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R A Nicholas

Publications and source records attributed to R A Nicholas.

At least 73 records · Page 4Linked to original sources
PubMed1994

Expression of a cloned P2Y purinergic receptor that couples to phospholipase C.

P2Y purinergic receptors previously have been shown to couple either to activation of phospholipase C through a pertussis toxin-insensitive mechanism or to inhibition of adenylyl cyclase through pertussis toxin-sensitive members of the G1 family of G proteins. These and other pharmacological data strongly suggest that multiple P2Y purinergic receptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] cloned a cDNA that, when expressed in frog oocytes, displayed the general pharmacological characteristics of a P2Y purinergic receptor but whose second messenger linkage was not resolved. We have now cloned the meleagrid (turkey) homologue of the previously cloned chick P2Y purinergic receptor and have stably expressed it in a heterologous human cell line (1321N1 astrocytoma cells) to establish its signaling properties. The purinergic receptor agonist 2-methylthio-ATP (2MeSATP) stimulated a marked activation of phospholipase C in 1321N1 cells stably expressing the meleagrid receptor. The order of potency of a series of analogues of ATP and ADP for stimulation of phospholipase C by the receptor expressed in 1321N1 cells [2MeSATP = 2-methylthio-ADP > adenosine 5'-O-(2-thio)diphosphate > ADP > 2-chloro-ATP = adenosine 5'-O-(3-thio)triphosphate > or = ATP > adenylyl-imidodiphosphate > UTP] was similar to that observed for P2Y purinergic receptors in turkey erythrocytes and many other tissues and was markedly different from those of the P2U and P2X purinergic receptor subtypes. Stimulation of inositol lipid hydrolysis by P2Y purinergic agonists was not affected by preincubation of cells with pertussis toxin. In contrast to its marked effects on phospholipase C activity, 2MeSATP caused only a small and variable inhibition of cAMP accumulation. Ribonuclease protection analysis of turkey tissues showed that this P2Y purinergic receptor is most highly expressed in blood and brain. Taken together, these results indicate that a phospholipase-C-activating P2Y purinergic receptor has been cloned and stably expressed in 1321N1 astrocytoma cells.

Animals↗
PubMed1993

Further studies of the application of live Salmonella enteritidis aroA vaccines in chickens.

A model was developed to simulate the lateral spread of Salmonella enteritidis infection among chickens. One group of newly hatched chicks was vaccinated orally with S enteritidis aroA. At three weeks old naive chickens were infected with a wild-type strain of S enteritidis and brought into contact with separate groups of aroA vaccinated chickens and unvaccinated control chickens. The vaccinated chickens were well protected against colonisation of the gut by the wild-type strain whereas the control group became heavily colonised. The IgG responses to a lipopolysaccharide extract of S enteritidis in the vaccinated chickens indicated a limitation of invasion from the gut. Chickens vaccinated orally at one day old with S enteritidis aroA were not protected against oral or intravenous challenge at eight weeks old with a wild-type strain of S typhimurium. A group of newly hatched female chicks was vaccinated orally with S enteritidis aroA and again at two weeks old. A second group also received oral booster doses at 16 and 18 weeks. When challenged intravenously with a wild-type strain of S enteritidis at 23 weeks old there was a significant reduction in the numbers of this strain in the spleens, livers, ovaries and caeca of both vaccinated groups. Booster vaccination at 16 and 18 weeks of age induced the greatest protection of the caeca.

Animals↗
PubMed1993

Identification of G alpha 11 as the phospholipase C-activating G-protein of turkey erythrocytes.

A 43 kDa phospholipase C-activating protein has been purified previously from turkey erythrocytes and shown to express immunological properties expected of that of the Gq family of G-protein alpha-subunits [Waldo, Boyer, Morris and Harden (1991) J. Biol. Chem. 266, 14217-14225]. Internal amino acid sequence has now been obtained from this protein which shares 50-100% sequence identity with sequences encoded by mammalian G alpha 11 and G alpha q cDNAs. To identify the purified protein unambiguously, it was necessary to compare its amino acid sequence with the sequence encoded by avian G-protein alpha-subunit cDNA. As such, mouse G alpha q was used as a probe to screen turkey brain and fetal-turkey blood cDNA libraries. A full-length cDNA was identified that encodes avian G alpha 11, on the basis of its 96-98% amino acid identity with mammalian G alpha 11. All eight peptides sequenced from the turkey erythrocyte phospholipase C-activating protein are completely contained within the deduced amino acid sequence of the avian G alpha 11 cDNA. Expression of this cDNA in Sf9 cells by using a baculovirus expression system resulted in the production of a 43 kDa protein that reacts strongly with antisera to the Gq family of G-protein alpha-subunits and activated purified avian phospholipase C in an AlF4(-)-dependent manner. Taken together, these results unambiguously identify the protein purified from turkey erythrocytes, on the basis of its capacity to activate avian phospholipase C, as G alpha 11.

