Biomedical subjects

R L Friedman

Publications and source records attributed to R L Friedman.

At least 55 records · Page 3Linked to original sources

PubMed1990

Isolation and characterization of the recA gene of Bordetella pertussis.

This report describes the detection and cloning of the Bordetella pertussis recA gene. Escherichia coli clones having recombinant plasmids containing the B. pertussis recA gene were isolated by complementing an E. coli RecA- mutant's inability to survive in the presence of methylmethanesulphonate (MMS). This gene was shown to complement the deficiency of E. coli RecA- strains to tolerate the DNA-damaging effects of both a chemical agent and ultraviolet light (u.v.). Deletion mapping experiments localized the gene to a 2.5 kb StuI-EcoRI fragment, and expression of the gene in E. coli resulted in the production of a 40 kD protein. These data strongly suggest that a region of the B. pertussis chromosome that encodes RecA-like activity has been isolated and cloned.

Blotting, Southern↗

PubMed1990

Adenylate cyclase toxin of Bordetella pertussis: production, purification, and partial characterization.

Bordetella pertussis produces a number of virulence determinants which contribute to its pathogenicity. One factor, the adenylate cyclase toxin (ACT), has been suggested to directly penetrate human phagocytes and disrupt their normal function by direct production of intracellular cyclic AMP (cAMP). Experiments evaluating the production of cell-associated ACT in liquid cultures of B. pertussis 504 demonstrated that the greatest activity was observed during mid-log-phase growth. Urea extracts of cells harvested during the time of maximal ACT production have been used to purify the toxin with both biological and enzymatic activities. ACT is a protein with an apparent molecular mass of 220 kDa and an isoelectric point of 7.0. The specific activity of purified ACT is 17,000 mumol of cAMP formed per mg per min. The the biological specific activity of purified ACT is 6,250 nmol of intracellular cAMP formed per mg per min in 2 x 10(6) S49 lymphoma cells per ml. Preparations containing 8 micrograms of ACT completely abrogated the chemiluminescence response of 2 x 10(6) human neutrophils per ml.

Adenylate Cyclase Toxin↗

PubMed1989

Development of a rapid diagnostic test for pertussis: direct detection of pertussis toxin in respiratory secretions.

Monoclonal antibodies (MAb) were produced against the specific Bordetella pertussis antigen pertussis toxin (PT). In preliminary studies, one MAb (IB12) was selected and used in an enzyme-linked dot blot immunoassay to evaluate the ability of the method to detect known amounts of PT in control experiments and to test its potential for direct detection of PT in nasopharyngeal secretion (NP) specimens from patients with confirmed cases of whooping cough. The dot blot assay was able to detect PT at levels as low as 10 ng per dot in either buffer or control NP specimens. The assay demonstrated specificity, reacting only with dot blots of whole B. pertussis and not Bordetella bronchiseptica, Bordetella parapertussis, or other bacterial strains. In preliminary studies, NP aspirate, swab, and wash specimens were compared. The specimen of choice was found to be the NP aspirate, for which 100% positive results were found in the assay. These initial studies suggest that the dot blot immunoassay in which a MAb is used for direct detection of PT in NP specimens may be useful as a rapid diagnostic test for pertussis.

Animals↗

PubMed1988

Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor.

Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'-untranslated region of 2.2 kb. The open reading frame encodes a 1,245-residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells.

Amino Acid Sequence↗

PubMed1988

Relaxation of bovine mesenteric artery induced by glyceryl trinitrate is attenuated by pertussis toxin.

Low concentrations (less than 1 nM) of glyceryl trinitrate (GTN) induced a considerable relaxation of bovine mesenteric arteries (BMA) brought to sustained contraction by the addition of phenylephrine. The concentration-response curve of GTN showed a biphasic pattern with a high and a low affinity component. Pretreatment with pertussis toxin (IAP) attenuated the high affinity relaxant component, but not the low affinity component or the relaxation induced by NaNO2. A polyclonal antibody to IAP counteracted the effect of the toxin on the GTN-response. Low concentrations of GTN increased the cGMP level in BMA via activation of the high affinity pathway. This effect was also inhibited in preparations pretreated with IAP. It is suggested from the present study that GTN induces relaxation of vascular smooth muscle via two separate pathways, the high affinity pathway might involve a receptor-complex interaction with a regulatory protein.

Animals↗

PubMed1988

Pertussis: the disease and new diagnostic methods.

Bordetella pertussis, the causative agent of whooping cough, produces an acute and chronic respiratory infection in infants and young children. B. pertussis is still a major health problem of young children throughout the world even though effective immunization against whooping cough is available. While predominantly a childhood disease, it has been reported also to be a cause of persistent cough in adults. This review discusses the numerous bacterial virulence factors that may play roles in the pathogenesis of pertussis and in immunity to infection. The present problems with pertussis diagnosis, recent advances, and future prospects for new and improved rapid diagnostics tests also are explored.

