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PubMed1981

beta-Galactosidase-linked immunoassay of prostaglandin F2 alpha.

A sensitive and specific enzyme immunoassay for prostaglandin F2 alpha was developed in which prostaglandin F2 alpha was labeled with beta-D-galactosidase. After competitive binding to antibody between enzyme-labeled and free prostaglandin F2 alpha, the immunoreactive product was precipitated by double antibody technique, and the enzyme activity of the precipitate was determined fluorometrically. The procedures allowed determination of 0.03-20 pmol of prostaglandin F2 alpha. The detection limit of 0.03 pmol was comparable with that of previously reported radioimmunoassays. Enzyme immunoassay and radioimmunoassay showed almost the same cross-reactivity with other prostaglandins and metabolites.

Binding, Competitive↗
PubMed2000

Lack of significant estrogenic or antiestrogenic activity of pyrethroid insecticides in three in vitro assays based on classic estrogen receptor alpha-mediated mechanisms.

Estrogenic and antiestrogenic activity of pyrethroid insecticides (d-trans-allethrin, cypermethrin, empenthrin, fenvalerate, imiprothrin, permethrin, d-phenothrin and prallethrin) was evaluated using a suite of three in vitro assays based on classic human estrogen receptor alpha (hER alpha)-mediated mechanisms. A mammalian cell-based luciferase reporter gene assay was developed for examining effects on hER alpha-mediated gene activation. hER alpha-independent effects on the gene activation were examined using control cells with constitutive luciferase activation by a herpes simplex virus thymidine kinase (HSV-TK) promoter for determining appropriate dose levels of test chemicals. Moreover, the test chemical-dependent interaction between hER alpha and a coactivator (transcriptional intermediary factor 2: TIF2) was analyzed by a yeast two-hybrid method, competitive binding to hER alpha being assayed by a fluorescence polarization method. Significant (p < 0.05) positive effects of estrogenic substances (E2/estradiol, diethylstilbestrol, and p-nonylphenol) were detected in all assays. An antiestrogen, 4-hydroxytamoxifen, significantly inhibited E2-mediated transactivation and interaction between hER alpha and TIF2 through hER alpha binding (p < 0.05). However, none of the pyrethroids tested showed significant (p < 0.05) estrogenic or antiestrogenic effects (100 nM-10 microM), indicating that they do not impact on the classic hER alpha-mediated activation pathway in vitro.

Binding, Competitive↗
PubMed1996

Endothelin-1 stimulates deoxyribonucleic acid synthesis and contraction in testicular peritubular myoid cells.

In the testis, endothelin-1 (ET-1) is produced by Sertoli cells, and it has been proposed to be a paracrine factor participating in the regulation of tubular and interstitial function. The response of purified testicular peritubular myoid cells (TPMC) to ET-1 was investigated in the present study. TPMC expressed a single class of high-affinity receptors that were shown by competitive binding experiments with sarafotoxin-6c to belong to the ETA subtype. The binding of ET-1 to TPMC was followed by rapid internalization of the receptor-ligand complex. ET-1 induced a prompt rise in intracellular Ca2+ concentration that was blunted in Ca(2+)-free medium and in the presence of Mn2+ or of voltage-operated-calcium-channel (VOC) blockers, indicating that both Ca2+ mobilization from intracellular stores and extracellular Ca2+ influx were involved. Thymidine uptake was promoted by ET-1 in a time-dependent manner, and the use of cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp] (BQ123) reduced the incorporation of thymidine. Protein kinase C (PKC) inhibition (100 nM calphostin C) abolished the ET-1 mitogenic effect. ET-1 also promoted TPMC contraction, as evaluated in collagen lattices, in a dose-related manner, with the half-maximal response observed at 1 nM. As in the case of mitogenesis, BQ123 blunted ET-1-induced contraction. PKC inhibition abolished ET-1-induced contraction. These findings indicate that ET-1 promotes DNA synthesis and contraction of TPMC and that both effects are mediated by PKC; they suggest as well that ET-1 may have a physiological role in the interaction between Sertoli cells and TPMC.

