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J Grassi

Publications and source records attributed to J Grassi.

At least 73 records · Page 4Linked to original sources
PubMed1995

Immunological studies of human constitutive cyclooxygenase (COX-1) using enzyme immunometric assay.

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.

Amino Acid Sequence↗
PubMed1995

Differential measurement of constitutive (COX-1) and inducible (COX-2) cyclooxygenase expression in human umbilical vein endothelial cells using specific immunometric enzyme immunoassays.

We have produced and characterized monoclonal antibodies (mAbs) directed against a specific carboxyterminal sequence of human cyclooxygenase-2 (residues 580-598). A rabbit polyclonal antiserum was also raised against another sequence of 10 amino acids (residues 570-581) not present in human constitutive cyclooxygenase-1. Affinity-purified polyclonal antibodies, coated on microtiter plates, were used as capture antibodies in a two-site immunometric assay, with an mAb-acetylcholinesterase conjugate used as tracer. The detection limit was 500 fmol/ml of peptide C3-COX2 (residues 570-595). The assay was specific for the cyclooxygenase-2 (COX-2) isoform, since no immunoreactivity could be detected in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). In contrast, extracts from cultured human umbilical vein endothelial cells challenged with 20 nM phorbol myristate acetate (PMA) showed an increase in COX-2 immunoreactivity related both to the increase in enzyme activity and the variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immunometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by measuring the immunoreactivity of the fractions obtained after molecular sieve chromatography of control and stimulated cell extracts, and corroborated the marked enhancement of COX-2 by comparison with COX-1. Treatment of PMA-activated cells with H-7 or actinomycin D totally abolished the COX-2 signal and had little effect on COX-1. No significant variation in COX-2 immunoreactivity was observed using the inactive isomer 4 alpha-PMA, even at 100 nM. These assays constitute the first quantitative analysis of constitutive COX-1 and of inducible COX-2 in nucleated cells at the protein level.

Amino Acid Sequence↗
PubMed1995

Immunometric assay of BN 52080, a heptapeptide C-terminal analogue of sorbin.

A novel type of enzyme immunometric assay has been developed for a heptapeptide, BN 52080. This compound is a short C-terminal analogue of sorbin and is under clinical evaluation for treatment of chronic diarrhea. In this solid-phase immobilized epitope immunoassay (SPIE-IA), the peptide is first immunologically bound to polyclonal antibodies adsorbed to a solid phase and then, after covalent immobilization with glutaraldehyde, is released from the antibody paratope by NaOH. The peptide linked to the solid phase is further quantified with a tracer consisting of the same antibodies purified by affinity chromatography and coupled to acetylcholinesterase. This assay has a detection limit of 10 pg/ml and is therefore five times more sensitive than competitive enzyme immunoassay using the same antibodies and BN 52080 coupled to acetylcholinesterase as tracer. The assay is specific and allows direct measurement of peptide in human plasma after subcutaneous or intravenous administration of 200 micrograms of BN 52080 to volunteers.

Amino Acid Sequence↗
PubMed1994

Enzyme immunometric assay for leukotriene C4.

An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.

Acetylcholinesterase↗
PubMed1994

A sensitive and specific enzyme immunoassay for cyclomaltoheptaose and some derivatives.

Antibodies were raised against cyclomaltoheptaose (beta-cyclodextrin), providing a highly sensitive enzyme immunoassay for beta-cyclodextrin and some derivatives with a detection limit of 120 pg/mL. Investigations of cross-reactivities with a wide variety of linear and cyclic maltooligosaccharides demonstrate that the antibodies are highly specific for cyclomaltoheptaose and a number of derivatives. The epitope is probably located on the secondary hydroxyl groups of the rim side. This enzyme immunoassay is shown to be suitable to detect cyclomaltoheptaose in urine and in plasma.

Animals↗
PubMed1994

Immunometric assay of low molecular weight haptens containing primary amino groups.

