Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop.

Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGß, hCGßn, hCGßcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGß-, 8 hCGα-, and 13 αß-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1%; epitope ß1), 12 with low (<1.0%; epitopes ß2/4), and 13 with high (>>1%; epitopes ß3/5) hLH cross-reactivity. The majority of hCGß epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of ß-sheet loops 1 and 3 (epitopes ß2-6; 27 mAbs) and the second around the cystine knot (e.g., epitopes ß1, ß7, and ß10; 9 mAbs). Four mAbs recognized epitopes on hCGßcf-only (e.g., epitopes ß11 and ß13) and six mAbs epitopes on the remote hCGß-carboxyl-terminal peptide (epitopes ß8 and ß9 corresponding to amino acids 135-144 and 111-116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGß-only, or hCG together with hCGß or hCG together with hCGß and hCGßcf. Sandwich assays that measure hCG plus hCGß and eventually hCGßcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGßcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope ß1 (Asp10, Asp60, and Gln89) in combination with epitopes ß2 or ß4 located at the top of ß-sheet loops 1 + 3 of hCGß involving aa hCGß20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes.

Omeprazole impairs the absorption of mycophenolate mofetil but not of enteric-coated mycophenolate sodium in healthy volunteers.

In 2 crossover studies, 12 healthy volunteers (6 male/6 female) received a single oral dose of mycophenolate mofetil (MMF) 1000 mg or an equimolar dose of enteric-coated mycophenolate sodium (EC-MPS) 720 mg fasting with and without coadministered omeprazole 20 mg bid. The plasma concentrations of mycophenolic acid (MPA) and of the inactive metabolite mycophenolic acid glucuronide (MPA-G) were measured by high-performance liquid chromatography (HPLC). In addition, dissolution of MMF 500 mg or EC-MPS 360 mg tablets was determined using an USP paddle apparatus in aqueous buffer of pH 1 to 7. The bioavailability of MPA following administration of MMF or EC-MPS was similar except for the time to peak concentration, which was longer in the EC-MPS group. Concomitant treatment with omeprazole lowered significantly C(max) and AUC(12h) of MPA following administration of MMF. The pharmacokinetics of EC-MPS was not affected. Dissolution of MMF in aqueous buffer decreased dramatically at pH above 4.5. The EC-MPS tablet was stable up to pH 5. Above, EC-MPS was quantitatively disintegrated and MPS quantitatively dissolved. There is strong evidence that impaired absorption of MMF with concomitant proton pump inhibitors is due to incomplete dissolution of MMF in the stomach at elevated pH.

Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels.

The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.3 channels by margatoxin was previously shown to prevent T cell activation and attenuate immune responses in vivo. In the present study, a triterpene natural product, correolide, was found to block Kv1.3 channels in human and miniswine T cells by electrophysiological characterization. T cell activation events, such as anti-CD3-induced calcium elevation, IL-2 production, and proliferation were inhibited by correolide in a dose-dependent manner. More potent analogs were evaluated for pharmacokinetic profiles and subsequently tested in a delayed-type hypersensitivity (DTH) response to tuberculin in the miniswine. Two compounds were dosed orally, iv, or im, and both compounds suppressed DTH responses, demonstrating that small molecule blockers of Kv1.3 channels can act as immunosuppressive agents in vivo. These studies establish correolide and its derivatives as novel immunosuppressants.

WIN 17317-3, a new high-affinity probe for voltage-gated sodium channels.

The iminodihydroquinoline WIN 17317-3 was previously shown to inhibit selectively the voltage-gated potassium channels, K(v)1.3 and K(v)1.4 [Hill, R. J., et al. (1995) Mol. Pharmacol. 48, 98-104; Nguyen, A., et al. (1996) Mol. Pharmacol. 50, 1672-1679]. Since these channels are found in brain, radiolabeled WIN 17317-3 was synthesized to probe neuronal K(v)1 channels. In rat brain synaptic membranes, [(3)H]WIN 17317-3 binds reversibly and saturably to a single class of high-affinity sites (K(d) 2.2 +/- 0.3 nM; B(max) 5.4 +/- 0.2 pmol/mg of protein). However, the interaction of [(3)H]WIN 17317-3 with brain membranes is not sensitive to any of several well-characterized potassium channel ligands. Rather, binding is modulated by numerous structurally unrelated sodium channel effectors (e.g., channel toxins, local anesthetics, antiarrhythmics, and cardiotonics). The potency and rank order of effectiveness of these agents in affecting [(3)H]WIN 17317-3 binding is consistent with their known abilities to modify sodium channel activity. Autoradiograms of rat brain sections indicate that the distribution of [(3)H]WIN 17317-3 binding sites is in excellent agreement with that of sodium channels. Furthermore, WIN 17317-3 inhibits sodium currents in CHO cells stably transfected with the rat brain IIA sodium channel with high affinity (K(i) 9 nM), as well as agonist-stimulated (22)Na uptake in this cell line. WIN 17317-3 interacts similarly with skeletal muscle sodium channels but is a weaker inhibitor of the cardiac sodium channel. Together, these results demonstrate that WIN 17317-3 is a new, high-affinity, subtype-selective ligand for sodium channels and is a potent blocker of brain IIA sodium channels.