Bordetella pertussis↗

PubMed1988

Effects of adenylate cyclase toxin from Bordetella pertussis on human neutrophil interactions with Coccidioides immitis and Staphylococcus aureus.

Bordetella pertussis extract that contained adenylate cyclase toxin produced large increases in human neutrophil cyclic AMP levels and inhibited their oxidative burst, as reflected by luminol-enhanced chemiluminescence and superoxide release. The adenylate cyclase toxin-containing extract blocked neutrophil-mediated inhibition of N-acetylglucosamine incorporation by arthroconidia of Coccidioides immitis in a dose-dependent fashion but had no effect on neutrophil phagocytosis of Candida glabrata and only a slight inhibitory effect on arthroconidial attachment. Neither purified pertussis toxin nor extracts from Bordetella mutants lacking the adenylate cyclase toxin affected neutrophil-mediated inhibition of arthroconidial N-acetylglucosamine incorporation. These studies indicate that adenylate cyclase toxin, alone or in concert with other B. pertussis-elaborated toxins, blocks neutrophil inhibition of arthroconidia, primarily by affecting neutrophil responses other than attachment or phagocytosis.

Acetylglucosamine↗

PubMed1987

Bordetella pertussis adenylate cyclase: isolation and purification by calmodulin-sepharose 4B chromatography.

Purified preparations of adenylate cyclase were obtained from crude urea extracts of Bordetella pertussis by a one-step calmodulin affinity chromatography technique. Diluted extract was loaded onto the column and washed, and adenylate cyclase was eluted with 10mM EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. A 104-fold purification was accomplished in one step. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the affinity-purified adenylate cyclase was dissociated into one major protein band with an apparent molecular weight of 60,000 and a minor band at 200,000. The affinity-purified adenylate cyclase was observed to have adenylate cyclase enzymatic activity which was activated by calmodulin, to bind 125I-calmodulin, and to be free of pertussis toxin as determined by in vivo and in vitro assays.

Adenylate Cyclase Toxin↗

PubMed1987

Bordetella pertussis adenylate cyclase: effects of affinity-purified adenylate cyclase on human polymorphonuclear leukocyte functions.

Affinity-purified adenylate cyclase (AC) of Bordetella pertussis, free of contaminating pertussis toxin, was demonstrated to have biological effects on human polymorphonuclear leukocytes (PMN). AC at doses of 25 and 50 micrograms/ml increased intracellular cAMP levels in the phagocytes 7.6- to 23.5-fold, respectively, above basal levels. AC inhibited PMN chemiluminescence, chemotaxis, and superoxide production in a dose-dependent manner. The 50% inhibitory dose for chemotaxis and chemiluminescence was 36.5 micrograms/ml; for superoxide generation it was 71.0 micrograms/ml. Although these PMN metabolic functions were impaired, no effect on phagocytic activity was observed.

Adenylate Cyclase Toxin↗

PubMed1985

Cloning of a gene whose expression is increased in scrapie and in senile plaques in human brain.

A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

Alzheimer Disease↗

PubMed1985

Mechanisms of 3-hydroxy-3-methylglutaryl coenzyme A reductase overaccumulation in three compactin-resistant cell lines.

We have isolated three mammalian cell lines which are resistant to compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The drug resistance in all three cell lines is due to an increase of HMG-CoA reductase activity. Two of the three cell lines overaccumulate HMG-CoA reductase messenger RNA when grown in the presence of compactin. DNA hybridization experiments indicate that both a baby hamster kidney-derived compactin-resistant cell line, C100, and a cell line derived from mouse 3T6 cells, 3T6-40, exhibit amplifications of the HMG-CoA reductase gene. A third compactin-resistant cell line derived from Chinese hamster ovary cells, ML100, does not exhibit an amplification of the HMG-CoA reductase gene, nor does it show an elevated level of HMG-CoA reductase mRNA, comparable to that seen in the other cell lines.

Animals↗

PubMed1985

Expression of human adenosine deaminase using a transmissable murine retrovirus vector system.

Human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) was expressed at high levels in cultured mouse cells using a transmissable murine retrovirus vector system. A cDNA clone encoding ADA has been inserted into a plasmid vector containing retroviral transcription and packaging signals as well as a selectable gene for G418 resistance. The constructions were transfected into psi 2 cells, which package the recombinant retroviral genomes into replication-defective virus particles. Isoenzyme analysis for ADA in G418-selected psi 2 cells showed at least 20-fold more human ADA activity than endogenous mouse ADA activity. A mouse T-cell lymphoma line, BL/VL3, was cocultured with transformed psi 2 cells producing human ADA, and some of the cocultured cells were selected for resistance to G418. Both G418-selected and unselected cocultured cells expressed human ADA activity at 25%-50% the level of the endogenous enzyme. Thus, efficient retroviral transduction of ADA expression was obtained.

Adenosine Deaminase↗

PubMed1985

Bordetella pertussis filamentous hemagglutinin gene: molecular cloning of a potential coding sequence.

