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Biomedical subjects

R Nichols

Publications and source records attributed to R Nichols.

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

A motif in human histidyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope.

In myositis, disease-specific autoantibodies may be directed against an aminoacyl-tRNA synthetase, usually histidyl-tRNA synthetase. To explore the basis for this phenomenon, we have made recombinant histidyl-tRNA synthetase in the baculovirus system. It was enzymatically active and recognized by human autoantibodies. A truncated protein lacking the first 60 amino acids was inactive as an antigen and as an enzyme. This region is within the first two exons, is predicted to have a coiled-coil configuration, and is found in some other synthetases but not in Escherichia coli or yeast histidyl-tRNA synthetase. Circular dichroism showed that the peptides from this region (amino acids 1-60 and 1-47) have the predicted high alpha-helical content, but smaller fragments (1-30, 14-45, and 31-60) do not. The peptides with a high alpha-helical content could inhibit autoantibodies almost completely, whereas the smaller peptides were unable to do so. The amino acid sequence of this coiled-coil domain in human histidyl-tRNA synthetase resembles the sequence of the extended this coiled-coil arm near the NH2 terminus of bacterial seryl-tRNA synthetase as well as similar regions in some eukaryotic aminoacyl-tRNA synthetases, raising the possibility that this domain serves a similar tRNA-stabilizing role and has been preserved from a common ancestor.

Amino Acid Sequence↗
PubMed1994

A universal nucleoside for use at ambiguous sites in DNA primers.

A non-discriminatory base analogue, or universal base, would be an invaluable component of oligonucleotide probes and primers for solving the design problems that arise as a result of the degeneracy of the genetic code, or when only fragmentary peptide sequence data are available. We have designed an alternative to previous universal nucleoside candidates, a new analogue, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (designated M; Fig. 1), which maximizes stacking while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex. Oligonucleotides containing M at several sites were used as primers for sequencing and the polymerase chain reaction. The sequencing primer d(5'-CGT AAM CAM AAM ACM AT-3') is as effective as the exact match d(5'-CGT AAT CAG AAA ACA AT-3'). It is also possible to sequence using a primer containing M at several contiguous positions, for example d(5'-CGT AAT MMM MMM MMM AT-3'). Melting curves show that duplexes formed on hybridization of the sequences d(5'-CCT TTT TMT TTT TGG-3') and d(5'-CCA AAA AXA AAA AGG-3'), where X is A, C, G or T, melted at a lower temperature than the corresponding duplexes containing only d(A.T) and d(C.G) base pairs, but showing little variation among different X bases (Tm range 3 degrees C).

Animals↗
PubMed1994

Callitachykinin I and II, two novel myotropic peptides isolated from the blowfly, Calliphora vomitoria, that have resemblances to tachykinins.

Two peptides, related to the locust myotropic peptides locustatachykinin I-IV, were isolated from the blowfly Calliphora vomitoria. Whole, frozen flies were used for extraction with acidified methanol. A cockroach hindgut muscle contraction bioassay was used for monitoring fractions during subsequent purification steps. A series of eight different high performance liquid chromatography column systems was required to obtain optically pure peptides. Two peptides were isolated and their sequences determined by Edman degradation and confirmed by mass spectrometry and chemical synthesis as APTAFYGVR-NH2 and GLGNNAFVGVR-NH2. They were named callitachykinin I and II. The peptides have sequence similarities to the locustatachykinins and vertebrate tachykinins. Both callitachykinins were recognized by an antiserum to locustatachykinin I in enzyme-linked immunosorbent assay (ELISA) tests and callitachykinin II was additionally recognized by an antiserum to the vertebrate tachykinin kassinin, suggesting that immunolabeling of blowfly neurons with these antisera is due to neuronal callitachykinins.

Amino Acid Sequence↗
PubMed1994

Assessment of patient satisfaction utilizing the American Pain Society's Quality Assurance Standards on acute and cancer-related pain.