Identification and biochemical characterization of a novel nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1.3.

A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest.

Dominant mutations confer resistance to the immunosuppressant, rapamycin, in variants of a T cell lymphoma.

Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is prevented.

Prevalence of antibodies to the core protein P17, a serological marker during HIV-1 infection.

Studies on monitoring the immune response to viral structural proteins during human immunodeficiency virus (HIV-1) infection have established the significance of antibodies to the core protein p24 during the progression of the disease. We have studied the prevalence of antibodies to the core protein p17 in order to study their diagnostic and prognostic significance in the pathogenesis of HIV-1. Full-length HIV-1 p17, molecularly cloned and expressed in Escherichia coli was purified by immunoaffinity chromatography using an HIV-1 p17-specific monoclonal antibody. A highly sensitive enzyme-linked immunoassay was developed using the purified recombinant p17 as the serological target to detect antibodies to p17. The results indicated that antibodies to p17 decline during progression of disease, with the decline being more dramatic as patients moved from asymptomatic to AIDS-related complex (ARC). Patient specimens deficient in p24 antibody, but having detectable levels of antibody to p17 were almost always positive for p24 antigen. Under these conditions, p17 antibody is an important serological marker because it provides a more consistent marker for core antigens during HIV-1 infection.

Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products.

mRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated. One clone, pM1-11, contains all three exons of human c-myc. The second clone, pM4-10, represents a relatively rare transcript that initiated in the first intron and includes the coding exons 2 and 3. The cDNA insert in pM1-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells. Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction. The protein produced in E. coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system. In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa. The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation. Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.

Conservation of capR (lon) DNA of Escherichia coli K-12 between distantly related species.

Mutations in the capR gene of Escherichia coli K-12 are responsible for a wide variety of phenotypic changes, including defects in cell division. Since this gene plays a critical role in cell division, it might be evolutionarily conserved. Of the DNAs examined by Southern analysis, capR probe sequences were found not only in other enterics but also in Caulobacter crescentus CB13 and the distantly related archebacterium Halobacterium halobium R1.

omp T: Escherichia coli K-12 structural gene for protein a (3b).

Chromosomal DNA from strain UT400, a previously described deletion mutant of Escherichia coli K-12 that lacks outer membrane protein a, failed to hybridize with plasmid DNA (pGGC110) containing the structural gene for protein a. We designate the genetic locus for protein a, located at approximately 12.5 min of the E. coli chromosome, ompT.

Methyl beta-glycosides of N-acetyl-6-O-(omega-aminoacyl)muramyl-L-alanyl-D-isoglutamines, and their conjugates with meningococcal group C polysaccharide.

Spacer arms 2.1-3.7 nm (21-37 A) long were prepared, and coupled with the methyl beta-glycoside of N-acetylmuramyl-L-alanyl-D-isoglutamine benzyl ester, to give blocked 6-acylates. Deprotection was effected with palladium chloride and triethyl-silane. Chemical conjugates of MDP-meningococcal group C polysaccharide were then synthesized, in attempts to enhance the immunogenicity of the polysaccharide antigen.

omega-Aminoalkyl beta-glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine, and their conjugates with meningococcal group C polysaccharide.

The 6-aminohexyl beta-glycoside of N-acetylmuramyl-L-alanyl-D-isoglutamine and its spacer-arm-linked analog (3.8 nm) were synthesized from 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-alpha-D-glucopyrano)-[2,1-d]-2- oxazoline, and coupled with meningococcal group C polysaccharide in attempts to enhance the immunogenicity of the polysaccharide antigen.

Design and preparation of affinity columns for the purification of eukaryotic messenger ribonucleic acid cap binding protein.

2',3'-O-[1-(2-Carboxyethyl) ethylidene]-7-methylguanosine 5'-diphosphate (5) and 7-(5-carboxypentyl) guanosine 5'-diphosphate (13) have been synthesized and immobilized on AH-Sepharose 4B to the extent of 17.4 and 36.6 mumol of ligand/g of gel, respectively. The affinity resins thus derives were employed in columns for the purificaton of 24K cap binding protein (CBP) from rabbit reticulocytes. Each resin was found to retain the protein of interest; elution of 24K CBP could then be effected by washing with 70 microM m7GDP. The 24K CBPs released from both columns were found to be active, both as judged by a cross-linking assay that utilized 10(4)-oxidized methyl-3H-labeled reovirus mRNA as a substrate for the protein and also by the ability of the isolated 24K CBP to stimulate the translocation of capped Sindbis virus mRNA in HeLa cell extracts.

Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.

A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.