiperoxidase pool cross react with reduced and alkylated apoenzymes. The antibodies inhibiting peroxidase activity do not bind to S-carboxymethyl of S-carboxamidomethylated apoenzyme derivatives as measured by absorption and competition of inhibition experiments. Glycopeptides derived from horseradish peroxidase also failed to bind anticatalytic antibodies. Antibodies that inhibit enzyme activity have specificity for noncarbohydrate conformation dependent antigenic determinants of horseradish peroxidase. Additional experiments probed the mechanism by which inhibitory antibody decreases the catalytic activity of horseradish peroxidase. Absorption spectra of horseradish peroxidase that has bound Fab fragments sufficient to cause 90% inhibition of the enzyme activity determined that the enzyme retained the ability to bind hydrogen peroxide. Thus, anticatalytic antibodies do not prevent the formation of the first enzyme-substrate intermediate but mediate their inhibitory effects by disrupting a later step in the reaction mechanism.

Antibody Specificity↗
PubMed1982

Homology of Plant Peroxidases: AN IMMUNOCHEMICAL APPROACH.

Antisera specific for the basic peroxidase from horseradish (Amoracea rusticana) were used to examine homology among horseradish peroxidase isoenzymes and among basic peroxidases from root plants. The antisera cross-reacted with all tested isoperoxidases when measured by both agar diffusion and quantitative precipitin reactions. Precipitin analyses provided quantitative measurements of homology among these plant peroxidases. The basic radish (Raphanus sativus L. cv. Cherry Belle) peroxidase had a high degree of homology (73 to 81%) with the basic peroxidase from horseradish. Turnip (Brassica rapa L. cv. Purple White Top Globe) and carrot (Daucus carota L. cv. Danvers) basic peroxidases showed less cross-reaction (49 to 54% and 41 to 46%, respectively). However, the cross-reactions of antisera with basic peroxidases from different plants were greater than were those observed with acidic horseradish isoenzymes (30 to 35%). These experiments suggest that basic peroxidase isoenzymes are strongly conserved during evolution and may indicate that the basic peroxidases catalyze reactions involved in specialized cellular functions. Anticatalytic assays were poor indicators of homology. Even though homology among isoperoxidases was detected by other immunological methods, antibodies inhibited only the catalytic activity of the basic peroxidase from radish.

Journal Article↗
PubMed1978

Canine pulmonary angiotensin-converting enzyme. Physicochemical, catalytic and immunological properties.

Antiontensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) has been solubilized from canine pulmonary particles and purified to apparent homogeneity. A value of approx. 140000 was estimated for the molecular weight of the native and the reduced, denatured forms of the enzyme. No free NH2-terminal residue was detected by the dansylation procedure. Carbohydrate accounted for 17% of the weight of the enzyme, and the major residues were galactose, mannose and N-acetylglucosamine with smaller amounts of sialic acid and fucose. Removal of sialic acid residues with neuraminidase did not alter enzymatic activity. The enzyme contained one molar equivalent of zinc. Addition of this metal reversed stimulation and inhibition of activity observed in the presence of Co2+ and Mn2+, respectively. Immunologic homology of pure dog and rabbit enzymes was demonstrable with goat antisera. Fab fragments and intact IgG antibodies displayed similar inhibition dose vs. response curves with homologous enzyme, whereas the fragments were poor inhibitors of heterologous activity compared to the holoantibodies. The canine glycoprotein was much less active than the rabbit preparation in catalyzing hydrolysis of Hip-His-Leu. In contrast, the two enzymes exhibited comparable kinetic parameters with angiotensin I as substrate.

Amino Acid Sequence↗
PubMed1978

Reversal of renovascular hypertension by antibodies specific for angiotensin-converting enzyme.

