GPCRDB: an information system for G protein-coupled receptors.

The GPCRDB is a G protein-coupled receptor (GPCR) database system aimed at the collection and dissemination of GPCR related data. It holds sequences, mutant data and ligand binding constants as primary (experimental) data. Computationally derived data such as multiple sequence alignments, three dimensional models, phylogenetic trees and two dimensional visualization tools are added to enhance the database's usefulness. The GPCRDB is an EU sponsored project aimed at building a generic molecular class specific database capable of dealing with highly heterogeneous data. GPCRs were chosen as test molecules because of their enormous importance for medical sciences and due to the availability of so much highly heterogeneous data. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm

Characterization of a reduction-sensitive factor from human plasma responsible for apparent false activity in competitive assays for antibody to hepatitis B core antigen.

Addition of reducing agents to competitive assays for antibody to hepatitis B core antigen (anti-HBc) eliminates apparent false reactivity of specimens obtained from individuals with no prior history of hepatitis B virus (HBV) infection and without other serological markers of HBV infection. We have purified and characterized a reduction-sensitive factor (RSF) isolated from the plasma of several volunteer blood donors. Column fractions were assayed fro anti-HBc by using a highly sensitive chemiluminescence assay with a detection of 0.15 Paul Ehrlich Institut units per ml at 50% inhibition. Gel filtration on Sephacryl S-300 indicated that reductant-sensitive samples possessed anti-HBc activity that was associated with immunoglobulin M (IgM), whereas reductant-stable activity was associated with IgG. Gel filtration followed by metal chelate affinity chromatography resulted in a 55-fold purification and demonstrated that RSF activity copurifies with IgM. RSF was recovered from a recombinant hepatitis B core antigen matrix and shown to be an IgM species by immunoblot. In addition, RSF activity coeluted with IgM protein from anti-mu-chain Sepharose. Discrepancies between enzyme immunoassay and radioimmunoassay procedures for anti-HBc (Corzyme and Corab, respectively: Abbott Laboratories, North Chicago, Ill.) appear to be due to the relative sensitivity of the enzyme immunoassay for IgM anti-HBc (sevenfold greater than the radioimmunoassay using a specific panel). The biological basis for the occurrence of low levels of nonspecific IgM anti-HBc reactivity in individuals not previously exposed to HBV remains to be elucidated.

Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen.

Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a negative population (n = 160) from assay cutoffs. With a selected population (n = 307), inclusion of reductant eliminated apparent anti-HBc activity in 54 of 81 samples in the 30 to 70% inhibition range. Reductant-stable anti-HBc samples were strongly associated with the presence of antibody to hepatitis B surface antigen (21 of 27). The association persisted below the detection limits of current assays to 0.3 to 0.4 Paul Ehrlich Institute units per ml. Only 1 of 54 reduction-sensitive borderline samples was confirmed to be positive for antibody to hepatitis B surface antigen. The modified procedures had unchanged or slightly improved sensitivity for immunoglobulin G (IgG)-associated anti-HBc activity. Although IgM anti-HBc detection was reduced from four- to eightfold in the presence of reductants, sensitivities remained at least twofold greater than tha of an enzyme immunoassay (Corzyme M; Abbott) designed to detect acute-phase levels of IgM anti-HBc. The use of reducing agents should significantly improve the reliability of anti-HBc testing, especially near assay cutoffs.

A chemiluminescent, microparticle-membrane capture immunoassay for the detection of antibody to hepatitis B core antigen.

A chemiluminescent, microparticle-membrane capture immunoassay (CLIA/MMC) for the detection of antibody to hepatitis B core antigen (anti-HBc) is described. The assay utilizes recombinant hepatitis B core antigen coupled to carboxylated latex microparticles. Human polyclonal IgG anti-HBc labelled with acridinium competes with antibody in the sample for a limited number of binding sites on the solid phase. After a 40 min incubation at 40 degrees C, the reaction mixture is transferred to a glass fiber capture membrane and washed. A chemiluminescent signal is produced by addition of alkaline peroxide and is quantitated on a semi-automated reader as described. The CLIA/MMC assay was compared with standard EIA and RIA procedures (Corzyme and Corab, respectively, Abbott Laboratories, North Chicago, IL). Assay sensitivities were RIA greater than CLIA/MMC greater than EIA. A population of 200 normal blood donors showed nearly identical distributions with the CLIA/MMC and RIA (mean = 11% inhibition, SD = 13% for both), compared with the EIA (mean = 13% inhibition, SD = 15%). With a selected plasma population (n = 307), the CLIA/MMC immunoassay showed an excellent correlation (r = 0.94) with both the EIA and RIA procedures. Association of anti-HBc reactivity near assay cutoffs with antibody to hepatitis B surface antigen suggested relative specificity in the order RIA greater than CLIA/MMC greater than EIA. The CLIA/MMC procedure, which can be readily automated, provides a non-istopic alternative to current EIA testing with performance more nearly equivalent to RIA.

Antiemetics in children receiving cancer chemotherapy: a double-blind prospective randomized study comparing metoclopramide with chlorpromazine.

This is the first prospective randomized study comparing commonly used antiemetics in children receiving cancer chemotherapy. We compared metoclopramide (MCP) with chlorpromazine (CLP), both administered in conventional doses, in 50 cancer patients aged 6 to 18 years who were receiving emetic chemotherapy. CLP proved significantly better than MCP in reducing both the frequency of vomiting (P less than .05) and the duration of nausea and vomiting (P less than .025). Extrapyramidal reactions (EPRs) were more common in MCP-treated patients. We conclude that, in the standard doses used, CLP is a better overall antiemetic than MCP for children receiving intensive chemotherapy. However, further prospective pediatric studies of antiemetic combinations are needed.

