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Biomedical subjects

C Brenner

Publications and source records attributed to C Brenner.

At least 91 records · Page 5Linked to original sources
PubMed1997

Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines.

Mycoplasmas are bacteria which can cause respiratory, arthritic, and urogenital diseases. During the early phase of infection, mycoplasmas usually induce an inflammatory response and a humoral response preferentially directed against their membrane-bound, surface-exposed lipoproteins. In this report, we describe the effects on immune cells of spiralin, a well-characterized mycoplasmal lipoprotein. Purified spiralin stimulated the in vitro proliferation of human peripheral blood mononuclear cells and murine splenocytes. The stimulation pathway was probably different from that followed by Escherichia coli lipopolysaccharide because the effect of spiralin was not abolished by polymyxin B. Comparison of the effects of whole, native spiralin with those induced by proteinase K-digested spiralin or by the C-terminal half of spiralin (peptide p[13.5]T) revealed that the first half of the protein, which contains the lipoylated N terminus, is responsible for the mitogenic activity. In contrast to whole spiralin, proteinase K-digested spiralin did not trigger murine B-cell differentiation and immunoglobulin G and M secretion. Stimulation of human or murine immune cells led to early secretion of proinflammatory cytokines (human tumor necrosis factor alpha and murine interleukin 1 or 6). Spiralin induced the T-cell-independent blastogenesis of murine B cells but did not stimulate T cells. Altogether, our data demonstrate that spiralin possesses potent immunostimulating activity, similar to that reported for lipoproteins of pathogenic gracilicutes (gram-negative eubacteria; e.g., Borrelia burgdorferi OspA and E. coli Braun lipoprotein), and are consistent with the fact that lipoproteins are major antigens during mycoplasma infections.

Animals↗
PubMed1997

Evaluation of the dyspeptic patient: a cost-utility study.

BACKGROUND: Traditionally, patients presenting with uncomplicated dyspepsia have been managed using empiric antisecretory therapy, followed by endoscopy in the event of persistent symptoms or complication. Since Helicobacter pylori is now accepted as an important and potentially reversible cause of ulcer disease, it is important to reevaluate the management of dyspepsia. The goal of this study is to evaluate seven outpatient strategies for the management of dyspeptic patients using a cost-utility analysis. METHODS: The study design was that of a cost-utility analysis. The model assumes that an adult patient with signs of dyspepsia but no signs of complication presents to the outpatient office of a primary care physician. Seven strategies are modeled: empiric antisecretory therapy; empiric H pylori eradication using oral omeprazole (20 mg [corrected] twice daily), clarithromycin (500 mg twice daily), and amoxicillin (1000 mg twice daily); use of either upper endoscopy, an upper gastrointestinal barium study (an upper GI), or the serum titer for H pylori as a diagnostic test to identify patients for H pylori eradication; or use of an initial diagnostic test followed by the serum titer for H pylori. The primary outcome was the cost per quality-adjusted life year (QALY) for each strategy for a 1-year period from presentation; secondary outcomes included the probability of symptomatic ulcer recurrence, cost per ulcer cure, and mortality. RESULTS: Three strategies were similarly cost-effective: empiric H pylori eradication ($1198 per QALY), use of a serum H pylori titer as an initial diagnostic test ($1214 per QALY), and empiric antisecretory therapy ($1288 per QALY). Empiric antisecretory therapy, however, was associated with significantly more symptomatic ulcer recurrences and deaths than any other strategy. CONCLUSIONS: This cost-utility analysis suggests that two strategies are reasonable for patients presenting with dyspepsia: (1) empiric H pylori eradication and (2) use of a serum H pylori titer to identify patients who might benefit from H pylori eradication. The latter strategy may be preferable because it is less likely to lead to antibiotic resistance. Strategies utilizing an upper GI or upper endoscopy (either with or without serum H pylori titer) or empiric antisecretory therapy do not improve outcomes and are associated with greater cost, morbidity, and/or mortality.

Adult↗
PubMed1996

Mycoplasmas and HIV infection: from epidemiology to their interaction with immune cells.

Mycoplasmas are possible HIV cofactors, contributing to the evolution of AIDS. Our knowledge about mycoplasma prevalence in HIV-infected subjects has considerably increased due the development of specific detection assays. A new mycoplasma, Mycoplasma penetrans, has been identified and has been shown to be associated with HIV infection, at least among individuals with homosexual practices. We and others investigated the properties of M. fermentans and M. penetrans concerning cell colonization, cell invasion and cytopathogenicity. The molecular components which are involved in the interaction between these bacteria and immune cells are beginning to be identified and characterized. Membrane lipoproteins of these wall-less prokaryotes are key components in their interaction with B cells and surface capsular material may contribute to their defense from the host immune response.

HIV↗
PubMed1996

Comparison of methaqualone excretion patterns using Abuscreen ONLINE and EMIT II immunoassays and GC/MS.