Goat antibodies developed against pure rabbit pulmonary angiotensin-converting enzyme (peptidyl dipeptidase, EC 3.4.15.1) were administered intravenously to rats with two-kidney Goldblatt hypertension and to normotensive animals. In the hypertensive model these antibodies were associated with a sustained, immune-dependent decrease of blood pressure to normal values. A smaller, but also immune-specific, reduction of blood pressure was observed in the normotensive group. The data suggest that heterologous antibody directed against angiotensin-converting enzyme may provide a biologically specific probe for examining the contribution of the renin-angiotensin system to normal and pathologic circulatory states.

Animals↗
PubMed1976

Pulmonary angiotensin-converting enzyme. Interspecies homology and inhibition by heterologous antibody in vivo.

Goat antibody against pure rabbit pulmonary angiotensin-converting enzyme was used to probe homology of converting enzymes from other species. Immunologically cross-reactive material was found in detergent-solubilized extracts of lung particles from rat, guinea pig, and dog by double immunodiffusion, radioimmunoassay, and inhibition of enzyme activity. No homology was demonstrable with bovine, frog, or chicken lung extracts. Antibodies from different individual goats yielded comparable estimates of homology by immunodiffusion and radioimmunoassay. In contrast, they varied greatly in extent and specificity of their inhibitory action on heterologous enzyme activity. The vasopressor effect of angiotensin I and the vasodepressor effect of bradykinin were diminished and potentiated, respectively, in rats treated with anti-rabbit enzyme antibody. A smaller but significant immune-dependent inhibition of the vasopressor response to angiotensin II was also observed.

Angiotensin II↗
PubMedDate not supplied

Population-based studies reveal differences in the allelic frequencies of two functionally significant human interleukin-4 receptor polymorphisms in several ethnic groups.

PURPOSE: The presence of functionally significant human interleukin-4 receptor sequence variants, Gln551Arg and Ile50Val, was examined in four anonymous New York State populations defined by ethnic origin. These variants were studied because they are associated with atopy or atopic asthma whose prevalence varies in different populations. METHODS: PCR/RFLP (Ile50Val) and PCR/allele-specific oligonucleotide hybridization (Gln551Arg) assays were developed to detect both polymorphisms in 855 newborn screening specimens. RESULTS: Arg551 was most frequently found in Blacks (allele frequency of 68%). However, the Ile50 allele was most common in Whites (allele frequency, 87%). Significantly more Blacks had chromosomes bearing both of the "enhanced signaling" variants (Ile50/Arg551). CONCLUSIONS: Enhanced IL-4R signaling is associated with increased IgE production (atopy). Therefore, our data suggest that the African American population may be at increased risk for diseases, including asthma, which are associated with atopy. These data also emphasize the importance of determining the frequencies of single nucleotide polymorphisms in different populations before drawing conclusions from allele association studies, since the background allele frequencies may be disparate between different populations.

Alleles↗
PubMedDate not supplied

The effect of 6% hydroxyethyl starch and desmopressin infusion on von Willebrand factor: ristocetin cofactor activity.

Hydroxyethyl starch is commonly used as a plasma volume expander in the surgical patient. Although it is generally considered a safe plasma substitution, some reports of an acquired von Willebrand's disease-like syndrome have been documented. To examine this further, von Willebrand factor: ristocetin cofactor activity (RCoF) was measured in two groups of patients perioperatively, following hydroxyethyl starch infusion and at 30, 60, and 240 minutes following either deamino-8-D-arginine vasopressin (DDAVP) (group I, n = 12) or saline (group II, n = 11). Following hydroxyethyl starch infusion, ristocetin cofactor activity decreased to 58 percent (group I) and 55 percent (group II) of their respective baseline values. After infusion of DDAVP, mean ristocetin cofactor activity in group I increased significantly to 95 percent at 30 minutes and 100 percent of baseline at 60 minutes. Mean ristocetin cofactor activity levels in group II, however, remained decreased, 69 percent and 57 percent of baseline at the same time points. There was no statistical difference between groups before or immediately after hydroxyethyl starch administration or at 240 minutes post-DDAVP or saline infusion. It is our conclusion that DDAVP is a safe therapy for the mild coagulapathy infrequently associated with hydroxyethyl starch administration.

Blood Coagulation Disorders↗