An evaluation of patient satisfaction with pain management is one component of a total quality assurance program on pain management recommended by the American Pain Society. This study utilized the patient satisfaction survey recommended by the Quality Assurance Committee of the American Pain Society and was conducted in an acute care, municipal hospital. Seventy-two medical-surgical patients were interviewed about their pain management. Data from the survey suggest that while patients experienced moderate-to-severe pain and had to wait relatively long periods of time for pain medications, in most cases they were satisfied with their overall pain management. Recommendations for conducting patient satisfaction surveys of pain management in acute care settings are reviewed, and methods for interpreting data from these types of surveys are discussed.

Acute Disease↗
PubMed1994

Allergic- and immune-associated diseases of the urinary tract.

Glomerular injury has a decided immunologic basis. Any infectious, inflammatory, neoplastic, or degenerative processes capable of sustained antigenic stimulation can induce immune-mediated glomerular injury. A variety of conditions and antigens, both endogenous and exogenous, are known to initiate immunologic glomerular damage. In many clinical situations, however, the precise antigenic source is occult and unrecognizable, and the glomerular disease is referred to as idiopathic.

Animals↗
PubMed1994

Tyrosine kinase activity in Pseudomonas aeruginosa.

Previous evidence showed that b- and a-type flagellins of Pseudomonas aeruginosa are modified in vivo by phosphorylation at tyrosine. This research was designed to demonstrate phosphorylation of flagellin at tyrosine in vitro. Evidence presented showed that flagellin is labelled by [gamma-32P]-ATP, but not by [alpha-32P]-ATP, when incubated with cell envelope fractions. Results suggested that autophosphorylation of a 42 kDa membrane protein occurred. No activity was detected in cytoplasmic fractions. Flagellin protein was identified by flagella-specific monoclonal antibody (mAb) and was labelled with anti-phosphotyrosine mAb. Confirmation of tyrosine kinase activity was shown by labelling of synthetic poly(Glu:Tyr) as a substrate with [gamma-32P]-ATP. Labelling of poly(Glu:Tyr) was heat sensitive and time dependent. Labelled phosphotyrosine was observed in partial acid hydrolysates of substrates. Using poly(Glu:Tyr) as substrate, tyrosine kinase activity was shown to be inhibited by sulphydryl reagents. It appears that tyrosine kinase and flagellin phosphorylation occur in several Pseudomonas spp. Location of phosphotyrosine in a conserved region of flagellin may serve as a cell signal so that intact flagellin is appropriately exported.

Adenosine Triphosphate↗
PubMed1994

Accumulation of glutamate by Salmonella typhimurium in response to osmotic stress.

Salmonella typhimurium accumulates glutamate in response to osmotic stress. Cells in aerobic exponential growth have an intracellular pool of approximately 125 nmol of glutamate mg of protein-1. When cells were grown in minimal medium with 500 mM NaCl, KCl, or sucrose, 290 to 430 nmol of glutamate was found to accumulate. Values were lower when cells were harvested in stationary phase. Cells were grown in conventional medium, harvested, washed, resuspended in the control medium or in medium with osmolytes, and aerated for 1 h. With aeration, glutamate was found to accumulate at levels comparable to those observed in exponential cultures. Antibiotics inhibiting protein synthesis did not affect glutamate accumulation when cells were aerated. Strains with mutations in glutamate synthase (glt) or in glutamate dehydrogenase (gdh) accumulated nearly normal levels of glutamate under these conditions. A double (gdh glt) mutant accumulated much less glutamate (63.9 nmol mg of protein-1), but a 1.9-fold excess accumulated when cells were aerated with osmotic stress. Methionine sulfone, an inhibitor of glutamate synthase, did not prevent accumulation of glutamate in cells aerated with osmotic stress. Glutamate dehydrogenase is thought to have minimum activity when ammonium is limiting. Resuspending cells with limiting ammonium reduced glutamate production but did not eliminate accumulation of excess glutamate when cells were osmotically stressed. Amino oxyacetic acid, an inhibitor of transamination reactions, did not prevent accumulation of excess glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1994

Concurrent illness and complications associated with canine hyperadrenocorticism.