A study was performed to compare the ONLINE and EMIT II immunoassays with gas chromatographic/mass spectrometric (GC/MS) analysis of methaqualone metabolites on urine using samples obtained from a clinical study. Urine was collected over a 72 h period from six healthy adults (4 male, 2 female) after oral dosing with 200 mg methaqualone (MTQ). Each urine sample was analyzed by ONLINE and EMIT II. The samples were then analyzed by GC/MS, hydrolyzed with beta-glucuronidase and again analyzed by GC/MS. Both immunoassays showed greater than 600 ng/ml concentrations of drug in each sample by the second void and remained highly positive for the rest of the 72 h. Unhydrolyzed samples analyzed by GC/MS showed both low concentrations of MTQ as well as its five major hydroxylated metabolites. The hydrolyzed samples analyzed by GC/MS showed high concentrations of the hydroxylated metabolites with the 2'-hydroxy and 3'-hydroxy metabolites being present at the highest concentrations, the 4'-hydroxy metabolite at a lower amount and the 6-hydroxy and 2-hydroxy metabolites at the lowest concentrations. The GC/MS data coupled with the antibody cross-reactivity data indicate that the major species in clinical samples that cross-react in both immunoassays are the conjugated forms of the hydroxylated metabolites of MTQ. Therefore when confirming by GC/MS after an immunoassay screen it would be prudent to confirm for the major hydroxylated metabolites as glucuronides of MTQ instead of the parent drug.

Adult↗
PubMed1996

Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter.

Plasmid DNA encoding the outer surface lipoprotein A (OspA) of Borrelia burgdorferi under the control of either strong eukaryotic/viral or its own bacterial promoter was injected intramuscularly (m. tibialis anterior) or intradermally into BALB/c and AKR/N mice. OspA-specific antibodies and OspA-reactive T helper 1 cells (Th1) were induced only with those plasmids containing the ospA structural gene including its own regulatory control region immediately upstream. In the absence of the ospA promoter, no or only marginal immune responses to OspA were obtained, even when strong eukaryotic promoter/enhancer elements were present. Together with the finding that the ospA promoter is active in a mouse B-lymphoma line, the data suggest that spirochetes are able to express at least part of their genes in the mammalian environment. Mice previously vaccinated with the relevant ospA plasmid DNA were protected against subsequent experimental challenge with a virulent strain of B. burgdorferi, as measured by the appearance of antibodies to a prominent protective epitope (LA-2) and the failure to re-isolate spirochetes from ear biopsies. In addition, C.B-17 severe-combined immunodeficient mice could be protected against infection by passive transfer of immune sera from ospA plasmid DNA-inoculated normal mice. Protective LA-2-related antibody titers obtained after repeated immunization persisted for 200 days and longer. This simple procedure of immunization using plasmid DNA consisting of a prokaryotic gene under the control of its own promoter holds great promise for the development of alternative subunit vaccines against bacterial infections, including Lyme disease. In addition, the availability of this novel prokaryotic promoter element now allows the study of the basis for the differential expression of bacterial genes in prokaryotic and eukaryotic environments.

Animals↗
PubMed1996

Purification of Mycoplasma gallisepticum membrane proteins p52, p67 (pMGA), and p77 by high-performance liquid chromatography.

The plasma membrane of the avian pathogen Mycoplasma gallisepticum contains about 200 polypeptides including the major lipoprotein pMGA. We have developed a simple and efficient procedure for the purification of three membrane proteins of this wall-less bacterium. Proteins were selectively extracted from isolated plasma membranes with the mild zwitterionic detergent (N-dodecyl-N,N-dimethylammonio) undecanoate (DDMAU) and subjected to size-exclusion chromatography (FPLC) in the presence of the same detergent. Two of the thus separated protein fractions were subjected to a third step involving an anion-exchange chromatography (HPLC), also in the presence of DDMAU, which led to the purification to homogeneity of p67, the major acyl protein of M. gallisepticum plasma membrane (yield, 40%; purification factor, 11), p52 (yield, 38%; purification factor, 20), and p77 (yield, approximately 45%; purification factor, 500). Analyses performed by Western blotting and crossed immunoelectrophoresis showed that the three purified proteins are distinct antigens. Furthermore, N-terminal sequencing confirmed that p67 is pMGA. The method described in this paper is simple, efficient, and nondenaturing; it provides pure proteins, at the milligram level for p52 and p67, and should prove easy to being scaled-up if necessary.

Antigens, Surface↗
PubMed1995

Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic alpha helix.

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic alpha helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne (Von Heijne, G. (1992) J. Mol. Biol. 225, 487-494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic alpha helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% alpha helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (> 250 microM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope.

Amino Acid Sequence↗
PubMed1995

Arg21 is the preferred kexin cleavage site in parathyroid-hormone-related protein.

