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Biomedical subjects

R A Nicholas

Publications and source records attributed to R A Nicholas.

At least 19 recordsLinked to original sources
PubMed1997

The 6H1 orphan receptor, claimed to be the p2y5 receptor, does not mediate nucleotide-promoted second messenger responses.

An orphan G protein-coupled receptor, termed 6H1, with approximately 30% sequence identity to P2Y receptors has been proposed to be a P2Y receptor (p2y5) based solely on a radioligand binding assay with [35S]dATP alphaS [Webb et al. (1997) Biochem. Biophys. Res. Commun. 219:105-110]. Previous work in our laboratory has shown that [35S]-dATP alphaS is not a general ligand for P2Y receptors, and thus inclusion of the p2y5 receptor in the family of P2Y receptors is questionable. To define unambiguously whether the p2y5 receptor is a P2Y receptor, we have cloned the turkey homologue of the chick p2y5 receptor. Sequence analysis indicated that the turkey receptor contains an additional 32 amino acids at its carboxy terminus compared to the published chick sequence. HA epitope-tagged turkey p2y5 receptors were stably expressed in 1321N1 human astrocytoma cells, and cells shown to express the HA-tagged p2y5 receptor by an intact cell-based ELISA were used to determine whether changes in second messenger levels occurred in response to a series of nucleotides. ATP, ADP, UTP, UDP, dATP alphaS, and A2P4 had no effect on either inositol phosphate or cyclic AMP concentrations in cells expressing the p2y5 receptor. Robust inositol phosphate and cyclic AMP responses occurred to other G protein-coupled receptors expressed in 1321N1 cells, which indicate that these cells contain all of the necessary signaling components to generate these second messenger responses. These data indicate that the 6H1/p2y5 receptor is not a member of the P2Y receptor family of signaling proteins.

Animals↗
PubMed1997

Personal use of drug samples by physicians and office staff.

CONTEXT: Pharmaceutical samples are commonly used in ambulatory care settings. There is limited research on their use or impact on health care providers and patients. OBJECTIVE: To determine the extent of personal use of drug samples over a 1-year period by physicians and medical office staff. DESIGN, SUBJECTS, AND SETTING: An anonymous cross-sectional survey of all physicians, resident physicians, nursing staff, and office staff in a family practice residency. MAIN OUTCOME MEASURE: Quantity of drug samples taken for personal or family use. RESULTS: Of 55 surveys issued, 53 (96%) were returned. A total of 230 separate drug samples were reported taken in amounts ranging from 1 dose to greater than 1 month's supply. Two respondents reported no use of drug samples, while 4 respondents reported taking more than 10 different samples. CONCLUSION: Drug samples are commonly taken by physicians and office staff for personal and family use. The ethical implications of this practice warrant further discussion.

Advertising↗
PubMed1997

Lack of nucleotide-promoted second messenger signaling responses in 1321N1 cells expressing the proposed P2Y receptor, p2y7.

A recently cloned G protein-coupled receptor (named the p2y7 receptor) with relatively low sequence identity to previously cloned P2Y receptors was proposed to be a member of this family of receptors on the basis of both a radioligand binding assay with [35S]dATP alphaS and an inositol phosphate response to ATP in COS-7 cells transiently transfected with receptor cDNA. Previous work in our laboratory has shown that [35S]dATP alphaS is not a general radioligand for the identification of P2Y receptors and that COS-7 cells express an endogenous P2Y receptor (P2Y2) that complicates the analysis of nucleotide-promoted inositol phosphate responses. Thus, data supporting inclusion of the p2y7 receptor in the P2Y family of receptors are equivocal. To determine unambiguously whether the p2y7 receptor is a P2Y receptor subtype, a p2y7 receptor bearing an epitope-tag at its NH2-terminus was expressed in 1321N1 cells and cell surface expression of the receptor was demonstrated by an intact cell-based ELISA. Cells shown to express epitope-tagged p2y7 receptors by ELISA were examined for their second messenger signaling properties in response to a range of nucleotides. ATP, UTP, ADP, UDP, and dATP alphaS had no effect on phospholipase C or adenylyl cyclase activities in cells expressing the p2y7 receptor. Experimental controls utilizing expression of other G protein-coupled receptors showed that 1321N1 cells displayed robust responses for each of these signaling pathways. These data, together with the low sequence identity of the p2y7 receptor to other P2Y receptors, indicate that the p2y7 is not a member of the P2Y family of signaling molecules.

