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D M Lawrence

Publications and source records attributed to D M Lawrence.

At least 37 records · Page 2Linked to original sources
PubMed1995

Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8.

Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3. We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2. Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J. A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5779-5783). By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly. These results suggested the presence of two polypeptides in this protein. Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB). The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes. The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences. The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A. These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases. The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus. Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases. The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues.

Amino Acid Sequence↗
PubMed1995

Autocatalytic processing of the 223-kDa protein of blueberry scorch carlavirus by a papain-like proteinase.

The first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine994 and histidine1075. A second possible proteinase domain, which contained cysteine895 and histidine984 residues with similar spacing but was otherwise less similar to the viral papain-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative helicase and the polymerase domains, possibly between or close to glycine1472 and alanine1473. Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine994, histidine1075, or glycine1472 abolished autoproteolysis in vitro, supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine895 and histidine984 residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine994 or histidine1075 were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine1472 signficantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine895 or histidine984 also were infectious, but resulted in delayed symptom expression in plants.

Amino Acid Sequence↗
PubMed1995

Identification of kappa opioid receptors in the immune system by indirect immunofluorescence.

A method to visualize the kappa opioid receptor is described that uses a high-affinity fluorescein-conjugated opioid ligand and indirect immunofluorescence with the phycoerythrin fluorophore to amplify the signal. The mouse thymoma cell line R1E/TL8x.1.G1.OUAr.1 (R1EGO), which expresses the kappa 1 but not mu or delta opioid receptors, was used as a positive control for fluorescence labeling. A fluorescein isothiocyanate-conjugated arylacetamide (FITC-AA) compound displaying high affinity for the kappa opioid receptor was synthesized. R1EGO cells were incubated with FITC-AA, in the absence or presence of the kappa-selective opioid antagonist nor-binaltorphimine (nor-BNI) as a competitor. By using fluorescence microscopy and flow cytometry, incubation of R1EGO cells with FITC-AA alone was not sufficient for the detection of specific staining of the kappa opioid receptor. To amplify the FITC-AA fluorescence, the fluorescein served as a hapten for subsequent antibody detection. R1EGO cells were incubated with FITC-AA, followed by biotinylated rabbit anti-fluorescein IgG and extravidin-conjugated R-phycoerythrin. By using this approach, R1EGO cells were stained with phycoerythrin-amplified FITC-AA, and the staining was displaced with nor-BNI. Flow cytometry showed that titrations of both FITC-AA and nor-BNI produced saturable concentration-dependent changes in the median phycoerythrin fluorescence intensity, with optimal staining at 30 microM FITC-AA. Up to 80% of the fluorescence above background was inhibited by nor-BNI. Freshly isolated thymocytes from C57BL/6ByJ mice also showed nor-BNI-sensitive staining with the FITC-AA amplification. This sensitive method of indirect phycoerythrin immunofluorescence can be used to amplify any fluorescein-conjugated opioid ligand for the detection of opioid receptors.

Acetamides↗
PubMed1995

Kappa opioid receptors expressed on three related thymoma cell lines. Differences in receptor-effector coupling.

The mouse thymoma R1.1 cell line was shown previously to express a single high-affinity kappa 1 opioid receptor that is negatively coupled through a pertussis toxin-sensitive G-protein to adenylyl cyclase. This study compared opioid receptor binding and inhibition of adenylyl cyclase activity in three unique derivatives of the R1.1 cell line. Membranes from the R1.G1 and R1E/TL8x.1.G1.OUAr.1 (R1EGO) cell lines bound both [3H]U69,593 and [3H](-)-bremazocine with similar affinities compared with R1.1 membranes, whereas membranes from the R1E/TL8x.1 (R1E) cell line did not possess any opioid binding sites, detected by radioreceptor binding. The Bmax values for [3H]U69,593 and [3H]-(-)-bremazocine binding to R1.G1 and R1EGO cell membranes were, respectively, 3- and 6-fold greater than those obtained with the parent R1.1 cell line. GTP and its nonhydrolyzable analog, Gpp(NH)p, inhibited [3H]U69,593 binding to all three cell lines. Stimulation of low-Km GTPase activity by the kappa-selective agonist (-)U50,488 was greatest in R1.G1 membranes, followed by R1EGO and R1.1. The maximal inhibition of forskolin-stimulated adenylyl cyclase activity by (-)U50,488 was 66 +/- 2% in R1.G1 and 49 +/- 2% in R1EGO, compared with 37 +/- 1% in R1.1 membranes. Whereas maximal inhibition of adenylyl cyclase activity did not correlate with receptor number among cell lines, the inhibition of cyclic AMP production did correlate with stimulation of low-Km GTPase activity. The R1.1 cell line and its derivatives, R1.G1 and R1EGO, express a similar type of kappa opioid receptor, which exhibits differences in coupling to G-proteins and to adenylyl cyclase among cell lines. These cell lines provide an excellent model system for studying the regulation of opioid receptor-adenylyl cyclase coupling efficiency.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1995

Health care diaries of young women.