Aluminum↗
PubMed1993

Penicillin-binding protein 1B from Escherichia coli contains a membrane association site in addition to its transmembrane anchor.

A working structural model of penicillin-binding protein 1B (PBP 1B) from Escherichia coli derived from previous data consists of a highly charged aminoterminal cytoplasmic tail, a 23-amino-acid hydrophobic transmembrane anchor, and a 758-amino-acid periplasmic domain. Using an engineered thrombin cleavage site, we have investigated the solubility properties of the periplasmic domain of PBP 1B. Twelve amino acids, comprised of the consensus thrombin cleavage site (LVPR decreases GS) and flanking glycine residues, were inserted into PBP 1B just past its putative transmembrane segment. To aid in purification, a hexa-histidine tag was also inserted at its amino terminus, and the engineered protein (PBP 1B-GT/H6) was purified and characterized. Inclusion of the thrombin cleavage site had no effect on the protein's intrinsic tryptophan fluorescence and affinity for [14C]penicillin G, indicating that the protein structure was not significantly perturbed. PBP 1B-GT/H6 was readily cleaved by thrombin at low thrombin/protein ratios to a protein with properties consistent with the removal of its cytoplasmic tail and transmembrane regions. Cleavage of the protein was dependent upon the presence of the thrombin cleavage site, and the thrombin-cleaved protein (PBP 1Bper) displayed an identical affinity for [14C] penicillin G binding as wild-type PBP 1B and uncleaved PBP 1B-GT/H6. [14C]Penicillin G-labeled PBP 1Bper eluted from a gel filtration column in the presence but not in the absence of 0.7% 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonic acid, and PBP 1Bper was found entirely in the membrane fraction of a thrombin digest of membranes containing overproduced PBP 1B-GT/H6. To further characterize this unusual solubility behavior, purified PBP 1Bper was reconstituted into lipid vesicles, which were then floated on a sucrose gradient. Floated vesicles contained > 95% of total 125I-penicillin V binding, indicating that PBP 1Bper directly associates with lipid membranes. These results strongly suggest that the periplasmic domain of PBP 1B associates with membranes independent of its amino terminal transmembrane region.

Amino Acid Sequence↗
PubMed1992

Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding.

All penicillin-binding proteins (PBPs) contain a conserved box of homology in the carboxyl-terminal half of their primary sequence that can be Lys-Thr-Gly, Lys-Ser-Gly, or His-Thr-Gly. Site-saturation mutagenesis was used to address the role of the lysine residue at this position (Lys213) in Escherichia coli PBP 5, a D-alanine carboxypeptidase enzyme. A soluble form of PBP 5 was used to replace Lys213 with 18 other amino acids, and the ability of these mutant proteins to bind [3H]penicillin G was assessed. Only the substitution of lysine with arginine resulted in a protein that was capable of forming a stable covalent complex with antibiotic. The affinity of [14C]penicillin G for the arginine mutant was 1.2-fold higher than for wild-type PBP 5 (4.4 versus 5.1 micrograms/ml for 20 min at 30 degrees C), and both proteins showed identical rates of hydrolysis of the [14C]penicilloyl-bound complex (t1/2 = 9.1 min). Surprisingly, the arginine-substituted protein was unable to catalyze D-alanine carboxypeptidase activity in vitro, which suggests that there is a substantial difference in the geometries of the peptide substrate and penicillin G within the active site of PBP 5.

Amino Acid Sequence↗
PubMed1992

Vaccination of chickens with chicken-derived Salmonella enteritidis phage type 4 aroA live oral Salmonella vaccines.