A new enzyme immunometric assay of small haptens containing primary amino groups (thyroxine, MW 777; substance P, MW 1347; endothelin, MW 2492) is described. The procedure involves different sequential steps: (1) immunocapture of the haptens (standard or sample) by monoclonal anti-hapten antibodies coated on 96-well microtiter plates; (2) cross-linking of haptens via their amino groups to the wells using homobifunctional reagents (glutaraldehyde or disuccinimidyl suberate); (3) denaturing treatments (HCl or methanol); (4) measurement of linked epitope using the same monoclonal anti-hapten antibodies labeled with acetylcholinesterase. A minimal detectable concentration in the 4-10 fmoL/mL range was observed. Each assay appeared to be 70-200 times more sensitive than conventional competitive enzyme immunoassay using the same monoclonal antibody-coated plate technology and acetylcholinesterase-hapten conjugates as enzymatic tracers. Precision and specificity were very satisfying. Good correlation was noted between this assay and the competitive assays performed for different biological samples (plasma, tissues, or supernatant cell culture).

Amines↗
PubMed1994

Biological activities of [Thr2]sarafotoxin-b, a synthetic analogue of sarafotoxin-b.

The 21 amino acid sarafotoxins (SRTX) c and d/e as well as endothelin-3 (ET-3) are known to be less toxic and weaker pharmacologically than the other isopeptides SRTX-a, SRTX-b and ET-1. Since SRTX-c, SRTX-d/e and ET-3 possess a Thr instead of a Ser at position 2, we investigated the possibility that this mutation could be responsible for the observed biological differences. Here we show that the synthetic [Thr2]SRTX-b has indeed a lower vasoconstriction efficacy (approximately 35%) in the rabbit aorta, but it is nearly as potent as SRTX-b in toxicity tests and in influencing contraction of the rat uterus. Using monoclonal antibodies directed against the structurally related endothelin-1, we also show that the antigenicity of the analogue is comparable to that of SRTX-b, suggesting that the overall structure of the two peptides is similar, despite the substitution at position 2. We suggest that the Thr2 substitution contributes to the lower activity of the 'weak' peptides in some systems; however, additional substitutions found in the 'weak' peptides of the ET/SRTX family most probably contribute to their low pharmacological activity.

Amino Acid Sequence↗
PubMed1994

Cholinesterases display genuine arylacylamidase activity but are totally devoid of intrinsic peptidase activities.

The purpose of this article was to evaluate the intrinsic character of arylacylamidase and peptidase activities that are often detected along with cholinesterase activities. Various pools of commercial or affinity-purified acetylcholinesterases (AChEs) were examined. Affinity-purified AChE displays esterase- and amidase-specific activities that are similarly enriched when compared with commercial AChE. By contrast, commercial AChE exhibits much higher tryptic-like and carboxypeptidase-specific activities than the affinity-purified enzyme. The parallel enrichment in esterase and arylacylamidase suggests that these two activities are copurified, whereas peptidases do not seem to behave similarly. We show that trypsinolysis or spontaneous degradation of affinity-purified AChE leads to the conversion of the 75-kDa monomer protein into two fragments of 50 and 25 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. However, these modifications are without effect on the esterase, arylacylamidase, and peptidase activities. This clearly shows that AChE does not behave as a zymogen of peptidases that would have been activated on autolysis of AChE. Immunoprecipitation of AChEs with a purified monoclonal antibody directed toward electric eel AChE totally separated the esterase and arylacylamidase activities (pellet) from peptidase activities (supernatant). The immunoprecipitated AChEs could be dissociated from the interaction with IgGs. These resolubilized AChE preparations have kept the same percentage of initial esterase and arylacylamidase activities but were totally devoid of peptidase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1994

Antipeptide polyclonal antibodies that recognize a substance P-binding site in mammalian tissues: a biochemical and immunocytochemical study.

We used complementary peptide methodology to obtain antibodies against the receptor for the neuropeptide substance P, specifically directed at the ligand-binding domain. Rabbits were immunized with two distinct peptides derived from the sequence of the RNA complementary to the mRNA for substance P. Binding experiments revealed that antipeptide polyclonal antibodies were able to recognize, through their paratope, a specific binding site on the rat parotid cell membranes. Substance P and antibodies competed for this binding site, because preincubation of membranes in the presence of substance P significantly reduced antibody binding, and conversely, preincubation of membranes in the presence of antibodies partly inhibited the binding of radioiodinated substance P. Immunocytochemical experiments performed on the rat cervical spinal cord show that the distribution of labeling by antibodies is similar to that observed by conventional autoradiography using 125I-substance P. Here again, control experiments demonstrated that antibodies and substance P were competing for the same binding site on the spinal cord. These biochemical and immunocytochemical data indicate that antipeptide antibodies recognize a substance P membrane binding site in nervous and nonnervous mammalian tissues. This site is likely to correspond to the NK1 specific receptor for substance P.