The purpose of this study was to describe young women's self-care activities related to health maintenance and symptom management. Seventy-six young adult women kept a daily health diary for 3 weeks. They reported a total of 1,213 health maintenance practices. The self-care interventions used to manage symptoms were varied and specific to the reported symptoms. The most frequently reported illness management activity was the use of over-the-counter medications. The self-care measures selected by these young women reflect deliberate approaches to their situations. The findings suggest that nurses in the community should use young adult health promoting and health maintaining behaviors as a basis for designing interventions.

Adult↗
PubMed1994

Nucleotide sequence of the carlavirus associated with blueberry scorch and similar diseases.

We have synthesized and mapped a library of cDNA clones representing the RNA genome of a strain of blueberry scorch carlavirus (BBScV) associated with a disease known locally, in New Jersey, U.S.A., as Sheep Pen Hill disease. The nucleotide sequence of that strain was determined to be 8514 residues, excluding the poly(A) tail. In addition, cDNA clones representing the 3' terminus of another strain of the virus from the same field were synthesized, mapped and sequenced. The overall identity between sequences of these two strains was approximately 90% spanning the 1634 residue overlap, confirming their identity as distinct strains and not simply different isolates of a single strain. Finally, the coat protein gene of a distinct strain of the virus, isolated from plants with blueberry scorch disease in the Puyallup Valley in Washington State, U.S.A., was cloned from total cDNA by PCR. Sequence analysis revealed that the strain from Washington was more divergent from the two New Jersey strains than they were from each other. Comparisons of these sequences with other carlavirus sequences indicated that BBScV is more closely related to lily symptomless virus and potato virus S than to potato virus M, Helenium virus S, carnation latent virus or poplar mosaic virus. BBScV and potato virus M shared approximately 54% nucleotide sequence identity overall.

Amino Acid Sequence↗
PubMed1994

Synthesis of infectious transcripts of blueberry scorch carlavirus in vitro.

Blueberry scorch carlavirus (BBScV) is a filamentous virus with a polyadenylated, positive-sense RNA genome. A near full-length cDNA clone of BBScV was constructed by assembly of clones from a cDNA library. To generate a full-length cDNA clone, the 5' terminus was mutagenized by PCR to introduce nucleotides present in the wild-type virus and not in the near full-length clone, and then fused directly to the T7 bacteriophage RNA polymerase promoter at the 5' terminus. Capped and uncapped BBScV transcripts were synthesized in vitro from the full-length cDNA clone. Capped transcripts were infectious, producing systemic symptoms identical to those caused by the wild-type virus following inoculation onto Chenopodium quinoa leaves. Uncapped transcripts were substantially less infectious than capped transcripts. This represents the first report of infectious transcripts for a member of the carlavirus group.

Base Sequence↗
PubMed1993

Young adult women: lifestyle and health locus of control.

A study of 76 young adult women, 38 nursing students and 38 non-nursing students, examined their lifestyle practices and health locus of control (HLOC). Findings revealed a significant difference between reported lifestyle practices and the career choice of these young adult women. The lifestyle practice areas in which the most notable differences occurred included: use of seat belts, frequency of alcohol use, frequency of junk food intake, use of illegal drugs and hours of sleep per night. While differences in HLOC were evident between nursing and non-nursing students, no relationship was found between a young woman's HLOC and her lifestyle practices. The differences in HLOC showed that nurses were more frequently pure internal whereas most non-nurses were found to be double externals. The pure chance category had the fewest number of respondents. The difference in lifestyle practices between these young adult women can be explained in part by curriculum variations, as can the difference in HLOC patterns.

Adult↗
PubMed1993

The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line is coupled to adenylyl cyclase through a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein.

The R1.1 mouse thymoma cell line expresses a high-affinity kappa opioid binding site. Opioid binding to this site is inhibited by guanine nucleotides, suggesting that the receptor is coupled to a guanine nucleotide-binding protein. Here, we present evidence that the kappa opioid binding site on R1.1 cell membranes is negatively coupled to adenylyl cyclase. The kappa-selective agonists (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl]benzeneacetamide methane-sulfonate hydrate [(-)-U50,488], (5 alpha,7 alpha, 8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxas- piro(4,5)dec-8-yl)benzeneacetamide (U69,593) and several dynorphin peptides inhibited basal and forskolin-stimulated cyclic AMP production by up to 40% in R1.1 cell membranes. The order of potency for the inhibition of adenylyl cyclase activity by opioid agonists correlated with their Ki values for the inhibition of [3H]U69,593 binding. Opioid-mediated inhibition of adenylyl cyclase activity was stereoselective, as (-)-U50,488 was more potent than the (+) isomer, and the inhibition was blocked by the kappa-selective antagonist nor-binaltorphimine. The opioid-mediated inhibition of adenylyl cyclase activity was also completely blocked by incubating R1.1 cells with Bordetella pertussis toxin (PTX). Incubation of R1.1 cell membranes with PTX and [adenylate-32P]NAD+ resulted in the exclusive labeling of a 41-kDa protein, as determined by separating the membrane proteins under reducing conditions on a SDS polyacrylamide gel, followed by autoradiography. These results suggest that a PTX-sensitive inhibitory guanine nucleotide-binding protein mediates the link between the thymoma kappa opioid receptor and adenylyl cyclase.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1993

Gastrointestinal trauma.