Amino Acid Sequence↗
PubMed1998

Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor.

Impaired insulin secretion is a hallmark in both type I and type II diabetic individuals. Whereas type I (insulin-dependent diabetes mellitus) implies ss-cell destruction, type II (non-insulin dependent diabetes mellitus), responsible for 75% of diabetic syndromes, involves diminished glucose-dependent secretion of insulin from pancreatic beta-cells. Although a clear demonstration of a direct effect of 17beta-estradiol on the pancreatic ss-cell is lacking, an in vivo insulinotropic effect has been suggested. In this report we describe the effects of 17beta-estradiol in mouse pancreatic ss-cells. 17beta-Estradiol, at physiological concentrations, closes K(ATP) channels, which are also targets for antidiabetic sulfonylureas, in a rapid and reversible manner. Furthermore, in synergy with glucose, 17beta-estradiol depolarizes the plasma membrane, eliciting electrical activity and intracellular calcium signals, which in turn enhance insulin secretion. These effects occur through a receptor located at the plasma membrane, distinct from the classic cytosolic estrogen receptor. Specific competitive binding and localization of 17beta-estradiol receptors at the plasma membrane was demonstrated using confocal reflective microscopy and immunocytochemistry. Gaining deeper knowledge of the effect induced by 17beta-estradiol may be important in order to better understand the hormonal regulation of insulin secretion and for the treatment of NIDDM. receptor.

ATP-Binding Cassette Transporters↗
PubMed1997

Prepro-neuropeptide Y mRNA and NPY binding sites in human inferior vagal ganglia.

The inferior vagal ganglia contain the cell bodies of centrally projecting vagal afferent neurones. Using in situ hybridization-histochemistry with a combination of two antisense neuropeptide Y (NPY) oligonucleotides, we have demonstrated that a population of human inferior vagal perikarya express mRNA encoding prepro-NPY, the precursor of NPY. In vitro receptor autoradiography, using both [125I]Bolton Hunter-NPY ([125I]BH-NPY, 15 pM) and [125I]peptide YY ([125I]PYY, 25 pM), enabled visualization of NPY binding sites. Competition binding with NPY (1 microM), PYY (1 microM) and [Leu31,Pro34]NPY (100 nM), suggest that both Y1 and Y2 receptor subtypes are present on human vagal afferent neurones. These observations suggest a potential role for NPY in neuromodulation of vagal transmission in humans.

Autoradiography↗
PubMed1999

Pulmonary natriuretic peptide system during rat development.

Maturational changes in the rat lung natriuretic peptide system were studied postnatally in 1-, 4-, and 22-day-old rats. Lung atrial natriuretic factor (ANF) content increased significantly from day 1 to day 4 (712+/-188 vs. 1905+/-520 pg/mg protein; p<0.01) but decreased to 532+/-41 pg/mg protein, on day 22. These changes paralleled ANF messenger RNA (mRNA) detected by reverse transcribed polymerase chain reaction (RT-PCR). Rat pulmonary development also was associated with quantitative and qualitative alterations in ANF receptors. Competitive-binding radioreceptor assays of lung membranes with 125I-ANF and increasing concentrations of unlabeled ANF revealed that the natriuretic peptide receptor-binding sites (Bmax) progressively increased with age from 112 +/-21 fmol/mg protein at day 1 to 211+/-16 (p<0.02) and 326+/-62 fmol/mg protein (p<0.04) in 4- and 22-day-old rats, respectively. Autoradiographic studies of 125I-tyr(0)CNP binding to lung sections revealed that the levels of the natriuretic peptide receptor B (NPR-B) were undetectable. On the other hand, binding of 125I-ANF increased with age, and the higher binding at 4 days was mainly due to increased density of the clearance receptor-C (NPR-C), and at 22 days due to increased natriuretic peptide receptor-A (NPR-A). The increase in natriuretic peptide binding was confirmed at the level of synthesis, where RT-PCR revealed that NPR-A mRNA significantly increased (p<0.01) in 22-day-old rats. In conclusion, these studies demonstrate that the rat pulmonary natriuretic peptide system is altered during development. The altered synthesis of lung natriuretic peptides and their receptors may play a role in the postnatal adaptation of pulmonary circulation.

