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PubMed1990

Effect of aging on alpha-1 adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain.

The effects of aging were examined on the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis in three brain regions. Tissue minces of thalamus, cerebral cortex and hippocampus from 3-, 18- and 28-month-old male Fischer 344 rats were prelabeled with [3H]myoinositol. Exposure of these prelabeled minces to phenylephrine and (-)-norepinephrine revealed that accumulation of [3H]inositol phosphates was selectively reduced by 20 to 30% in the thalamus and cerebral cortex of the oldest age group. Analysis of concentration-response and competition binding curves indicated that this decrease was due to diminished agonist efficacy rather than diminished receptor affinity. The reduction in responsiveness to phenylephrine and (-)-norepinephrine in the cerebral cortex and the lack of any changes in the hippocampus parallel previously reported changes in the density of alpha-1 adrenergic receptors with aging. These data indicate that the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis is reduced in some, but not all, brain regions of aged Fischer 344 rats.

Adrenergic alpha-Agonists↗
PubMed1991

Characterization of a new monoclonal antibody (TR-19) against human transferrin receptor and its application in topographic study.

A new monoclonal antibody TR-19 (IgG2a) directed to the human transferrin receptor (CD71) was prepared after immunization of BALB/c mice with human non-T, non-B ALL cell line REH-6. This monoclonal antibody (mAb) reacted in immunofluorescence with all human cell lines tested. The reactivity with human peripheral blood leukocytes was observed only after stimulation with phytohemagglutinin (PHA). The protein precipitated by mAb TR-19 has an apparent molecular weight of 180 kDa under nonreducing and 90 kDa under reducing conditions. Sequential immunoprecipitation showed that mAb TR-19 recognizes the same antigen as the reference anti-transferrin receptor mAb OKT9. The mAb TR-19 coprecipitates the radioiodinated transferrin as a complex with its receptor. The mAb TR-19 with other three mAbs (OKT9, 5E9 and MEM-75) was used in the topographic study of the transferrin receptor. By competitive binding radioimmunoassay it was found that these mAbs recognize two distinct antigenic sites: One is specified by mAbs TR-19 and OKT9 and the second by mAbs 5E9 and MEM-75.

Animals↗
PubMed1991

Increased plasma noncortisol glucocorticoid activity in open-angle glaucoma.

Total biologic plasma glucocorticoid activity of normal, ocular hypertensive, and open-angle glaucoma patients was compared using a glucocorticoid receptor-based competitive binding assay. Multiple linear-regression analysis was used to adjust for the effects of significant ocular and nonocular variables, including therapy for glaucoma. The glaucoma patients had significantly greater plasma glucocorticoid activities than did normal subjects. A comparison of receptor-based assay values to values obtained with a cortisol radioimmunoassay showed that significant amounts of biologic glucocorticoid activity in the plasma of the glaucoma patients could not be explained by cortisol alone. In the normal and ocular hypertensive groups, however, virtually all of the plasma glucocorticoid activity could be accounted for by cortisol. These results suggest that in open-angle glaucoma patients, noncortisol glucocorticoids are responsible for elevating biologic plasma glucocorticoid activity. Thus, open-angle glaucoma may be associated with a disturbance of the hypothalamic-pituitary-adrenal axis that produces increased plasma levels of both cortisol and other noncortisol glucocorticoids.

Aged↗
PubMed1990

Chronic isoproterenol administration causes altered beta adrenoceptor-Gs-coupling in guinea pig lung.