Two strains of Salmonella enteritidis phage type 4 isolated from chickens, SeLA5 and Se267, were virulent in Balb/c mice by intraperitoneal (i.p.) infection. In 18-20-day-old chickens given 10(7) c.f.u. intravenously (i.v.), both strains caused some deaths. One-day-old chicks infected orally with 10(9) c.f.u. of either strain also suffered limited mortality. Strain SeLA5 was the more virulent by the oral route. AroA mutants SeLA5 aroA and Se267 aroA were reduced in i.p. virulence by up to six logs in the Balb/c mouse model, did not kill 18-20-day-old chickens when injected i.v. and did not multiply in the tissues. Oral vaccination of chicks with either 10(9) c.f.u. at one-day-old or 10(5) c.f.u. at seven-day intervals until day 21 with vaccine strains SeLA5 aroA or Se267 aroA conferred significant protection against intravenous challenge with virulent strain Se109 NalR and reduced intestinal shedding following oral challenge. Seroconversion following oral vaccination was not seen with the lower dose regimen and was low with the higher dose. Seroconversion following oral challenge was marked in unvaccinated birds but much lower in vaccinated birds, suggesting that oral vaccination had prevented invasion by the challenge strain.

Administration, Oral↗
PubMed1991

Detection of antibody to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs.

Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect IgG antibodies to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs. Better discrimination and more consistent results were obtained between eggs from experimentally infected and uninfected hens by using saline-dilution of yolk rather than chloroform extraction. Threshold absorbance values were determined in three salmonella-free flocks, and on the basis of these results ELISA optical density values greater than 0.25 were considered to be positive for antibodies to salmonella. Four flocks with a history of salmonella infection were examined; three contained birds which were seropositive for S enteritidis by ELISA and from which S enteritidis was isolated, and a large proportion of eggs from these birds contained antibody to S enteritidis. Eggs from the fourth flock had no detectable antibody, although serum antibody was detected in some birds. No salmonellae were isolated from the yolks of the eggs from any of the four flocks.

Animals↗
PubMed1991

Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.

Animals↗
PubMed1991

Treatment of acute mountain sickness: hyperbaric versus oxygen therapy.

STUDY OBJECTIVES: To compare the benefits of simulated descent in a hyperbaric chamber with those of supplementary oxygen for the treatment of acute mountain sickness. DESIGN: A prospective study. SETTING: The Snake River Health Clinic in Keystone, Colorado, which has an altitude of 2,850 m (9,300 ft). TYPE OF PARTICIPANTS: Twenty-four patients who presented with acute mountain sickness. INTERVENTIONS: A simulated descent of 1,432 m (4,600 ft) was attained by placing the patients in a fabric hyperbaric chamber and pressurizing the chamber to 120 mm Hg (2.3 PSI) above ambient pressure. Patients were randomly assigned to either the hyperbaric treatment or treatment with 4 L of oxygen given by facemask; both treatments lasted for two hours. MEASUREMENTS AND MAIN RESULTS: Mean arterial oxygen saturation (SaO2) increased 7% (84 +/- 2% to 91 +/- 1%) with pressurization and 14% (83 +/- 4% to 96 +/- 1%) with oxygen during treatment over pretreatment levels. Symptoms of acute mountain sickness decreased as rapidly with pressurization as with oxygen treatment, despite significantly higher SaO2 in the oxygen-treated group during treatment. Symptomatic improvement was retained in both groups at least one hour after treatment. CONCLUSION: Simulated descent in a fabric hyperbaric chamber is as effective as oxygen therapy for the immediate relief of acute mountain sickness.

Acute Disease↗
PubMed1990

Inactivated canine parvovirus vaccines: an alternative method for assessment of potency.

Groups of three-week-old chickens were given graded doses of inactivated canine parvovirus vaccines. Blood samples were taken three weeks later and the sera examined by ELISA for antibodies to canine parvovirus. Reproducible, linear, log-dose serological responses were observed, enabling the potency of the vaccines to be compared. The simultaneous administration of other components of canine multivalent vaccines appeared to reduce the response to the parvovirus component. When its results have been correlated with the degree of protection in dogs this test could be used to assess the potency of inactivated canine parvovirus vaccines.

Animals↗
PubMed1990

Vaccination of chickens with a Salmonella enteritidis aroA live oral Salmonella vaccine.

