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Biomedical subjects

R A Nicholas

Publications and source records attributed to R A Nicholas.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1996

Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor.

Observation that the G protein-coupled P2U receptor (P2Y2 receptor) is activated by UTP as well as ATP provided the first indication that a class of uridine nucleotide-responsive receptors might exist. This hypothesis was confirmed by our identification of a uridine nucleotide-specific receptor on C6-2B rat glioma cells and by the recent cloning of two uridine nucleotide-responsive receptors, the P2Y6 receptor [J. Biol. Chem. 270:26152-26158 (1995)] and the P2Y4 receptor [J. Biol. Chem. 270:30849-30852 (1995) and J. Biol. Chem. 270:30845-30848 (1995)]. The relative nucleotide selectivities of these uridine nucleotide-activated receptors have not been established. Therefore, we cloned and expressed the P2Y6 and P2Y4 receptors in 1321N1 human astrocytoma cells and compared their relative selectivities for UDP, UTP, and other uridine and adenine nucleotides with that of the P2Y2 receptor expressed in the same cells. These comparisons were made by measuring inositol phosphate accumulation under conditions in which the initial purity and stability of agonists were rigidly ensured and quantitatively assessed. The data indicate that the P2Y2 receptor is activated with similar potencies by ATP and UTP but not by ADP or UDP; the P2Y6 receptor is activated most potently by UDP but weakly by UTP, ATP, and ADP; and the P2Y4 receptor is activated most potently by UTP, less potently by ATP, and not at all by nucleotide diphosphates. Furthermore, the P2Y6 receptor, which displays a uridine nucleotide selectivity essentially identical to that of the uridine nucleotide-specific receptor in C6-2B cells, was shown to be natively expressed in C6-2B cells and to account for the uridine nucleotide responses originally identified in these cells. These results define the uridine nucleotide selectivity of three phospholipase C-linked receptors: a receptor that is selectively activated by UDP (P2Y6 receptor), selectively activated by UTP (P2Y4 receptor), and activated by UTP and ATP but not by diphosphate nucleotides (P2Y2 receptor).

Adenosine Triphosphate↗
PubMed1995

P2-purinergic receptors: subtype-associated signaling responses and structure.

Extracellular adenine nucleotides interact with P2-purinergic receptors to regulate a broad range of physiological processes. These receptors include the P2Y-, P2U-, P2T-, P2X-, and P2Z-purinergic receptor subtypes. This review focuses on the first three of these receptor subtypes, which couple to G proteins and regulate the inositol lipid, cyclic AMP, and other second-messenger signaling cascades. Both pharmacological data and the occurrence of selectivity of coupling to second-messenger pathways indicate the existence of multiple members in several of the classes of P2-purinergic receptor subtypes. Complementary DNAs cloned for P2Y- and P2U-purinergic receptors predict proteins with seven transmembrane-spanning motifs, typical of that of other G protein-linked receptors.

Adenine Nucleotides↗
PubMed1995

How well do older persons tolerate moderate altitude?

We studied the physiologic and clinical responses to moderate altitude in 97 older men and women (aged 59 to 83 years) over 5 days in Vail, Colorado, at an elevation of 2,500 m (8,200 ft). The incidence of acute mountain sickness was 16%, which is slightly lower than that reported for younger persons. The occurrence of symptoms of acute mountain sickness did not parallel arterial oxygen saturation or spirometric or blood pressure measurements. Chronic diseases were present in percentages typical for ambulatory elderly persons: 19 (20%) had coronary artery disease, 33 (34%) had hypertension, and 9 (9%) had lung disease. Despite this, no adverse signs or symptoms occurred in our subjects during their stay at this altitude. Our findings suggest that persons with preexisting, generally asymptomatic, cardiovascular or pulmonary disease can safely visit moderate altitudes.

Acute Disease↗
PubMed1994

Molecular cloning and characterization of a novel beta-adrenergic receptor.