Complications associated with canine HAC include thromboembolism, infection, diabetic mellitus, pancreatitis, or growing pituitary tumor. The serious consequences of the multisystemic effects of cortisol excess show the need for careful examination and monitoring of animal patients with HAC so that the many associated complications may be recognized, treated, and in some cases prevented.

Acute Disease↗
PubMed1993

Spatial and temporal expression identify dromyosuppressin as a brain-gut peptide in Drosophila melanogaster.

The Drosophila dromyosuppressin peptide (TDVDHVFLRFamide) is a member of a family of peptides containing the common C-terminal sequence-RFamide. Dromyosuppressin shares a high degree of sequence homology with leucomyosuppressin isolated from cockroach (pEDVDHVFLRFamide) and identity with neomyosuppressin isolated from fleshfly. By means of sequence-specific antisera, the cellular expression pattern of dromyosuppressin immunoreactive material was determined for all stages of Drosophila development. Dromyosuppressin immunoreactivity first appears in two cells of the medial protocerebrum in embryos. The larval stage is characterized by an increase in the number of dromyosuppressin immunoreactive cells in the brain and the first appearance of cellular expression in the ventral ganglion. Immunoreactive fibers extend from the medial protocerebrum cells into the ventral ganglion. Relative to the larval stage, the pupal and adult stages are marked by an increase in the number of immunoreactive cells in the central nervous system and an increase in the arborization of immunoreactive fibers extending from these cells. Immunoreactivity is present in larvae in two cells near the anus; in the adult gut, expression is observed in two cells in the rectum and immunoreactive fibers in the crop that appear to extend from the central nervous system. In general, the number of cells containing dromyosuppressin immunoreactive material increases throughout Drosophila development. However, expression in three cells is restricted to specific developmental periods. These data identify dromyosuppressin as a brain-gut peptide regulated at both a cellular and developmental level.

Amino Acid Sequence↗
PubMed1993

Identification and cloning of GP-3 from rat pancreatic acinar zymogen granules as a glycosylated membrane-associated lipase.

The protein components of highly purified secretory granule membranes and the granule contents from rat exocrine pancreas were characterized by two-dimensional polyacrylamide gel electrophoresis, protein staining, lectin absorption, and Western blotting with anti-secretory protein antibodies. NH2-terminal amino acid sequence was obtained for a approximately 53-kDa glycoprotein denoted GP-3, present only in granule membrane preparations where it was resistant to washing with Na2CO3 and KBr. The sequence of this protein showed homology to pancreatic lipase but was distinct from the NH2-terminal sequence of a 50-kDa content protein presumed to be secretory lipase. Polymerase chain reaction amplification with degenerate oligonucleotide primers to GP-3 and secretory lipase gave partial length subclones that were used to isolate clones from a rat pancreas cDNA library. Dideoxy sequencing of full-length subclones of GP-3 revealed the predicted amino acid sequence for a mature protein of 452 amino acids with a potential N-linked glycosylation site and a deglycosylated molecular weight of 50,860. The GP-3 sequence possesses the serine esterase consensus sequence G-X-S-X-G centered around Ser154 and the catalytic state triad Asp178-His265-Ser154 characteristic of pancreatic lipases. Northern blot analysis of various rat tissues showed GP-3 expression solely in pancreas. Comparison of GP-3 nucleotide and amino acid sequence, along with pancreatic lipases of various species including rat, shows extensive homologies to both proteins and reveals an underlying diversity in the pancreatic lipase family. Close homology is observed between GP-3 and a lipase molecule previously isolated from mouse cytotoxic T cells.

Amino Acid Sequence↗
PubMed1993

Titration of histidine 62 in R67 dihydrofolate reductase is linked to a tetramer<-->two-dimer equilibrium.