Chronic administration of isoproterenol caused major changes in the molecular and functional characteristics of lung beta adrenoceptors. The dose of isoproterenol delivered by the minipumps over 7 days corresponded to approximately 60 micrograms.kg-1h-1. The agonist treatment produced an 80% decrease in the number of beta adrenoceptors in lung membranes. However, a significant proportion of the remaining receptors could still form complexes with the nucleotide regulatory protein (Gs) in the presence of agonist. This was demonstrated by the persistence of high affinity sites (42% versus 53% in controls, P less than .01) in isoproterenol competition binding curves and of the tight binding of isoproterenol in the presence of the reagent N-ethylmaleimide (44% versus 60% in controls, P less than .01). However, part of the receptor population displaying high affinity toward the agonist was insensitive to guanosine 5'-triphosphate. These changes were associated with a decrease in the Gs activity, as demonstrated by the lower degree of cholera toxin-mediated adenosine diphosphate ribosylation. Moreover, chronic against treatment caused a 50% reduction in the maximal relaxing effect of isoproterenol in the isolated lung parenchymal strip, whereas the tissue sensitivity to beta adrenoceptor stimulation was unchanged. It is concluded that chronic administration of isoproterenol to guinea pigs caused marked changes in beta adrenoceptor function in the lung tissue. The molecular events involved in beta adrenoceptor desensitization by chronic in vivo administration of relatively low catecholamine doses appear to differ from those reported for cultured cells, as well as those mediated by acute in vivo catecholamine administration.

Animals↗
PubMed1979

Denervation-induced changes in alpha and beta adrenergic receptors of the rat submandibular gland.

Denervation resulted in a marked increase in the beta adrenergic response and isoproterenol-stimulated cyclic adenosine 3':5'-monophosphate accumulation of dispersed cells prepared from a rat submandibular gland. This increase in beta adrenergic response was paralleled by an increase in the density of beta adrenergic receptors in membranes prepared from these glands. Denervation also produced a moderate increase in alpha adrenergic receptor density in membranes prepared from whole glands. However, the alpha adrenergic response in cells, epinephrine-induced release of potassium, appeared unaltered by denervation. Furthermore, membranes prepared from denervated dispersed cells did not show the increase in alpha adrenergic receptor density seen in membranes from an intact denervated gland. These data demonstrate that removal of noradrenergic nerve terminals affects alpha and beta adrenergic receptors differently. While it is clear that beta adrenergic membrane receptors participate in denervation-induced supersensitivity, the changes in alpha adrenergic membrane receptors are more complex and may not contribute to the supersensitivity seen after denervation. On the basis of competitive binding studies, the adrenergic receptors in membranes from intact submandibular glands were subclassified as beta-1 and alpha-2. Denervation did not alter the binding characteristics of the alpha-2 receptors in this gland, demonstrating that alpha-2 adrenergic membrane receptors can be postsynaptic in this adrenergically innervated tissue.

Animals↗
PubMed1990

Prolactin receptors in the primate choroid plexus.

The presence of prolactin receptors in the choroid plexus of primates has until now been assumed to be based on observations made on lower vertebrate animal models. An in vitro autoradiographic technique employing the principles of competitive binding was used to demonstrate the presence of specific prolactin binding sites in the choroid plexus of monkeys. Thus the primate choroid plexus resembles that of lower vertebrate mammals in that it possesses the prerequisite receptor for a prolactin blood-to-CSF transport mechanism.

Animals↗
PubMed1985

Monoclonal antibodies to a 28,000 mol. wt protein antigen of Mycobacterium leprae.

Monoclonal antibodies (MoAb) have been used to analyse a protein antigen from Mycobacterium leprae with a subunit mol. wt of 28,000 daltons. Three different patterns of species specificity were observed with two antibodies being specific for M. leprae, two partially specific, and one broadly cross-reactive amongst all mycobacteria. Competitive binding and sandwich assays demonstrated that the specific and partially specific antibodies recognized closely related regions of the molecule while the cross-reactive antibody recognized a spatially separate epitope on the same polypeptide chain. Identification of specific and cross-reactive epitopes on a single antigenic molecule may be of considerable importance for understanding the functioning of the cell-mediated immune system during leprosy infection and the use of MoAb for such analyses is discussed.

Antibodies, Monoclonal↗
PubMed1986

Distinct epitopes on the T cell antigen receptor of HPB-ALL tumor cells identified by monoclonal antibodies.

