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D R Nelson

Publications and source records attributed to D R Nelson.

At least 109 records · Page 6Linked to original sources
PubMed1996

Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations of oxidative phosphorylation and AAC expression.

ADP/ATP transport is the terminal step of oxidative phosphorylation in mitochondria. In this paper seven mutants of AAC2 from Saccharomyces cerevisiae are studied on the cellular and mitochondrial level. Six conspicuously located arginines were mutated into mostly neutral residues [Nelson, D. R., Lawson, J. E., Klingenberg, M., & Douglas, M. G. (1993) J. Mol. Biol. 230, 1159-1170]. R96A, R96H, R204L, and R294A are located in the second transmembrane helix of each repeat while R252I, R253I, and R254I are in the arginine triplet of the last domain. All six arginine residues are conserved in all known ADP/ATP carrier sequences. At the cellular level, oxidative phosphorylation in R96H and R294A retains 8% of the wild-type rate, but it is virtually zero in the other mutants. However, cytochrome c, a parameter of oxidative capacity, remains at 4-42% of wt. The weak coordination of respiratory chain and AAC expression indicates that respiration is needed also for other purposes. In mitochondria the AAC-linked ATP synthesis is measured and segregated by using the AAC inhibitor bongkrekate (BKA). Only the R96H and R294A mutants express a significant rate of AAC-dependent ATP synthesis amounting to 2-18% of the plasmid-borne wild-type AAC2 mitochondria. In all other mutants it is virtually zero. However, respiratory capacity and cytochrome c content are reduced only by 20-70%. Whereas in immunoblots the presence of AAC is detected in all mutant mitochondria, by quantitative ELISA no AAC can be measured down to 0.05 mumol of AAC dimer/g of protein in R96A and R204L, whereas in R96H, R2521, R2531, and R254I the content is around 0.2 and in R294A the content is 0.46 as compared to 0.6 in the plasmid wild type. Also the [3H]CAT and [3H]BKA binding is virtually zero in some mutants and closely parallels the ELISA-determined AAC content, indicating that the mutations did not affect the inhibitor binding site. The turnover of AAC [V(ATP)/AAC content] in oxidative phosphorylation is reduced to 10% or 20% except for the two intrahelical mutants R96H and R294A. In the three Arg triplet mutants, it is nearly zero. In conclusion, the first two intrahelical arginines R96 and R204, are essential for expression but probably also for the activity of AAC. R294A still retains good transport activity and remarkably high expression of AAC. All arginines in the triplet 252, 253, 254 are essential. Extrapolation of the in vitro phosphorylation rates to the cellular level by the cytochrome c factor reveals a large discrepancy to the in vivo rates in particular for R294A. This indicates that these mutations render the AAC more sensitive to the regulatory intracellular ATP/ADP ratio than the wt AAC.

Amino Acid Sequence↗
PubMed1996

Probing the role of positive residues in the ADP/ATP carrier from yeast. The effect of six arginine mutations on transport and the four ATP versus ADP exchange modes.

Mutagenesis of three intrahelical arginines, R96, R204, or R294, and of each member of the arginine triplet R252, R253, R254 into neutral residues had resulted in a strong suppression of oxidative phosphorylation in cells and isolated mitochondria [Müller, V., Basset, G., Nelson, D. R., & Klingenberg, M. (1996) Biochemistry 35, 16132-16143]. Here we determine the transport activity of wild-type and mutant AAC in reconstituted proteoliposomes using a new rapid removal-stop method without relying on the inhibitor stop which can be compromised by mutations. The basic electroneutral ADP/ADP exchange activity is strongly or totally suppressed in six out of seven of these mutations, with the exception of R294A, which retains nearly wild-type activity. Carboxyatractylate (CAT) inhibits the ADP/ATP exchange rate only to 3-10% in wild type and R294A and up to 40% in other mutants, whereas bongkrekic acid (BKA) inhibits 50% (wild type and R294A) and 90% (other mutants). Consequently, AAC is preferentially reconstituted with the matrix surface outside. All these mutations drastically change activity distribution among the four exchange modes ADP/ADP, ADP/ATP, ATP/ADP, and ATP/ATP. Whereas in wild-type AAC the homo ATP/ATP exchange is twice as high as the ADP/ADP exchange, in mutants it is 10 to 15 times lower. Similarly, the hetero ATP/ADP exchange in wild-type AAC is higher than the ADP/ ATP exchange, but in mutants it is several times lower. Thus, these mutations afflict the ATP-linked modes, in particular those linked to external ATP. The inhibition of oxidative phosphorylation is thus explained by the suppression of ATP export versus ADP import mode. The "extra"-inhibition of oxidative phosphorylation in mutant cells is explained by the extreme shift in mutants in favour of ATP import versus ADP export. Besides structural changes, the mutant effects indicate electrostatic interactions of these arginines with the anionic substrates. The loss of one positive charge raises the translocation barrier the more negative the substrate, i.e. more for ATP4- than for ADP3-. In none of these arginine mutants was the binding of CAT or BKA abolished.