R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded protein that confers clinical resistance to the antibacterial drug trimethoprim. To determine whether an acidic titration in kinetic pH profiles is related to titration of histidines 62, 162, 262, and 362, the stability of tetrameric R67 DHFR has been monitored as a function of pH. For the pH range 5-8, tetrameric R67 DHFR reversibly dissociates into dimers, as monitored by ultracentrifugation and molecular sieving techniques. From the crystal structures of dimeric and tetrameric R67 DHFR [Matthews et al. (1986) Biochemistry 25, 4194-4204] (Narayana, Matthews, and Xuong, personal communication), symmetry-related histidines 62, 162, 262, and 362 occur at the two dimer-dimer interfaces and protonation of these residues could destabilize tetrameric R67 DHFR. Ionization of these histidines was confirmed by monitoring the chemical shifts of the C2 proton in NMR experiments, and best fits of an incomplete titration curve yield a pKa of 6.77. Since tryptophans 38, 138, 238, and 338 also occur at the dimer-dimer interfaces, fluorescence additionally monitors the tetramer-two dimers equilibrium. When fluorescence was monitored over the pH range 5-8, a protein concentration dependence of fluorescence was observed and global fitting of three titration curves yielded Kd = 9.72 nM and pKa = 6.84 for the linked reactions: [formula: see text] Modification of H62, H162, H262, and H362 by diethyl pyrocarbonate stabilizes dimeric R67 DHFR and causes a 200-600-fold decrease in catalytic efficiency. Decreased catalytic activity in dimeric R67 DHFR is presumably due to loss of the putative single active site pore found in tetrameric R67 DHFR.

Chromatography, Gel↗
PubMed1993

Immunocytochemistry of sequence-related neuropeptides in Drosophila.

Based on structure, activity, and expression, the Drosophila drosulfakinin I peptide (DSK I; FDDY(OSO3H)GHMRFamide) is similar to the vertebrate peptide, cholecystokinin. Dromyosuppressin (DMS; TDVDHVFLRFamide) is an abundant peptide isolated from adult Drosophila which shares a high degree of sequence homology with peptides isolated from chicken, cockroach, fleshfly, and locust. DSK I and DMS, encoded by different precursors, have similar expression patterns in larval brain tissue; each localizes to cells in the anterior and medial protocerebrum. Because of the precedence for coexistence of neural messengers, it was of interest to determine the cellular expression patterns relative to one another. The question of whether the two peptides were expressed in the same cells was resolved using an immunofluorescent double-labeling technique developed for sequence-specific antisera raised in separate animals of the same species. Double labeling was done using a combination of indirect and direct immunofluorescence. DSK I and DMS were shown to localize to different cells in close proximity to one another in the larval brain. The non-overlapping expression patterns of these peptides illustrate the complete lack of cross-staining with this technique.

Amino Acid Sequence↗
PubMed1993

Blood pressure and life style in the People's Republic of China: three samples in the INTERSALT Study.

In INTERSALT, the International Cooperative Study on Electrolytes and Blood Pressure, three centres located in the People's Republic of China (PRC) had distinctive patterns of BP and of life style variables that, in INTERSALT overall, were found to relate to BP. The PRC centres had low body mass index (BMI) and alcohol consumption but high urinary sodium and sodium/potassium ratio (Na/K). Compared with 45 other INTERSALT centres, average BMI was 22.7 vs. 25.4 and alcohol consumption was 32% vs. 61%, with heavy drinking 3% vs. 14%. However, Na/K in the PRC was 6.7 vs. 3.2 in the other centres. This combination of factors may underlie the BP pattern observed. While mean BP in the PRC was lower than in the 45 centres (-7.0 mmHg SBP, -5.6 mmHg for DBP), this was counterbalanced by other findings. Upward slope of systolic pressure with age was 45% greater for the PRC than the other centres and 24% greater for diastolic pressure. As a result, although percentage hypertensive for ages 20-49 years for the PRC was half that in the 45 centres (6% vs. 12%), by age 50-59 years the prevalence was 31% in the PRC and 38% in the other centres. Hypertension prevalence in Tianjin, with highest Na/K in INTERSALT (7.6) was 40% for ages 50-59 years. While sample size in individual centres does not provide power to demonstrate aetiological relationships, these findings permit formulation of the following hypothesis: although lower body mass and lower alcohol intake may counteract to some degree impact of high salt intake, such intake over decades eventually takes its toll on BP.

Adult↗