The T cell antigen receptor is a approximately 90,000 dalton disulfide linked heterodimer that is non-covalently associated with the CD3 complex. Prior studies have demonstrated that anti-CD3 or -Ti antibodies can mimic antigen and induce cellular proliferation and the secretion of lymphokines. An early event in activation via CD3/Ti is a rapid increase in concentration of intracellular Ca2+ levels. In the present studies, we have produced a panel of monoclonal antibodies (MAb) against the Ti expressed on HPB-ALL tumor cells. All MAb immunoprecipitate a approximately 90,000 dalton disulfide linked heterodimer and induced co-modulation of Ti and CD3. On the basis of competitive binding studies, four distinct epitopes on the Ti of HPB-ALL were identified with MAb L38, L39, L41, and L42. These epitopes were additionally discriminated on the basis of reactivity with normal polyclonal T cell populations and functional effects on HPB-ALL. L39 reacted with a monomorphic epitope present on approximately 2 to 5% of peripheral blood T lymphocytes from all donors examined and was specifically mitogenic for peripheral blood T cells expressing this epitope. L39+ T cells in blood included both CD4+ and CD8+ lymphocytes. In contrast, L38, L41, and L42 failed to react with peripheral blood T cells and were not mitogenic for peripheral blood lymphocytes. Anti-Leu-4, L38, L39, and L41 MAb all induced a rapid increase in (Ca2+)i in HPB-ALL tumor cells, similar to previous findings with anti-CD3 and anti-Ti MAb against various tumor cells and peripheral blood T cells. In contrast, L42 MAb did not induce a substantial increase in (Ca2+)i. Failure of L42 to induce a substantial increased (Ca2+)i could not be attributed to the apparent titer, avidity, or isotype of the antibody. These findings suggest that induction of increased (Ca2+)i upon binding of Ti is epitope dependent. Furthermore, these data demonstrate that several distinct public and private epitopes can be identified on the T cell antigen receptor.

Antibodies, Monoclonal↗
PubMed1986

Characterization of carcinoembryonic antigen-specific monoclonal antibodies and specific carcinoembryonic antigen assay in sera of patients.

Six murine monoclonal antibodies (mAbs) reactive with carcinoembryonic antigen (CEA) were prepared. Four of these, CA204, CA205, CA206 and CA208, were specific to purified CEA whereas the other two, NA201 and NA203, were also reactive with the non-specific cross-reacting antigen (NCA). These mAbs were all IgG1, except one IgG2a (CA206), with high affinities for CEA. NA203, CA204 and CA208 appear to define three different epitopes on the CEA molecule as determined by competitive binding assay. These mAbs also reacted with CEA-producing cells. The treatment of the cells with tunicamycin did not affect the binding of the mAbs to CEA-producing cells. None of the above mAbs bound to CEA-related antigens, NCA-2, alpha 1-acid glycoprotein, or blood group antigens. The combination of CA208 (solid phase) and CA204 (tracer) was used to construct a sensitive CEA-specific sandwich ELISA to detect CEA in the sera of patients with various malignant and non-malignant diseases. Particularly when CEA values were low in sera from non-cancerous patients, the above mAbs sandwich assay showed reduced background reactivity with NCA-like substances and permitted the detection of CEA at a level as low as 1 ng/ml.

Antibodies, Monoclonal↗
PubMed1986

Effect of pentosan polysulphate administration in man on the formation of covalent complexes between heparin cofactor II and thrombin generated in plasma by contact activation.

An assay for the quantification of pentosan polysulphate (PPS) in plasma is described. As PPS has been shown to potentiate thrombin inhibition by the second heparin cofactor (HC II), the principle of this assay was to measure the formation of covalent complexes between HC II and the thrombin generated in plasma after contact activation and recalcification. The complexes were quantified by using purified 125I-HC II added to the plasma as a tracer and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The assay was sensitive to low PPS concentrations (limit of detection: 0.1 microgram/ml) and therefore suitable for the measurement of PPS in plasma after its administration to man. The clearance of PPS was studied in 3 subjects receiving respectively 10, 50 and 100 mg intravenously (IV) and in 3 subjects receiving 35 mg subcutaneously (SC). PPS was still detectable 8 h after 50 and 100 mg IV and 6 h after 35 mg SC. The activated partial thromboplastin time (APTT) was, in comparison, relatively insensitive but for concentrations above 1 microgram/ml the values derived from the APTT and from the SDS-PAGE method fitted. The results were also in general agreement with those reported by McGregor et al. (5) who used a sensitive competitive binding assay. This indicates that low concentrations of PPS previously measured chemically are also pharmacologically active in plasma.