Animals↗
PubMed1997

Evaluation of the effect of endothelin-1 and characterization of the selective endothelin a receptor antagonist PD155080 in the prostate.

PURPOSE: To evaluate the contractile effect of endothelin-1 (ET-1) on prostatic urethral pressure and to characterize the effect of the selective ETA receptor antagonist PD155080 on ET-1 mediated prostatic urethral pressure. MATERIALS AND METHODS: The effect of intravenous ET-1 administration on canine urethral pressure was determined in the presence and absence of PD155080. The affinity of PD155080 for endothelin-mediated contraction was determined using antagonist dissociation studies. Saturation and competition binding studies were performed using [125I] ET-1 in both human and canine prostate. RESULTS: ET-1 bolus injection elicited shallow and prolonged increases the prostatic urethral pressure. Pretreatment with PD155080 totally abolished the urethral contractile response to ET-1. Specific [125I] ET-1 binding was saturable and of high affinity. Two ET receptor subtypes (ETA receptor, ETB receptor) have been identified in human prostate. The ratio of ETA to ETB receptors was approximately 1.5:1 in both human and canine prostates. Isometric tension studies revealed that PD155080 shifted the ET-1 dose-response curves to the right and exhibited no effect on the ETB receptor selective agonist sarafotoxin dose-response curves. CONCLUSION: ET-1 mediates prostate smooth muscle tone and may play a role in the pathophysiology and treatment of benign prostatic hyperplasia (BPH).

Animals↗
PubMed1999

Regulation of cholecystokinin-mediated amylase secretion by leptin in rat pancreatic acinar tumor cell line AR42J.

Expression of the long form of the leptin receptor, the isoform that is considered to have full signaling capability, has been reported in the central nervous system and several peripheral cell types. However, only a few cell lines have been shown to express the long form of the receptor. AR42J, a cell line derived from azaserine-treated rat pancreas, is a common model for pancreatic acinar cell secretion. In this study, the presence of leptin-receptor variants and leptin action was evaluated in this cell line. Messenger RNAs for both the long and a short form of the leptin receptor were detected by reverse transcription-polymerase chain reaction (RT-PCR) in AR42J cells, and authenticity of the receptor was confirmed by DNA sequencing. Competitive binding studies demonstrated that binding of radiolabeled leptin was specific and did not cross-react with cholecystokinin (CCK). Biologic effects of leptin on amylase release and intracellular calcium mobilization were further assessed in the presence and the absence of CCK, a known pancreatic secretagogue. Although leptin alone (< or =200 ng/ml) did not affect basal amylase release, it inhibited amylase release stimulated by 1 nM CCK by 48%. Leptin alone had no significant effect on calcium mobilization. However, pretreatment of leptin (10 and 100 ng/ml) enhanced calcium responses stimulated by CCK. These data demonstrate that the rat pancreatic tumor cell line AR42J expresses a functional form of the leptin receptor that modulates the action of CCK in calcium mobilization and amylase release.

Amylases↗
PubMed1980

Substrate-labeled fluorescent immunoassay for phenobarbital.