Adenine Nucleotides↗
PubMed1997

A mutational analysis of residues essential for ligand recognition at the human P2Y1 receptor.

We conducted a mutational analysis of residues potentially involved in the adenine nucleotide binding pocket of the human P2Y1 receptor. Mutated receptors were expressed in COS-7 cells with an epitope tag that permitted confirmation of expression in the plasma membrane, and agonist-promoted inositol phosphate accumulation was assessed as a measure of receptor activity. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7 predicted by molecular modeling to be involved in ligand recognition were replaced with alanine and, in some cases, by other amino acids. The potent P2Y1 receptor agonist 2-methylthio-ATP (2-MeSATP) had no activity in cells expressing the R128A, R310A, and S314A mutant receptors, and a markedly reduced potency of 2-MeSATP was observed with the K280A and Q307A mutants. These results suggest that residues on the exofacial side of TM3 and TM7 are critical determinants of the ATP binding pocket. In contrast, there was no change in the potency or maximal effect of 2-MeSATP with the S317A mutant receptor. Alanine replacement of F131, H132, Y136, F226, or H277 resulted in mutant receptors that exhibited a 7-18-fold reduction in potency compared with that observed with the wild-type receptor. These residues thus seem to subserve a less important modulatory role in ligand binding to the P2Y1 receptor. Because changes in the potency of 2-methylthio-ADP and 2-(hexylthio)-AMP paralleled the changes in potency of 2-MeSATP at these mutant receptors, the beta- and gamma-phosphates of the adenine nucleotides seem to be less important than the alpha-phosphate in ligand/P2Y1 receptor interactions. However, T221A and T222A mutant receptors exhibited much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors, respectively) than in diphosphate or monophosphate potency. This result may be indicative of a greater role of these TM5 residues in gamma-phosphate recognition. Taken together, the results suggest that the adenosine and alpha-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the exofacial side of the human P2Y1 receptor.

Adenosine Monophosphate↗
PubMed1997

Cloning and characterization of the ponA gene encoding penicillin-binding protein 1 from Neisseria gonorrhoeae and Neisseria meningitidis.

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.

Amino Acid Sequence↗
PubMed1996

A guanine nucleotide-independent inwardly rectifying cation permeability is associated with P2Y1 receptor expression in Xenopus oocytes.

The functional properties of the G protein-coupled P2Y1 receptor were investigated in Xenopus oocytes. Incubation of oocytes expressing either the human or turkey P2Y1 receptor with adenine nucleotide agonists resulted in an increase in Cl- current and activation of a novel cation current with an inwardly rectifying current-voltage relationship. Activation of either the human P2Y2 (P2U-purinergic) or M1 muscarinic receptor expressed in oocytes resulted in an increase in Cl- current similar to that observed in P2Y1 receptor-expressing oocytes but had no effect on cation current. P2 receptor agonists stimulated both the cation current and Cl- current in P2Y1 receptor-expressing oocytes with EC50 values and an order of potency (2-methylthioadenosine diphosphate > 2-methylthioadenosine triphosphate (2MeSATP) > ATP > UTP) that were similar to those previously observed for activation of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human or turkey P2Y1 receptor. The P2Y receptor antagonists suramin and pyridoxal phosphate 6-azophenyl-2'-4'-disulfonic acid both shifted to the right the concentration-response relationship for 2MeSATP for stimulation of oocyte currents. Although injection of oocytes with either GDPbetaS (guanyl-5'-yl thiophosphate) or GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) resulted in loss of adenine nucleotide-promoted Cl- channel activation, neither guanine nucleotide altered the 2MeSATP-stimulated cation current. These data are consistent with the view that activation of the novel cation current by the P2Y1 receptor does not involve a G protein. Tail current analysis of the novel P2Y1 receptor-associated cation conductance revealed that the open channel current-voltage relationship was outwardly rectifying with a reversal potential of -38 mV for the turkey P2Y1 receptor and -36 mV for the human P2Y1 receptor. Replacement of Na+ with K+ ions in the bathing solution produced a shift in reversal potential to near zero mV, but significant outward rectification remained. The cation current was not permeable to either Ca2+ or Ba2+ and exhibited steady-state inactivation at holding potentials below -60 mV. These results indicate that the P2Y1 receptor exhibits both metabotropic properties and a novel G protein-independent ionotropic response when expressed in Xenopus oocytes.