Adult↗
PubMed1987

Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium.

We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.

Alkaline Phosphatase↗
PubMed1988

Study of the epitope structure of purified Dac G I and Lol p I, the major allergens of Dactylis glomerata and Lolium perenne pollens, using monoclonal antibodies.

The use of mAb allowed us to further analyze the cross-reactivity between purified Dac g I and Lol p I, the major allergens of Dactylis glomerata (cocksfoot) and Lolium perenne (Rye grass), respectively. It was first shown, using IEF, followed by immunoprinting, that serum IgE antibodies from most grass-sensitive patients recognize both Dac g I and Lol p I. Second, three different anti-Lol p I mAb, 290A-167, 348A-6, and 539A-6, and one anti-Dac g I mAb, P3B2 were all shown to react with Dac g I and Lol p I, indicating that the two molecules share common epitopes. Epitope specificity of the mAb was determined by competitive binding inhibition of a given labeled mAb to solid phase fixed Dac g I or Lol p I by the mAb. The results indicated that the four mAb are directed against four different and non-overlapping epitopes present on both allergens. Using double-binding RIA, our data strongly suggest that the common epitopes are not repetitive on both molecules. In addition to their similar physicochemical characteristics, such as isolectric points and m.w., Dac g I and Lol p I share four identical epitopes. Binding inhibition of human IgE to Lol p I and Dac g I by the mAb was also assessed. The results indicated that each mAb was able to inhibit such reactions to variable degree but no additive inhibition was observed when two mAb of different specificities were used in combination, suggesting that the human IgE binding site is partially shared by each epitope recognized by the four mAb.

Allergens↗
PubMed1989

[Comparative studies on monoclonal antibody KM10 and anti-CEA monoclonal antibodies].

The specificity of KM10 was evaluated in comparison with newly developed anti-CEA monoclonal antibodies (A10, B9, JA4, AH3). Both KM10 and all anti-CEA monoclonal antibodies reacted with CEA in ELISA system, and with adenocarcinoma of the stomach, colon, and pancreas in the immunohistochemical assay. B9, JA4, and AH3 were suggested to react with CEA related antigens, such as NCA and BGPI, whereas KM10 and A10 were suggested to recognize the distinctive part of CEA. The antigenic determinant of CEA reactive with KM10 and A10 was revealed to be protein moiety after enzyme treatment. The competitive binding inhibition assay, however, indicated that epitopes of KM10 and A10 were different each other. Enzyme immunoassay using both KM10 and A10 could detect CEA. These findings showed the possible use of both KM10 and A10 for clinical diagnosis and treatment by means of targeting for the distinctive part of CEA.

Adenocarcinoma↗
PubMed1989

Carboxyl-terminal modification of a gastrin releasing peptide derivative generates potent antagonists.

Gastrin releasing peptide (GRP) is a 27-residue peptide hormone which is analogous to the amphibian peptide bombesin. GRP serves a variety of physiological functions and has been implicated as an autocrine factor in the growth regulation of small cell lung cancer cells. We have developed a series of potent GRP antagonists by modification of the COOH terminus of N-acetyl-GRP-20-27. The most potent member of this series, N-acetyl-GRP-20-26-OCH2CH3, exhibits an IC50 of 4 nM in a competitive binding inhibition assay. This compound blocks GRP-stimulated mitogenesis in Swiss 3T3 mouse fibroblasts, inhibits GRP-dependent release of gastrin in vitro, and blocks GRP-induced elevation of [Ca2+]i in H345 small cell lung cancer cells. These results demonstrate that while residues 20-27 of GRP influence binding of the parent peptide to its receptor, the COOH-terminal amino acid is primarily responsible for triggering the subsequent biological response.