An assay for the anticonvulsant drug phenobarbital (PB) has been developed that is based on the principles of the substrate-labeled fluorescent immunoassay. A fluorogenic enzyme substrate, galactosyl umbelliferone, was covalently linked to a derivative of PB. The labeled drug, galactosyl umbelliferone-PB (GUPB), is nonfluorescent under conditions of the assay; however, hydrolysis of the galactosyl moiety by bacterial beta-galactosidase yields a fluorescent product. When GUPB is bound by antibody to PB, it is not a substrate for enzymatic hydrolysis. Thus, only GUPB not bound to antibody is hydrolyzed. In competitive binding reactions, using a fixed concentration of GUPB and a limiting amount of antibody, the PB in serum and the GUPB compete for antibody-binding sites. The fluorescence produced upon enzymatic hydrolysis of unbound GUPB is directly proportional to the concentration of PB. Unknown serum levels of PB are determined from a standard curve of fluorescent intensity versus standard PB concentrations. The assay is specific, sensitive, and easy to perform. It is carried out by adding the equivalent of 2 microliters of serum standard or unknown directly to a cuvette containing 3 ml of a buffered solution of antibody and enzyme. One-hundred microliters of GUPB is added, and the fluorescence intensity is measured after a fixed time (any time from 5 to 90 min). Using clinical specimens, our assay correlated well with a commercial enzyme immunoassay (correlation coefficient = 0.97) and had an interassay precision of less than 7%.

Antibodies↗
PubMed1985

Therapeutic potential of monoclonal antibodies to the leukocyte-common antigen. Synergy and interference in complement-mediated lysis.

A series of rat monoclonal antibodies against the human leukocyte-common antigen were isolated and, by means of competitive binding assays with purified antigen, two distinct groups were defined that recognize different epitopes of the molecule. None of these antibodies were lytic with human complement, but when antibodies against each of these two epitopes were used in combination, synergistic lysis with human complement could be obtained. Synergistic lysis was only seen when each antibody of the pair was of the IgG2b subclass. IgG2a antibodies could not synergize, and in fact could interfere with lysis obtained by the pair of IgG2b antibodies. Although synergy has so far only been studied for rat monoclonal antibodies we also show that it is possible with mouse antibodies or with combinations of mouse and rat antibodies. The information about the principles of synergy and its interference should provide a rationale for using well-planned cocktails of monoclonal antibodies for therapy rather than a shotgun polyclonal antiserum.

Animals↗
PubMed1985

Characterization of major structural proteins of measles virus with monoclonal antibodies.

We have prepared and characterized monoclonal antibodies against five major structural proteins, i.e. the HA, P, NP, F and M proteins, of measles virus. At least three non-overlapping antigenic sites were delineated on the HA protein, three on the P, four on the NP, four on the F and five on the M proteins by competitive binding assays. Antigenic sites on the HA and F proteins roughly represented functional domains defined by serological tests. The reactivity of monoclonal antibodies with various measles virus strains including those from subacute sclerosing panencephalitis (SSPE) and other members of the morbillivirus family was studied by immunofluorescence. A monoclonal antibody or set of monoclonal antibodies to each of the antigenic sites showed a characteristic pattern of cross-reactivity with heterologous strains. The HA and NP proteins were antigenically the most variable, followed by the F and M proteins, while the P protein was relatively stable. None of the 14 anti-M monoclonal antibodies reacted with non-virus-producing SSPE cells, strongly suggesting the absence of M protein in these cells.

Antibodies, Monoclonal↗
PubMed1986

Antigenic comparison of envelope protein E between Japanese encephalitis virus and some other flaviviruses using monoclonal antibodies.

The antigenic relationships between Japanese encephalitis (JE) virus and several other flaviviruses have been investigated using anti-JE virus monoclonal antibodies (MAbs). Seventeen MAbs directed against envelope protein E of JE virus were characterized and divided into eight MAb groups based on reactivity patterns in haemagglutination inhibition test, neutralization (N) test, ELISA and competitive binding assay with JE virus. The results suggest the existence of at least eight epitopes on the E protein of JE viruses. Analysis of cross-reactivity of the antibodies with several other flaviviruses indicated these findings JE virus, belonging to West Nile (WN) subgroup, is antigenically closely related to viruses in the same subgroup, i.e. Murray Valley encephalitis (MVE), WN and St. Louis encephalitis (SLE) viruses. Of these three viruses, JE virus has the closest relationship with MVE virus. WN virus is relatively close to JE virus, whereas SLE virus is the least closely related. Dengue viruses types 1 and 2, which belong to another subgroup of flaviviruses, show markedly less antigenic homology to JE virus. One of the critical N sites on the E protein showed JE virus specificity. Some cross-reactive antibodies which did not neutralize JE virus showed low but significant N activity against several other flaviviruses. Mixtures of several MAbs, which showed different reactivity patterns, potentiated the N activity against not only JE virus but also other members of the WN subgroup of flaviviruses, namely MVE, WN and SLE viruses.