Animals↗
PubMed1996

A comparison of serological tests and gross lung pathology for detecting contagious bovine pleuropneumonia in two groups of Italian cattle.

Western and dot blotting techniques were compared with complement fixation tests (CFT), indirect enzyme-linked immunosorbent assays (ELISA), mycoplasma culture and gross lung pathology to detect Mycoplasma mycoides subspecies mycoides SC, the cause of contagious bovine pleuropneumonia (CBPP), in two groups of Italian cattle. None of the animals showed any clinical signs before slaughter. In group A, seven of the 20 cattle had characteristic lung lesions of acute and chronic CBPP but only six were positive by CFT. Western blotting detected antibody in eight of the animals, of which six had lesions and significant CFT titres (> 50 per cent fixation at a serum dilution of 1/10) and two had neither. In group B, seven of the 17 cattle had lesions characteristic of CBPP, and 12 were seropositive by CFT. Western blotting detected antibody in 13 of the animals including one which had a negative CFT titre. The ELISA was less sensitive than either CFT or Western blotting, detecting antibody in five animals in group A and nine animals in group B. The dot blotting test correlated well with Western blotting but gave a small number of ambiguous results. The causative organism was isolated from four of the 20 cattle in group A and six of the 17 cattle in group B.

Animals↗
PubMed1996

Random mutagenesis of the sheep Na,K-ATPase alpha1 subunit generating the ouabain-resistant mutant L793P.

The polymerase chain reaction was used to randomly mutagenize a cDNA cassette encoding amino acids 691-946 of the sheep Na,K-ATPase alpha subunit. The mutagenized cassettes were used to replace the wild-type region in the full-length cDNA, and pools of mutants were transfected into HeLa cells. After the generation of resistant cells via selection in 0.5 microM ouabain, polymerase chain reaction was used to amplify the mutagenized cassette from the genomic DNA of the stable transfectants. Sequence analysis of the polymerase chain reaction product revealed three amino acid substitutions: I729V, L793P, and K836R. Subsequent site-directed mutagenesis experiments showed that only L793P was important for resistance. To elucidate the role of L793 in ouabain inhibition, additional mutations at this position were prepared. L793A and L793I mutants were constructed and expressed in HeLa cells. Only L793A survived selection using ouabain, which suggested that resistance is not due to the specific substitution of leucine with proline. To explore the mechanism of resistance, apparent affinities of the L793P mutant for sodium and potassium were compared to the wild-type HeLa pump. Although the apparent affinities were comparable for sodium, the mutant had a 2-fold higher apparent affinity for potassium. This suggests that the mechanism of ouabain insensitivity of L793P is due to a perturbation in the region of the enzyme that may include the K+ binding site.

Amino Acid Sequence↗
PubMed1996

Molecular cloning, expression and regulatory activity of G alpha 11- and beta gamma-subunit-stimulated phospholipase C-beta from avian erythrocytes.

A turkey erythrocyte phospholipase C (PLC) has been instrumental in delineating the role of G-proteins in receptor-regulated inositol lipid signalling. This isoenzyme is uniquely regulated both by alpha-subunits of the Gq family and by G-protein beta gamma-subunits. A 4819 bp cDNA encoding this PLC has been cloned from a turkey erythrocyte cDNA library. The open reading frame of this cDNA encodes a 1211-amino-acid protein (calculated molecular mass 139050 Da) that contains amino acid sequences of 16 peptides sequenced from the turkey erythrocyte PLC. The predicted sequence of the turkey PLC shows considerable similarity with the sequences of previously cloned members of the PLC-beta family, with the highest identity (71%) shared with PLC-beta 2 and lesser identities observed with PLC-beta 1 (49%), PLC-beta 3 (46%) and PLC-beta 4 (37%). The largest differences in sequence between the turkey PLC-beta and other PLC-beta isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-beta 2, which differ in their regulation by G-protein alpha-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in alpha q activation of these proteins. Recombinant turkey PLC-beta was purified to homogeneity following expression from a recombinant baculovirus in Sf9 insect cells. The immunoreactivity and mobility on SDS/PAGE of the recombinant enzyme were the same as observed with native turkey erythrocyte PLC-beta. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-beta in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with G alpha 11 or G-protein beta gamma-subunits. The turkey PLC-beta was more sensitive to activation by G alpha 11 than was PLC-beta 2, and was more sensitive to activation by beta gamma-subunits than either PLC-beta 2 or PLC-beta 1.