In an attempt to isolate new G protein-coupled receptors from turkey erythrocytes, reverse transcribed polymerase chain reaction was performed on fetal turkey blood RNA using degenerate primers based on conserved sequences present in seven transmembrane receptors. An open reading frame in one of the clones, designated 4C (497 base pairs), displayed approximately 50-60% identity to all of the previously cloned beta-adrenergic receptors (beta-ARs). A lambda-DASH turkey genomic library was screened with a probe generated from the partial 4C cDNA, and the gene encoding this receptor was localized to a 3.5-kilobase pair HindIII fragment. Ribonuclease protection analysis of turkey lung mRNA indicated that the 3' end of the coding sequence of the 4C gene, like beta 3-AR, was interrupted by an intron. To obtain the cDNA sequence of 4C, RNA-polymerase chain reaction was performed using primers complementary to regions identified by ribonuclease protection analysis to be present in 4C mRNA. Comparison of the genomic and cDNA sequences of 4C indicated that the first exon encodes 414 amino acids of the protein, the second exon (68 base pairs) encodes an additional 12 residues followed by a stop codon, and the third exon is composed of 3'-untranslated sequence. The 4C receptor was transiently expressed in COS-1 cells, and the apparent affinities of a series of beta-AR agonists and antagonists were determined using [125I]iodocyanopindolol. As implicated by its amino acid sequence, 4C displayed a pharmacological selectivity that was consistent with that of a beta-AR but distinct from other cloned beta-ARs. Isoproterenol, epinephrine, and norepinephrine stimulated cyclic AMP accumulation in a concentration-dependent manner in mouse L cells stably expressing the 4C receptor. No effect on phospholipase C activity was observed. Ribonuclease protection assays indicated that 4C mRNA exhibits a broad tissue distribution, which suggests that it may play an important role in avian physiology.

Adenylyl Cyclases↗
PubMed1994

High yield expression of the Neurospora crassa plasma membrane H(+)-ATPase in Saccharomyces cerevisiae.

A simple system for high yield expression of the neurospora plasma membrane H(+)-ATPase is described. Two neurospora H(+)-ATPase cDNAs differing only in a few bases preceding the coding region were cloned into a high copy number yeast expression vector under the control of the constitutive promoter of the yeast plasma membrane H(+)-ATPase, and the resulting plasmids were used to transform Saccharomyces cerevisiae strain RS-72, which requires a plasmid-borne functional plasma membrane H(+)-ATPase for growth in glucose medium (Villalba, J. M., Palmgren, M. G., Berberian, G. E., Ferguson, C., and Serrano, R. (1992) J. Biol. Chem. 267, 12341-12349. Both plasmids supported growth of the cells, indicating that the neurospora ATPase is expressed in functional form in yeast. Western blots of membranes from the transformants confirmed that the neurospora ATPase is expressed in the yeast cells, with production in the range of several percent of the yeast membrane protein. Importantly, when the expressed, recombinant neurospora ATPase molecules are solubilized from the membranes with lysolecithin and subjected to glycerol gradient centrifugation, they migrate to a position indistinguishable from that of the native ATPase and display a comparable specific ATPase activity, indicating that the great majority of the recombinant neurospora ATPase molecules produced in yeast fold in a natural manner. This expression system thus appears to be ideal for site-directed mutagenesis studies of the neurospora ATPase molecule.

Adenosine Triphosphate↗
PubMed1994

Observations on a broiler breeder flock naturally infected with Salmonella enteritidis phage type 4.

Bacteriological and serological studies in a broiler breeder flock naturally infected with Salmonella enteritidis indicated that the infection had become established by about 20 to 22 weeks of age. By 35 weeks, 70 per cent of the birds were positive by ELISA and at 39 weeks, 39 per cent were estimated to be carrying the infection as judged by culture post mortem. By 50 weeks, post mortem culture showed 17 per cent to be infected and 85 per cent reacted to the ELISA. Over the period of observation at the laboratory, combined cloacal swabbing and post mortem culture showed that at least 12.5 per cent of the birds were carrying the infection and 95.7 per cent reacted serologically. As the level of infection apparently declined with age it may have been present at too low a level to detect in the unincubated eggs and newly hatched chicks which were cultured. However, maternal antibody was present in 81 per cent of the eggs and 23 per cent of the chicks and it may have affected the colonisation of the organism and the ability to isolate S enteritidis.

Animals↗