A mouse-virulent strain of Salmonella enteritidis, Se795 (LD50 less than 10 organisms for mice), was non-virulent for 12-day-old chickens given 10(6) cfu intravenously; the organisms were cleared from liver and spleen by day 14 as measured by direct plating and by day 21 by enrichment. An Se795aroA mutant, CU58, was also cleared from liver and spleen by day 14 after intravenous inoculation of 10(7) cfu. Day-old chicks vaccinated orally with either one dose of 10(9) CU58 at 1 day of age, 10(7) at 1 and 14 days, or 10(5) at 1 and 7 days followed by 10(9) at 14 and 21 days of age, were challenged orally with a nalidixic acid resistant variant of the virulent phage type 4 S. enteritidis strain 109. All vaccinated groups showed a reduction in faecal shedding of the challenge. Chickens given four doses of CU58 showed a significant reduction of cfu in liver, spleen and faeces following intravenous challenge with virulent strain 109. Intramuscular vaccination with 10(9) cfu of Aro strain CU58 at 1 day of age gave no protection against oral challenge with virulent strain 109. Serum antibody production to LPS (ELISA) was minimal in all vaccinated birds. The results indicate that oral vaccination with Aro- S. enteritidis can confer protection to day old chicks against virulent S. enteritidis.

Animals↗
PubMed1990

Molecular biology of mammalian amino acid receptors.

The amino acid receptor proteins are ubiquitous transducers of most excitatory and inhibitory synaptic transmission in the brain. In July 1987 two reports appeared describing the molecular cloning of a pair of subunits of the GABAA receptor (7) and one subunit of the glycine receptor (13). These papers sparked wide interest and led quickly to the concept of a ligand-gated receptor-ion channel superfamily that includes nicotinic acetylcholine receptors as well as certain amino acid receptors. The identification of additional subunits of each receptor followed; with the recent cloning of a kainate receptor subunit (14), only the NMDA receptor remains elusive. Several disciplines have been brought to bear on these receptor clones, including in situ hybridization and functional expression in Xenopus laevis oocytes and mammalian cell lines. In this review we compare cloning strategies that have been used for amino acid receptors and discuss structural similarities among the receptor subunits. Two findings that have arisen from molecular cloning and expression of these receptors receive special attention. First, the molecular heterogeneity of GABAA receptors is larger than expected from pharmacological studies of native receptors. Second, although the native receptors are thought to be heterooligomers, much like the model proposed for the nicotinic receptors, some individual amino acid receptor subunits can form functional receptor channels, presumably in a homomeric configuration. This review focuses, therefore, on what we have learned from cloning efforts about amino acid receptors and what might lie ahead in this field.

Amino Acid Sequence↗
PubMed1990

Homomeric GluR1 excitatory amino acid receptors expressed in Xenopus oocytes.

The GluR1 cDNA clone encodes a functional excitatory amino acid receptor (Hollmann et al., Nature 342: 643-648 (1989]. We have studied the pharmacological properties of this homomeric (single subunit) receptor expressed in Xenopus oocytes and compared these properties with those of receptors encoded by rat forebrain mRNA. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate, quisqualate, and glutamate were partial agonists at both GluR1 and forebrain non-N-methyl-D-aspartate (-NMDA) receptors. The potency of the agonists kainate, domoate, and glutamate was higher, and that of the antagonists 6-cyano-7-nitro-quinoxalinedione and 6,7-dichloro-3-hydroxy-2-quinoxaline carboxylic acid lower, for GluR1 receptors as compared with forebrain non-NMDA receptors. The GluR1 receptor differed strikingly from forebrain-derived non-NMDA receptors, however, in that it exhibited slow, calcium-dependent desensitization. Thus, most properties of the GluR1 receptor are similar but not identical to those of non-NMDA receptors expressed from forebrain mRNA. These results indicate that the ligand recognition sites on GluR1 homomeric receptors are subtly different from those of non-NMDA receptors expressed from a mixture of forebrain mRNA.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1989

Serological and bacteriological investigations of chickens from flocks naturally infected with Salmonella enteritidis.

Groups of 10 birds were obtained from four flocks which had shown evidence of natural salmonella infection. S enteritidis had been isolated from three flocks and S typhimurium from the fourth. Each bird was housed in a separate cage and blood samples and cloacal swabs were taken weekly to follow the course of natural infection. After four weeks the birds were killed and examined post mortem. The isolation of Salmonella species could not be related to the serological results. In individual birds the rapid slide test and tube agglutination test could not be relied upon to detect infection; the microantiglobulin test and the enzyme-linked immunosorbent assay (ELISA) were more sensitive than the other tests and detected some infected birds that were negative by the rapid slide and tube agglutination tests, and also showed high titres in some birds from which Salmonella species could not be isolated post mortem. Sera obtained from two flocks which had a history of natural S enteritidis infection were evaluated by all the tests; evidence of infection was found with the microantiglobulin and ELISA tests but not with the other tests.

Agglutination Tests↗