Antibodies, Monoclonal↗
PubMed1986

Analysis of the respiratory syncytial virus fusion protein using monoclonal and polyclonal antibodies.

At least four distinct epitopes are described on the respiratory syncytial virus fusion protein (VP70) using 13 monoclonal antibodies in solid-phase competitive binding studies. Two, and possibly three, fusion-inhibiting epitopes, one non-fusion-inhibiting neutralizing epitope, and one non-neutralizing epitope are described. All but the latter site demonstrated partial overlap, suggesting possible topographical proximity of these epitopes. Polyclonal rabbit sera to VP70, which neutralized virus but did not inhibit fusion of infected cells, blocked the binding of all fusion-inhibiting monoclonal antibodies to VP70 in the solid-phase assay but did not inhibit their effect in vitro. Western blot analysis of these monoclonal antibodies demonstrates that one fusion-inhibiting epitope is localized on the 48K fragment of VP70 and is resistant to denaturation by heat, 2-mercaptoethanol and SDS.

Antibodies, Monoclonal↗
PubMed1988

Monoclonal antibodies to the E1 and E2 glycoproteins of Sindbis virus: definition of epitopes and efficiency of protection from fatal encephalitis.

Protection of mice from fatal neuroadapted Sindbis virus encephalitis can be accomplished by passive transfer of monoclonal antibodies (MAbs) to either the E1 or E2 glycoprotein of Sindbis virus. Both neutralizing and non-neutralizing MAbs can be protective. To define further the characteristics of MAbs that provide protection from fatal disease, antigenic epitopes on the E1 and E2 glycoproteins were identified using a competitive binding enzyme immunoassay. Four distinct epitopes on E1 and three on E2 were defined. MAbs to all E1 epitopes, both neutralizing (three) and non-neutralizing (one) protected mice from fatal encephalitis. MAbs to the E2 neutralizing epitopes (two) protected mice from fatal encephalitis while those to the non-neutralizing epitope did not. The efficiency of protection from fatal Sindbis virus encephalitis of four neutralizing and non-neutralizing protective anti-E1 and anti-E2 MAbs representing different epitopes was compared. The neutralizing MAbs (against epitopes E2-ab, E2-c and E1-c) gave 50% protection at lower doses (2 to 20 micrograms) than the non-neutralizing MAb representing epitope E1-e (150 micrograms) when given before virus challenge. When given after virus challenge, MAbs to E2-ab and E2-c protected at lower doses (0.03 to 0.3 micrograms) than did either MAbs to E1-c (greater than 100 micrograms) or E1-e (10 micrograms). The MAbs to E1-e, E2-ab and E2-c were required in larger amounts to afford protection before than after challenge, while the opposite was true for MAb to E1-c.

Animals↗
PubMed1989

Characterization of monoclonal antibodies against four structural proteins of rinderpest virus.