Parathyroid-hormone-related protein (PTHrP) contains several potential sites for proteolytic processing. Although there is considerable evidence for the existence of cleaved products in vivo, little is known about the post-translational processing of PTHrP. We have used purified kexin (Kex2) protease to identify which cleavage sites in recombinant PTHrP(1-141) might be of physiological significance. Cleavage products were identified by N-terminal sequencing. Kex2 preferentially cleaved PTHrP(1-141) carboxy to the triplet arginine site Arg-Arg-Arg21 with a Km of 3.3 +/- 1.7 microM and a kcat of 6 +/- 1.2 s-1. Substitution of alanine for Arg19 resulted in substantially reduced conversion, while no detectable cleavage occurred when alanine was substituted for either Arg20 or Arg21. In contrast, the degree of Kex2 cleavage at Arg21 in PTHrP(1-34) was lower. No detectable cleavage occurred in an unrelated synthetic peptide containing both double and triple arginine sites. Low levels of cleavage also took place carboxy to Lys-Arg97, Lys-Arg105, Arg-Arg106 and Thr-Arg108. Cleavage carboxy to Lys-Arg105, the best of these minor sites, occurred with a Km of 8.4 +/- 2.7 microM and a kcat of 0.8 +/- 0.2 s-1. These studies indicate that the preferred Kex2 cleavage site in PTHrP(1-141) is carboxy to Arg-Arg-Arg21, which effectively destroys its parathyroid-hormone-like biological activity. Cleavage of this site by Kex2-related mammalian convertases in vivo may be an important mechanism for full elaboration of the non-parathyroid-hormone-like paracrine actions of PTHrP in a tissue-specific manner.

Arginine↗
PubMed1995

Evaluation of the efficacy of zwitterionic dodecyl carboxybetaine surfactants for the extraction and the separation of mycoplasma membrane protein antigens.

The ability to extract mycoplasma membrane protein antigens using the alkyl carboxybetaine surfactants (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC = 4.3 mM) and (N-dodecyl-N,N-dimethylammonio)undecanoate (DDMAU, CMC = 0.13 mM) was assessed by protein titration and SDS-PAGE analysis. The maximum yields of membrane protein solubilization ranged from 20 to 90%, depending upon both the mycoplasma membrane investigated and the surfactant used. In five of six cases, the extraction was optimal for surfactant concentrations of ca. 25 mM. DDMAB displayed a higher efficiency in membrane protein extraction. The order of efficiency for both surfactants was Spiroplasma melliferum > Acholaplasma laidlawii > Mycoplasma gallisepticum. In contrast, DDMAU proved much more selective. The order of selectivity was M. gallisepticum > S. melliferum > A. laidlawii. The highest selectivity was recorded for the major proteins p67 and spiralin of M. gallisepticum and S. melliferum, respectively. For p67, notably, DDMAU proved superior to 10 other surfactants. Dot immunobinding and crossed immunoelectrophoresis analyses showed that both dodecyl carboxybetaines were suitable as membrane protein-solubilizing agents in immunological techniques. Furthermore, these surfactants did not exhibit effects adverse to the activity of A. laidlawii membrane NADH oxidase. One promising application of DDMAU is the separation of membrane proteins by ion-exchange HPLC as illustrated by the good resolution of M. gallisepticum membrane proteins and purification of p67 to almost homogeneity. These data show that dodecyl carboxybetaine surfactants are useful for the extraction of mycoplasma membrane antigens under mild conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Bacterial↗
PubMed1995

Molecular cloning and immunological characterization of a novel linear-plasmid-encoded gene, pG, of Borrelia burgdorferi expressed only in vivo.

Previously we have found that sera from immunocompetent mice infected either naturally by ticks or experimentally with low numbers of Borrelia burgdorferi ZS7 bacteria lack OspA- and OspB-specific antibodies but confer optimal protection on severe combined immunodeficiency mice against challenge with spirochetes (U.E. Schaible, L. Gern, R. Wallich, M. D. Kramer, M. Prester, and M. M. Simon, Immunol. Lett. 36:219-226, 1993). We have now used the latter immune sera to identify new spirochetal structures with relevance for protection from an expression library of the virulent European strain B. burgdorferi ZS7. Here we report the cloning and characterization of a novel lipoprotein, designated pG, the gene for which is located on a 48-kb linear plasmid. Sequence analysis of the pG gene revealed an open reading frame encoding a putative lipoprotein of 196 amino acids with a calculated molecular mass of 22 kDa and a consensus cleavage sequence (Leu-X-Y-Z-Cys) recognized by signal peptidase II. Restriction fragment length polymorphism analyses of pG derived from independent B. burgdorferi isolates from different geographic areas revealed that the gene is species specific, with, however, extensive genotypic heterogeneity. Comparison of the protein sequence of pG with those of other known B. burgdorferi outer surface lipoproteins (OspA to OspF and P27) demonstrated that pG is most related to OspF. Furthermore, the upstream region of pG exhibited extensive sequence homology (> 94%) with the ospEF promoter region. Mouse immune sera to recombinant pG did not recognize a corresponding molecule in lysates of in vitro-propagated ZS7 spirochetes. However, experimental or natural infection of mice with ZS7 resulted in the induction of antibodies with reactivity for pG and the potential to delay the development of clinical arthritis. Together with the finding that sera from Lyme disease patients also contain antibodies to pG, our data suggest that the pG gene is preferentially expressed in the mammal environment.