We have constructed an expression library of Bordetella pertussis DNA sequences, cloned in Escherichia coli. Random DNA fragments were inserted into the beta-galactosidase gene of bacteriophage lambda gt11 and plaques were screened with antibodies directed against filamentous hemagglutinin. The antigen-positive clone obtained expressed B. pertussis sequences as polypeptides fused to beta-galactosidase. The fusion polypeptides reacted both with antibodies to beta-galactosidase and with antibodies to filamentous hemagglutinin. The positive clone contains B. pertussis DNA sequences as determined by specific hybridization to Bordetella DNA.

Base Sequence↗

PubMed1984

Induction of human mRNAs by interferon.

We have cloned a set of seven cDNAs corresponding to mRNAs induced by type I interferon in human cells, and are using these probes to characterize the response to interferon in detail. We have studied the kinetics of induction of each mRNA in three different human cell lines, the sensitivity of induction to cycloheximide, the effect of withdrawing interferon and the response of a receptor-positive interferon-resistant cell line.

Cell Line↗

PubMed1984

Clozapine blocks disruptive and discriminative stimulus effects of quipazine.

Clozapine was tested in two serotonin-dependent behavioral measures. One group of rats was trained to discriminate the serotonin agonist, quipazine, from saline in a two-lever operant choice task. Pretreatment with clozapine completely blocked the discrimination of quipazine. Another group of rats was trained to bar press for milk on a variable interval schedule of reinforcement. Quipazine decreased the response rate in these animals and pretreatment with clozapine completely reversed this effect. Thus, clozapine acted as a serotonin antagonist in both measures of serotonin function.

Animals↗

PubMed1984

Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells.

Eighteen cDNAs, cloned from interferon-treated T98G neuroblastoma cells, correspond to seven different mRNAs induced up to 40-fold by interferon. One codes for metallothionein II and another for a class I HLA. The others do not code for proteins of known sequence. In the continued presence of interferon, accumulation of the mRNAs continues for about 1 day but ceases whenever interferon is removed. Once induced, the mRNAs are stable. Synthesis of new proteins is not required for induction. The rate of transcription of one of the genes doubles 5 min after treatment with interferon and reaches a maximum by 60 min. This rate begins to fall after 4-6 hr, reaching the uninduced level by 8-12 hr. Since the mRNA continues to accumulate after 8-12 hr, posttranscriptional events must also play a role in increasing its level.

Animals↗

PubMed1984

Multicenter study of family physician prescribing.

The objective of this study was to examine differences in prescribing characteristics among four Iowa family practice offices, each associated with family practice residency programs. This prospective study collected data over a four-month period, utilizing duplicate, carbon-copy prescriptions. The prescriptions were tabulated according to individual drug and therapeutic categories. Differences in prescribing frequency among offices were analyzed using chi-square 2 X 2 contingency tables. The number of prescriptions written at each office (designated A through D) were as follows: A, 1,034; B, 1,449; C, 2,965; and D, 2,335. The most frequently prescribed drug category was systemic antibiotics, followed by cough, cold, or allergy products, analgesic and anti-inflammatory drugs or muscle relaxants, diuretics, and topical anti-infectives. There were statistically significant differences in the frequencies of these categories among offices. The most frequently prescribed drug was amoxicillin at offices A, B, and C, and erythromycin at office D. There were statistically significant differences in the frequencies of the top ten drugs at each office. From these data the family practice faculty and clinical pharmacists can identify therapeutic areas that may require additional educational emphasis for the resident.

Drug Prescriptions↗

PubMed1984

Discriminative stimulus properties of quipazine: mediation by serotonin2 binding sites.

The relative abilities of putative serotonin (5-HT) antagonists to block the quipazine discriminative cue were compared with their relative potencies to compete for the 5-HT1 and 5-HT2 binding sites in the brain. Male Sprague-Dawley rats were trained to discriminate 2.5 mg/kg of quipazine from saline. Once training was complete, antagonists were administered 90 min before a 5-min test with 1 mg/kg of quipazine, a dose which gave 75% responding on the quipazine lever. A dose-response curve was generated for each antagonist and an ID50 value (dose that inhibits responding to 50% on the quipazine lever) was determined by log-logit analysis. The binding of [3H]-5-HT to the 5-HT1 site and of [3H]spiperone to the 5-HT2 site was determined in crude membranes prepared from frontal cortex ( [3H]spiperone) or pons-medulla ( [3H]-5-HT) of naive rats. IC50 values for the antagonists were determined by log-logit analyses of competition binding curves. The ID50 values for the blockade of the quipazine discrimination by the 5-HT antagonists correlated significantly with their affinities for the 5-HT2 binding site (r = 0.87). In contrast, no correlation existed between effects on the behavioral measure and affinities for the 5-HT1 site (r = 0.15). These results suggest that the quipazine cue is mediated by an action at central 5-HT2 sites.

Animals↗