The incidence of gastrointestinal trauma is low in comparison with solid organ injury to the abdomen. The most commonly injured organs are the small bowel and colon. Knowledge of the mechanism of injury alerts the nurse to areas of potential injury and guides the clinical examination. Because of the delayed presentation of these injuries, the nurse must have a high degree of suspicion for the patient who presents with the following clinical findings: bruising of the abdomen, abdominal tenderness or guarding, leukocytosis and elevated amylase and lipase, absent or decreased bowel sounds, and abdominal distention. Morbidity and mortality are directly related to the failure to treat the injuries early and the number of associated injuries. Monitoring of the hemodynamic, respiratory, and metabolic status, along with fluid and electrolyte balance, are key in the management of patients. Surveillance for signs of infection is mandatory for preventing sepsis in these types of injuries. Maintenance of skin integrity is a major concern and requires vigilant nursing care and, in some instances, innovative ways to manage the drainage from wounds and drains. Continuous monitoring and surveillance of the patient with trauma to the gastrointestinal tract will alert the nurse to the injury and prevent complications. These include hemorrhage, abscess, fistula, peritonitis, pancreatitis, esophageal stricture, and wound problems.

Critical Illness↗
PubMed1992

kappa-Opioid binding sites on a murine lymphoma cell line.

As a first step in determining whether any subset of lymphocytes expresses opioid receptors, membranes prepared from mouse lymphoma cell lines were screened for [3H]naloxone binding sites. Membranes from the R1.1 cell line specifically bound [3H]naloxone. The Hill coefficient for [3H]naloxone binding was 0.93 +/- 0.18, and nonlinear regression analysis indicated that a one-site model was the best fit of the [3H[naloxone saturation binding data. Low concentrations of kappa-selective opioids, but neither mu nor delta opioids, inhibited [3H]naloxone binding. Saturation binding studies with the kappa-selective compound [3H]U69,593 revealed a single binding site with a KD value of 0.204 +/- 0.039 nM and a Bmax value of 31.7 +/- 3.1 fmol/mg of membrane protein. The Hill coefficient for [3H]U69,593 binding was 1.03 +/- 0.11, indicative of a single site. Time courses for the association and dissociation of [3H]U69,593 binding at 25 degrees C exhibited properties consistent with a single class of binding sites. Low concentrations of kappa-selective opioids, including dynorphin peptides, inhibited [3H]U69,593 binding, while high concentrations of mu opioids were needed to inhibit binding, and the delta-selective ligands were ineffective at concentrations up to 10 microM. Stereoselectivity of the binding site was demonstrated by the finding that the Ki value for (-)-pentazocine in inhibiting [3H]U69,593 binding was 25 times less than for the (+)-isomer. Based on its high affinity for U69,593, alpha-neo-endorphin, and dynorphin B, the kappa opioid binding site on R1.1 cell membranes belongs to the kappa 1b subtype. As observed with brain kappa opioid binding sites, sodium inhibited [3H]U69,593 binding to R1.1 cell membranes in a concentration-dependent manner. These data demonstrate that the murine lymphoma cell line R1.1 expresses kappa opioid binding sites that are very similar to brain kappa opioid binding sites.

Animals↗
PubMed1992

Changes in vestibulo-ocular reflex (VOR) anticipate changes in vergence angle in monkey.

The axis of head rotation is usually different from the axis of eye rotation. Geometrical considerations show that the eyes translate with respect to visual targets located near the head. In order to accurately stabilize retinal images against this translation, the vestibulo-ocular reflex (VOR) must be modulated inversely with the target's distance. The neural correlate of viewing distance used to modulate the VOR is not known. Since vergence angle is related inversely to viewing distance, an efference copy of instantaneous vergence angle or proprioceptive afferent information from extraocular muscles could be used to adjust the VOR. To examine this hypothesis, we compared the time-course of changes in the VOR with the time-course of changes in vergence angle. The VOR was induced by briefly rotating monkeys about a vertical axis at different times during the execution of vergence eye movements. We found that the amplitude of the VOR changed systematically during the course of a vergence eye movement. On average, the changes in the amplitude of vestibular induced eye movements anticipated changes in vergence angle by 50 msec, but in some instances, up to 200 msec of anticipation was observed. These data suggest that a central command signal rather than an afferent or efferent copy of vergence eye position was used to modulate the VOR.

Animals↗