Amino Acid Sequence↗
PubMed1993

One-step site-directed mutagenesis of the Kex2 protease oxyanion hole.

BACKGROUND: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis. Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-alpha-factor, the precursor of the alpha-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction. Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the 'oxyanion hole'. RESULTS: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast. Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion. Transformants expressing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate. The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro. We found that, with three peptides that are good substrates of wild-type Kex2, the k(cal) of the Asp 314 enzyme was reduced approximately 4500-fold and its K(M) approximately 4-fold, relative to the wild-type enzyme. For the peptide substrate corresponding to the cleavage site of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduced only 125-fold, while the K(m) was increased 3-fold. Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo - by the intramolecular cleavage that removes its amino-terminal pro-domain - occurs at an unchanged rate. CONCLUSIONS: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site. Aspartate at the oxyanion hole appears to confer k(caf) discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding.

Journal Article↗
PubMed1993

The synthesis of inhibitors for processing proteinases and their action on the Kex2 proteinase of yeast.

Peptidyl chloromethane and sulphonium salts containing multiple Arg and Lys residues were synthesized as potential inhibitors of prohormone and pro-protein processing proteinases. The potencies of these compounds were assayed by measuring the kinetics of inactivation of the yeast Kex2 proteinase, the prototype of a growing family of eukaryotic precursor processing proteinases. The most potent inhibitor, Pro-Nvl-Tyr-Lys-Arg-chloromethane, was based on cleavage sites in the natural Kex2 substrate pro-alpha-factor. This inhibitor exhibited a Ki of 3.7 nM and a second-order inactivation rate constant (k2/Ki) of 1.3 x 10(7) M-1.s-1 comparable with the value of kcat./Km obtained with Kex2 for the corresponding peptidyl methylcoumarinylamide substrate. The enzyme exhibited sensitivity to the other peptidyl chloromethanes over a range of concentrations, depending on peptide sequence and alpha-amino decanoylation, but was completely resistant to peptidyl sulphonium salts. Kinetics of inactivation by these new inhibitors of a set of 'control' proteinases, including members of both the trypsin and subtilisin families, underscored the apparent specificity of the compounds most active against Kex2 proteinase.

Amino Acid Chloromethyl Ketones↗
PubMed1993

Overdose of risperidone.

Risperidone is an investigational antipsychotic agent currently being tested in an international multicenter drug trial. We report the first case of a risperidone overdose in an apparent suicide attempt. This was without serious clinical side effects and manifested mainly as ECG abnormalities.

Adult↗
PubMed1992

Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease.

The prohormone-processing Kex2 protease of the budding yeast Saccharomyces cerevisiae can be converted from an intracellular membrane protein to a soluble, secreted, and active form by deletion of the transmembrane domain and C-terminal tail. One such molecule was purified to near homogeneity from the culture medium of an overexpressing yeast strain. Amino acid sequence analysis revealed that the N terminus of mature Kex2 protease is created by a potentially autoproteolytic cleavage at Lys108-Arg109, prior to the domain homologous to subtilisin, followed by trimming of Leu-Pro and Val-Pro dipeptides by the Ste13 dipeptidyl aminopeptidase. Kinetic parameters were examined using fluorogenic peptidyl-methylcoumarin amide substrates. Initial burst titration indicated that the preparation was entirely active. Measurements of dependence of activity on pH yielded a simple curve suggesting titration of a single ionizable group. Activity was half-maximal at pH 5.7 and nearly constant from pH 6.5 to 9.5. Discrimination between substrates was as great as 360-fold in Km and 130-fold in kcat. Substrates with a Lys-Arg dipeptide preceding the cleaved bond were preferred, having kcat/Km values up to 1.1 x 10(7) sec-1.M-1. The enzyme cleaved substrates having Arg-Arg, Pro-Arg, Ala-Arg, and Thr-Arg with increased Km but with unchanged kcat. In contrast, the enzyme displayed a dramatically lower kcat for a Lys-Lys substrate with a smaller increase in Km. Thus the two residues preceding the cleaved bond may play distinct roles in the selectivity of binding and cleavage of prohormone substrates.

Amino Acid Sequence↗