Amino Acid Sequence↗
PubMed1994

Two-site immunoradiometric assay of chicken acetylcholinesterase: active and inactive molecular forms in brain and muscle.

Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC 3.1.1.7). Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C-131, which is conformation dependent and preferentially recognizes active AChE. We have set up two-site immunoradiometric assays, using an immobilized capture antibody, C-6 or C-131, and a radiolabeled antibody, 125I-C-54. The C-6/C-54 assay quantifies the totality of inactive and active AChE subunits: It detects 10(-3) Ellman unit (approximately 40 pg of protein) and yields a linear response up to at least 25 10(-3) Ellman units. An analysis of gradient fractions, using C-6/C-54 and C-131/C-54 assays as well as activity determination, shows that the A12 and G4 forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G2 and G1 forms. Both active and inactive G2 and G1 forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G1 at D1.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1994

Subpicogram determination of oxytocin by an enzyme immunoassay using acetylcholinesterase as label.

The pure tetrameric form of Acetylcholinesterase (EC-3.1.1.7) from the electric eel electrophorus electricus has been covalently coupled to oxytocin. This conjugate has been used as tracer in a heterologous competitive immunoassay. Microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody were used to separate bound and free moieties of the tracer. Acetylcholinesterase activity bound to the solid phase was measured by a colorimetric assay. The minimum detectable concentration was 0.075 pg/well (ie 1.5 pg/ml) and precision was less than 8% at concentration above 0.15 pg/well. An extraction step improved sensitivity up to 10 times with good recoveries. To assess the validity of this assay, basal levels of oxytocin were measured during the oestrous cycle of a cow.

Acetylcholinesterase↗
PubMed1994

Species specificity and organ, cellular and subcellular localization of the 100 kDa Ras GTPase activating protein.

A p100-GAP isoform, generated by an alternative splicing mechanism that eliminates the 180 hydrophobic amino acids at the amino terminus of p120-GAP, has been described in human placenta, in addition to the known p120GAP and neurofibromin. This p100-GAP possesses full Ras-GTPase stimulating activity. p120-GAP is ubiquitously localized in the cytosol while the localization of p100-GAP is unknown. Here we have explored the precise localization of p100-GAP and show that p100-GAP is present only in extracts of primate placenta. It is abundant in both human and Maccaca Rhesus placentae, where it is present in far larger amounts than p120-GAP. The p100-GAP is species-specific since it was not detected in the placenta of pig, sheep, mouse or rat. p100-GAP was also found to be organ-specific, since it was not detectable in organs other than the placenta. In this connection, we substantiated our previous finding that p100-GAP is mainly localized in the trophoblasts. Both subcellular trophoblast fractionation and immunofluorescence analyses showed that this protein was distributed between the cytosol, plasma membrane and a fraction bound to the nucleus, but not inside it. This highly restrictive specificity of p100-GAP localization in relation to species, organ and cell type, confirms the extreme singularity of this protein, and strongly suggests a particular specific function in the trophoblast.

Amino Acid Sequence↗
PubMed1994

Total interleukin-6 in plasma measured by immunoassay.

Determinations of total cytokine concentration in biological fluids by immunoassays face two major problems: the biochemical heterogeneity of the analyte and the interference of cytokine-binding proteins. We developed an ultrasensitive enzyme immunoassay for interleukin-6 (IL-6), using monoclonal antibodies and acetylcholinesterase as the tracer enzyme. The antibodies recognized recombinant and glycosylated forms of IL-6 equally. The antibodies measured dimeric recombinant IL-6, yet we could not detect IL-6 oligomers in plasma samples. We investigated the potential interference of soluble IL-6 receptor (sIL-6R), which is present at high concentrations in plasma samples (1 to 2 nmol/L). Heat treatment of the sample obviated the sIL-6R interference. Using calibrators in a plasma matrix, we demonstrated by fractionation, dilution, and recovery experiments that the immunoassay accurately measured total IL-6 in both normal and pathological serum and plasma samples.