Amino Acid Sequence↗
PubMed1996

Localization of a putative second membrane association site in penicillin-binding protein 1B of Escherichia coli.

We have shown previously that the periplasmic domain of penicillin-binding protein 1B (PBP 1Bper; residues 90-844) from Escherichia coli is insoluble in the absence of detergents, and can be reconstituted into liposomes [Nicholas, Lamson and Schultz (1993) J. Biol. Chem. 268, 5632-5641]. These data suggested that native PBP 1B contains a membrane association site in addition to its N-terminal transmembrane anchor. We have studied the membrane topology of PBP 1B in greater detail by assessing detergent binding and solubility in the absence of detergents for PBP 1Bper and a set of proteolytic fragments of PBP 1B. PBP 1Bper was shown by three independent methods to bind to detergent micelles, which strongly suggests that the periplasmic domain interacts with the hydrophobic milieu of membrane bilayers. Digestion with high weight ratios of thrombin of purified PBP 1B containing an engineered thrombin cleavage site on the periplasmic side of the transmembrane anchor generated four fragments in addition to PBP 1Bper that varied in size from 71 to 48 kDa. In contrast to PBP 1Bper, all fragments of 67 kDa and smaller were eluted from a gel-filtration column in the absence of detergents and did not bind to detergent micelles. The N-terminal sequences of the four fragments were determined, allowing the cleavage sites to be located in the primary sequence of PBP 1B. These data localize the membrane association site of PBP 1B to a region comprising the first 163 amino acids of the periplasmic domain, which falls within the putative transglycosylase domain. Lipid modification does not appear to be the mechanism by which PBP 1Bper associates with membranes.

Amino Acid Sequence↗
PubMed1996

Pharmacological and second messenger signalling selectivities of cloned P2Y receptors.

1. Four different phospholipase C (PLC)-activating P2Y receptors have been cloned and stably expressed in 1321N1 human astrocytoma cells. These include the human homologues of the P2Y1, P2Y2 and P2Y4 receptors and the rat homologue of the P2Y6 receptor. 2. The nucleotide selectivities of these four receptors have been compared directly by measuring inositol phosphate accumulation in response to nucleotides under conditions in which the initial purity and stability of agonist was rigidly assured and quantitatively assessed. 3. The P2Y1 receptor is specific for adenine nucleotides and slightly more sensitive to disphosphates than triphosphates. When expressed in 1321N1 astrocytoma cells, it couples selectively to the stimulation of PLC and not to the inhibition of adenylyl cyclase. 4. The P2Y2 receptor is activated by UTP and ATP with similar potency and is not activated by nucleoside diphosphates. Diadenosine terraphosphate is a potent agonist at this receptor. 5. The P2Y4 receptor is highly selective for UTP over ATP and is not activated by nucleoside disphosphates. 6. The P2Y6 receptor is activated most potently by UDP, but weakly or not at all by UTP, ADP and ATP. The P2Y6 receptor appears to be identical to the uridine nucleotide-specific receptor previously characterized in C6-2B rat glioma cells. 7. We have identified a P2Y receptor on C6 glioma cells that inhibits adenylyl cyclase but has no effect on PLC. This receptor exhibits a pharmacological selectivity similar but not identical to that of the P2Y1 receptor. When the P2Y1 receptor was expressed in these C6 cells, it conferred an inositol lipid signalling response to adenine nucleotides that was pharmacologically identical to that of the P2Y1 receptor. Thus, the P2Y receptor of C6 glioma cells represents an additional receptor that exhibits the classical pharmacological selectivity of a P2Y1-R, but which couples to adenylyl cyclase rather than to PLC.

Adenosine Triphosphate↗
PubMed1996

Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor.