Twenty-four monoclonal antibodies (MAbs) against rinderpest virus (RPV) were established and characterized by several serological tests. Of the 24 MAbs. 10 recognized the nucleoprotein (NP), six the phosphoprotein (P), four the haemagglutinin (H), and two the fusion (F) protein as determined by radioimmunoprecipitation assay. The specificities of the remaining two MAbs could not be determined. From a competitive binding assay using MAbs against each structural protein, at least five, four and two separate antigenic sites were identified on the NP, P and H proteins, respectively. MAbs against the H protein neutralized the infectivity of the virus, but those against the F protein were only neutralizing in the presence of guinea-pig complement. The reactivities of each of the MAbs for other strains of morbillivirus were tested using an indirect immunofluorescent antibody assay. The MAbs against four out five antigenic sites on the NP showed cross-reactivity amongst all the strains of morbillivirus tested whereas the fifth antibody reacted only with RPV. Of the antibodies specific for the P protein, the antibody against one site was cross-reactive with all the strains of RPV, measles virus (MV) and canine distemper virus (CDV), the antibody against another site was reactive with RPV and MV but not with CDV, and the antibodies against the other two sites were specific for RPV.

Antibodies, Monoclonal↗
PubMed1989

Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies.

Competitive binding studies and antigen capture assays were done with monoclonal antibodies (MAbs) raised against hog cholera virus (HCV) to map the corresponding epitopes. A model was constructed in which the 13 epitopes were situated in four distinct antigenic domains: A, B, C and D. Domain A was subdivided into A1, A2 and A3. The functional relevance of this model was assessed by the characterization of pestivirus strains, by neutralization studies with the MAbs, and by isolation of variants that escaped neutralization. The topographical arrangement of the epitopes, as constructed in the model, was corroborated by the functional assays. The MAbs that defined domains A1 and A2 recognized all 94 HCV strains tested. Domains A3, B, C and D varied among the HCV strains. Neutralization was observed with MAbs defining domains A1, B and C. Synergistic neutralization occurred using MAbs against domains A1 and B, and A1 and C, but not within the domains. With MAbs defining A1, B or C, variants could be isolated that escaped neutralization and immunostaining by these MAbs.

Antibodies, Monoclonal↗
PubMed1989

Location of neutralizing epitopes on the fusion protein of Newcastle disease virus strain Beaudette C.

A panel of eight neutralizing monoclonal antibodies (MAbs) against the fusion (F) protein of Newcastle disease virus (NDV) has been shown to locate a major antigenic site on the basis of competitive binding assay and additivity index studies. Five epitopes (A1 to A5) have been located within this site on the F protein of the Beaudette C strain of NDV on the basis of cross-resistance plaque assays of MAb-resistant mutants raised against these MAbs. Epitopes A1, A4 and A5 are distinct; epitope A2 partially overlaps epitope A3. Nucleotide sequence analysis of the F genes of MAb-resistant mutants showed that each predicted single amino acid substitutions ranging from amino acid residues 157 to 171 for epitope A4 and at residues 72, 78, 79 and 343 for epitopes A1, A2, A3 and A5 respectively. These locations indicate that both the F1 and F2 fragments are involved in the formation of a single antigenic site and suggest the involvement of extensive protein folding in the active form of this F protein.

Amino Acid Sequence↗
PubMed1989

Characterization of a new virus-neutralizing epitope that denotes a sequential determinant on the rabies virus glycoprotein.

Two new monoclonal antibodies (MAbs) derived from mice immunized with the Pitman-Moore (PM) strain of rabies virus were used to identify and characterize two unique antigenic determinants on the rabies virus glycoprotein. One of the determinants, which defined an additional antigenic site on the rabies virus glycoprotein, was delineated as a distinct epitope by the newly generated MAb, 6-15C4, in competitive binding studies and by comparative antigenic analysis of neutralization-resistant variant viruses. Both antigenic determinants were compared with the five previously described antigenic sites which bind virus-neutralizing antibodies on the challenge virus standard (CVS) and Evelyn-Rokitnicki-Abelseth (ERA) strain glycoproteins. The results presented in this communication show that the 6-15C4 epitope is the first epitope described in the rabies virus glycoprotein that does not depend on the native conformation of the glycoprotein for binding virus-neutralizing antibody. These data suggest that it may be possible to generate a synthetic peptide vaccine against rabies.

Animals↗