Angiotensin-converting enzyme in epithelial and neuroepithelial cells.

Angiotensin-converting enzyme (CE) occurs in three types of cell: endothelial, epithelial, and neuroepithelial. In all three, it appears to be bound to plasma membrane. With antisera to the human enzyme, CE is demonstrated in paraffin sections on the apical surface of epithelial cells in the proximal tubule of the kidney, the mucosa of the small intestine, the syncytial trophoblast of the placenta, and the choroid plexus. Epithelial CE is characteristically found on microvillous surfaces in contact with an effluent, well placed to act on substrate in flux. In the brain, CE occurs in nerve fibers and terminals, mainly mesiobasally and in basal ganglia. Mesiobasal CE coincides with other components of the renin-angiotensin system (RAS) in the choroid/ventricular fluid, the subfornical organ, and the magnocellular neurosecretory system of the hypothalamus. Extrapyramidal CE, however, may not be related to the RAS. In the substantia nigra and the globus pallidus, the enzyme has the same cellular distribution as two putative neuromodulators, substance P and enkephalin, the latter a known substrate of CE.

Measurement of human converting enzyme level by direct radioimmunoassay.

CE was purified from human lung, and antisera were raised in rabbits. Antisera inhibited the activity of the purified enzyme from lung and kidney and the plasma CE of normal persons and sarcoid patients. With antisera at a titer of 1:100,000, a sensitive, direct RIA was developed. CE purified from lung or kidney and CE present in normal and in sarcoid plasma gave parallel logit-log displacement lines, suggesting immunological identity. The level of CE in normal human plasma was 400 +/- 131 ng/ml. In untreated sarcoid patients, the enzyme level and activity increased in parallel. There was a negative correlation (r = -0.81) between enzyme level and diffusing capacity of the lung for CO in sarcoid patients. Synthetic inhibitors such as captopril or MK 421 did not interfere with the RIA, permitting enzyme levels to be monitored in patients undergoing acute inhibitor therapy. During administration of MK 421, CE activity was negligible and plasma levels of CE did not change. In contrast, renin activity increased eightfold during the inhibitor therapy.

Hydrolysis of enkephalins by human converting enzyme and localization of the enzyme in neuronal components of the brain.

Homogeneous human CE hydrolyzed Met5-enkephalin fastest of the biologically active substrates tested. The inactivation of Met5-enkephalin was inhibited by antiserum to Ce and by specific inhibitor of CE (MK 421; I50 = 8 x 10(-9)M). Direct radioimmunoassay of CE extracted from basal ganglia indicated immunological identity of lung, kidney, and brain CE. Immunocytochemically, CE was demonstrated in the choroid plexus, where the enzyme appears to be bound, as in kidney and gut, to the surface of the epithelial cell bathed by luminal fluid. CE immunoreactivity in brain parenchyma was demonstrated in neuronal elements in the hippocampus, hypothalamus, neocortex, globus pallidus, and substantia nigra.

Inhibition of human converting enzyme in vitro by a novel tripeptide analog.

We have studied inhibition of homogeneous human converting enzyme by a new inhibitor, a ketomethylene derivative of the blocked tripeptide substrate, Bz-Phe-Gly-Pro (ketoACE). KetoACE inhibited the hydrolysis of Hip-His-Leu and Hip-Phe-Arg at different concentrations (I50 values were 4 X 10(-8) M and 2 X 10(-7) M, respectively). Kinetic studies indicated that ketoACE inhibits the hydrolysis of both substrates by a similar, non-competitive mechanism. At the lowest enzyme concentration tested, using 3H-Hip-Gly-Gly as substrate, the I50 of ketoACE was 6 X 10(-9) M. KetoACE protected a functional tyrosine residue in the active site of human converting enzyme from modification with N-acetylimidazole. It is proposed that there are alternate (hydrophobic) binding sites for both inhibitors and substrates in the active site of human converting enzyme. It should be possible to develop other high-affinity inhibitors of this class that bind to hydrophobic sites and do not require metal binding via a sulfhydryl group.

Purification and characterization of human converting enzyme (kininase II).

We purified peptidyl-dipeptidase (converting enzyme, EC 3.4.15.1) to homogeneity from the membrane fraction of human lung and for comparison, from human and hog kidney. The membrane-bound lung enzyme was purified 1800-fold with 19% yield, and the kidney enzyme 640-fold with 10% yield. The specific activities with Bz-Gly-His-Leu were 81 mumol/min/mg for the lung and 65 for the kidney enzyme. The lung enzyme was homogeneous in gel electrophoresis with Mr = 155,000 and Sw,20 = 8.0 in ultracentrifugation. Antibodies elicited against lung or kidney enzyme cross-reacted with enzyme from other organ, but not with the hog enzyme. In isoelectric focusing both human enzymes had a major form with a pI of 5.2. The lung preparation also contained more acidic forms (pI = 4--5), which were eliminated by treatment with neuraminidase. Lung and kidney converting enzyme hydrolyzed bradykinin, angiotensin I, and enkephalins and had similar kinetic constants. Bradykinin was the best substrate, as indicated by its kcat/Km, but Met5-enkephalin had the highest turnover number. The hydrolysis of Bz-Gly-His-Leu was inhibited by captopril (SQ 14225) competitively, and by Keto-ACE, a non-peptide derivative of Bz-Phe-Gly-Pro, non-competitively.