1. The coding sequence of the P2Y1-purinoceptor was cloned from a human genomic library. 2. The open reading frame encodes a protein of 373 amino acids that is 83% identical to the previously cloned chick and turkey P2Y1-purinoceptor and is > or = 95% homologous to the recently cloned rat, mouse, and bovine P2Y1-purinoceptors. 3. The human P2Y1-purinoceptor was stably expressed in 1321N1 human astrocytoma cells using a retroviral vector. Although the P2Y1-purinoceptor agonist, 2MeSATP, had no effect on inositol phosphate accumulation in cells infected with the P2Y1-purinoceptor virus. No effect of 2MeSATP on cyclic AMP accumulation was observed in P2Y1-receptor-expressing 1321N1 cells. 4. The pharmacological selectively of 18 purinoceptor agonists was established for the expressed human P2Y1-purinoceptor. 2MeSATp was more potent than ATP but less potent than 2MeSADP. ADP also was more potent than ATP. A similar maximal effect was observed with most agonists tested. However, alpha, beta-MeATP had no effect and 3'-NH2-3'-deoxyATP and A2P4 were partial agonists. The order of potency of agonists for activation of the turkey P2Y1-purinoceptor, also stably expressed in 1321N1 cells, was identical to that observed for the human P2Y1-purinoceptor. 5. C6 glioma cells express a P2Y-purinoceptor that inhibits adenylyl cyclase but does not activate phospholipase C. Expression of the human P2Y1-purinoceptor in C6 cells conferred 2MeSATP-stimulated inositol lipid hydrolysis to these cells. The phospholipase C-activating human P2Y1-purinoceptor could be delineated from the endogenous P2Y-purinoceptor of C6 glioma cells by use of the P2-purinoceptor antagonist, PPADS, which blocks the P2Y1-purinoceptor but does not block the endogenous P2Y-purinoceptor of C6 cells. P2-purinoceptor agonists also exhibited differential selectivities for activation of these two P2Y-purinoceptors.

Amino Acid Sequence↗
PubMed1996

Development and application of enzyme-linked immunosorbent assay for specific detection of Salmonella enteritidis infections in chickens based on antibodies to SEF14 fimbrial antigen.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to the SEF14 fimbrial antigen (SEF14-DAS ELISA) and was evaluated for its use in the specific detection of chicken flocks infected with Salmonella enteritidis. The SEF14-DAS ELISA successfully discriminated between chickens experimentally infected with S. enteritidis and those infected with S. panama or S. typhimurium, although the SEF14 responses in adult birds infected with S. enteritidis were detectable but low. In contrast, ELISAs used to detect antibodies to lipopolysaccharide (LPS) and flagella were unable to discriminate between the infected groups of chicks and adult birds infected with different Salmonella serotypes. LPS and flagellar responses were low and variable in chicks, whereas in adult hens they were found to be consistently strong. When flocks naturally infected with S. enteritidis were tested by the SEF14-DAS ELISA and ELISAs to detect LPS and flagellar antibodies, it was found that they could all identify the infected flocks, although there was little correlation between individual serum samples. The study shows that the SEF14-DAS ELISA may offer advantages over existing assays with comparable sensitivities coupled with higher specificities for the serological detection of S. enteritidis-infected chicken flocks.

Animals↗
PubMed1996

Quinolones: a practical review of clinical uses, dosing considerations, and drug interactions.

A review of the literature on quinolones reveals numerous clinically relevant points regarding indications, dosing considerations, and drug interactions. Quinolones are useful in the treatment of several infectious diseases. Unfortunately, indiscriminate use of these valuable antimicrobials has resulted in increased patterns of resistance. It is important to consider carefully the site of infection and the potential pathogens in each patient before dosing. Quinolones have excellent oral absorption, with peak serum concentrations approaching those achieved with intravenous administration. When prescribing quinolones, the dose should be based on estimated creatinine clearance. Quinolones are associated with several clinically significant drug interactions. Some of these agents are well-documented inhibitors of hepatic metabolism of theophylline, caffeine, and warfarin. It has been well documented that divalent and trivalent cations in antacids, sucralfate, and some other products significantly reduce the absorption of quinolones. Avoidance or proper management of these interactions is required to ensure optimal safety and efficacy.

Anti-Infective Agents↗
PubMed1996

Intussusception.

Intussusception is the invagination of one bowel segment into another. It is an emergent condition that most commonly affects infants between five and nine months of age, but it can also occur in other age groups. The etiology is usually idiopathic in infants five to nine months of age; neonates, older children and adults more commonly have lead points such as a Meckel's diverticulum or a neoplasm. Early diagnosis is essential to avoid treatment delays, which can increase morbidity and mortality. It has been reported that patients with intussusception present with abdominal pain, vomiting and bloody stools, but this classic triad is often absent. More commonly, lethargy and irritability are the presenting signs. A rectal examination, with testing for occult blood, is an important part of the evaluation and is frequently positive. Barium enema is the gold standard for diagnosis and also has therapeutic potential for reducing the intussusception. Ultrasound is an accurate, low-risk screening tool when performed and interpreted by an experienced ultrasonographer. Surgical reduction is performed if nonoperative reduction is contraindicated or unsuccessful, or if a lead point is suspected.

Humans↗