Antibodies, Monoclonal↗
PubMed1993

Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxyl-terminal region of the cyclooxygenase-2.

Cyclooxygenase (Cox) exists in two forms in human endothelial cells (HUVEC). We have raised antibodies that recognize the sequence of the carboxyl-terminal portion of the human Cox-2 (C)-NASSSRSGLD-DINPTVLLK. Cyclooxygenase activity of HUVEC challenged with interleukin 1 alpha or a phorbol ester increased in parallel with the mass of a protein doublet analyzed by Western blot using antibodies directed against the Cox-2 peptide; a monoclonal antibody directed against Cox-1 showed a small change in protein mass. A 35S-labeled protein doublet with a molecular mass of approximately 70,000 daltons was immunoprecipitated with the anti-Cox-2 antiserum in L-[35S] methionine-labeled cells stimulated with interleukin 1 alpha. This protein was not recovered by pretreating the antiserum with the Cox-2 peptide before immunoprecipitation. A minor variation in 35S-immunoprecipitated protein was obtained with the polyclonal anti-Cox-1 antibody. Both immunoprecipitated Cox-1 and Cox-2 possessed cyclooxygenase activity that was inhibited by flurbiprofen. Endoglycosidase H treatment of immunoprecipitated Cox-2 proteins caused a decline in the apparent molecular size similar to that observed with immunoprecipitated Cox-1 or sheep cyclooxygenase but did not suppress the doublet. These results show by direct protein measurement that HUVEC synthesize the novel Cox-2 under appropriate stimulation, with little changes of Cox-1.

Amino Acid Sequence↗
PubMed1993

The role of the preserved sequences of Dam methylase.

We have undertaken a site directed mutational analysis of two of the preserved regions in the amino acid sequence of Dam methylase in order to characterize their role. Mutations in region IV (sequence DPPY) abolish catalytic activity and greatly affect AdoMet crosslinking. Mutants in region III display a lowered specific activity with an unchanged AdoMet crosslinking capacity. We have also made a series of deletions both at the N and C terminal parts of the protein, which have been found to provide inactive enzyme. We discuss the significance of these results for the understanding of the functional properties of the enzyme.

Amino Acid Sequence↗
PubMed1993

Enzyme immunometric assay for endothelin using tandem monoclonal antibodies.

Seven distinct mouse monoclonal antibodies (mAbs) directed against human endothelin-1 (ET-1) have been obtained. On the basis of specificity studies performed with competitive immunoassays and of complementary binding studies, these mAbs were classified in two groups. mAbs of group A (Endo-4, -5, -6 and -10) were shown to be directed against the N terminal loop while those of group B (Endo-2, -8 and -18) recognized the C terminal part of the peptide. A pair of monoclonal antibodies with optimal properties for a two-site immunometric assay were selected and the test was performed in 96-well microtiter plates coated with one mAb (Endo-18), while another mAb (Endo-4) covalently labeled with enzyme acetylcholinesterase was used as tracer. Under optimal conditions, the assay appeared to be very sensitive since concentrations as low as 1 pg/ml could be significantly detected. The precision was also very good with a coefficient of variation below 10% from 3 to 250 pg/ml. The assay was specific for mature endothelin presenting no cross-reactivity with the precursor Big ET-1. On the other hand, strong cross-reactivity was observed with other ET-1-related peptides, including ET-2, ET-3, VIC peptide and sarafotoxin 6-b. The assay permitted specific determination of ET-1 in supernatants of cultured endothelial cells and the validity of the test was demonstrated by HPLC fractionation experiments. In addition, the assay also appeared to be suitable for direct determination of ET-1 in plasma. Studies performed with plasma from healthy subjects revealed that circulating levels of ET-1 are below or close to the detection limit of the method (< 8 pg/ml).

Amino Acid Sequence↗