Amino Acid Sequence↗
PubMed1989

Opioid receptor selectivity of peptide models of beta-endorphin.

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic alpha-helical domain (residues 13-29) in beta-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for mu- and delta-opioid receptors than for kappa opioid receptors. Relative to beta-endorphin, the peptide models were 2-3 times more potent in the mu and kappa receptor binding assays, and about equipotent in the delta receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to beta-endorphin and the other was twice as potent. Like beta-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by mu receptors and not kappa receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of beta-endorphin.

Amino Acid Sequence↗
PubMed1985

Epitope-specific antibody response to murine hepatitis virus-4 (strain JHM).

Monoclonal hybridoma antibodies to the structural proteins of murine hepatitis virus-4, strain JHM (MHV-4) were used in a competition binding enzyme immunoassay to analyze at the epitope level the antibody response of mice after infection with MHV-4. Colonized mice often had pre-existing MHV antibodies directed against epitopes on the E2 glycoprotein, the E1 glycoprotein, and the nucleocapsid protein. These mice generated a secondary antibody response after virus inoculation, reaching peak levels 7 days after infection. In contrast, Nude/+ mice raised in a pathogen-free colony had no detectable circulating MHV antibodies and generated a primary antibody response which gradually increased to peak levels 14 to 28 days after infection. Kinetics of antibody responses against specific epitopes usually correlated well with measured total virus-specific antibody responses, but variation was observed. Mice injected with three antigenically distinct strains of MHV made antibody responses to conserved epitopes but not to an antigenic determinant absent in these strains. Measurement of epitope-specific responses in a polyclonal population of viral specific antibodies is feasible and a valuable adjunct in understanding viral immunity.

Animals↗
PubMed1989

[Enzyme immunoassay for thromboxane B2].

A sensitive and specific enzyme immunoassay for TXB2, the stable degradation product of thromboxane A2, was developed, in which the hapten molecule was labeled with pure peroxidase. After competitive binding to antibody between enzyme-labeled and free TXB2, the immunoreactive product was precipitated by double antibody technique, and the enzyme activity of the precipitate was determined spectrophotometrically. The procedures allowed a determination of 3-200 pg TXB2/tube (0.081 to 5.4 nmol/l).

Animals↗
PubMed1989

Immunologic significance of diverse specificity of monoclonal antibodies against mannans of Candida albicans.

Two agglutinating IgM mAb against mannan Ag of Candida albicans strains were investigated for their specificity. The agglutinating patterns of both mAb with a panel of stationary phase of yeast cells of standard strains did not match those of any known polyclonal antibody (PAb) factors. The reactive patterns of both mAb for a given panel of 202 isolates of seven Candida species and the mode of competitive binding between mAb and a PAb factor, as determined by a combination of direct and indirect immunofluorescence staining, demonstrated that the two mAb were a part of PAb factor 4 (a prescribed reactive pattern of adsorbed PAb) but were different from each other in their specificity. The thereby designated mAb 4b and 4c were tested under PAb factor 4-positive Candida strains and further division into two to three serotypes of each species were made. 1H-Nuclear magnetic resonance spectra (500 MHz) of purified neutral mannans from strains of each serotype showed that although the 1H-nuclear magnetic resonance spectra of mannans from two subtypes of C. albicans serotype A and from each of two serotypes of Candida guilliermondii and Candida glabrata were identical or similar, the two mAb were still able to distinguish their fine determinant structures. Our findings suggest that mAb with entirely identical specificity cannot be produced against even the same determinant groups of mannan. In addition, the fact that microheterogeneity may occur without limit in the mannans of the strains suggests that antibodies with unlimited diverse specificities are produced directed against these antigenic varieties as well.

Agglutination Tests↗