Arginine↗
PubMed1996

Genomic cloning and protein expression of a novel rat brain cytochrome P-450 CYP2D18* catalyzing imipramine N-demethylation.

We have previously reported the isolation of two cDNA clones, designated 2d-29 and 2d-35, which have identical open reading frames and code for a novel brain cytochrome P-450 (P-450) belonging to the CYP2D subfamily, and noted that the mRNA of clone 2d-35 seems to be expressed in the brain but not in the liver (1). Although the deduced amino acid sequence of these clones differs from that of the liver CYP2D4 by only 5 amino acids distributed in the C-terminal region, this new P-450 cDNA clone contained a unique 5'-extension, and we posit in this report by analysis of a genomic clone that this 5'-untranslated sequence is derived from a gene distinct from that of CYP2D4. Thus, this novel P-450 was named P-450 2D18 according to the recommended nomenclature (2). The expressibility of this cDNA was confirmed by in vitro translation using a reticulocyte system, and protein expression was performed using COS-M6 cells. Immunoblot analysis showed a cross-reacting band of the predicted size range with anti-P-450 2D6 antiserum, which was not seen in control cells. Furthermore, the CYP2D18-expressed COS cell lysate showed N-demethylation activity toward imipramine, whereas another brain P-450 CYP4F6-expressed COS cell lysate showed 10-hydroxylation activity. This is the first report that associates an individual P-450 isozyme in brain with a particular metabolic alteration of the antidepressant imipramine.

Animals↗
PubMed1996

Deletion of the nuclear gene encoding the mitochondrial citrate transport protein from Saccharomyces cerevisiae.

The nuclear gene encoding the mitochondrial citrate transport protein (i.e., CTP1) has been deleted from a haploid yeast strain. The stable yeast deletion strain was constructed by homologous recombination of the HIS3 gene at the CTP1 gene locus. Deletion of the CTP was confirmed by PCR. Immunoblot analysis provided the first quantitative estimate of the level of the CTP in wild-type yeast mitochondria and indicated the absence of expressed CTP in mitochondria isolated from the deletion strain. Deletion of CTP1 did not lead to a phenotype on any carbon source tested, indicating that CTP1 is not an essential gene. This suggests that either known alternative pathways are able to produce sufficient acetyl-CoA to support biosynthetic reactions, or there exists a second CTP gene. The ability of the deletion strain to serve as a host for the correct targeting and overexpression of a mutated CTP was then demonstrated. These studies provide a system which permits the use of site-directed mutagenesis to examine both CTP targeting to mitochondria, as well as the molecular basis underlying CTP function. Moreover, this system will not only facilitate the study of the yeast CTP, but also CTPs expressed from the cDNAs of higher eukaryotes.

Carrier Proteins↗
PubMed1996

The yeast ADP/ATP carrier. Mutagenesis and second-site revertants.

Results of mutagenesis and selection of spontaneous second-site revertants of the yeast ADP/ATP carrier AAC2 is described. Currently, 50 mutants have been made in AAC2 at 35 locations. Yeast carrying mutations at K38, K48, R96, D149, R152, R204, D249, R252, R253, R254 and R294 are all unable to grow on glycerol. Seven of these mutants have yielded second-site revertants when plated on rich yeast media containing glycerol and ethanol. The R96 mutants and the R254 and R253 mutants produce similar changes in the AAC2 molecule because the same sites are affected by their revertant mutations. This system of mutations and revertants is now poised to yield insights into the dynamics of ADP and ATP transport, and mitochondrial carrier structure in general.

Amino Acid Sequence↗
PubMed1996

Comparison of intravenous ketorolac, meperidine, and both (balanced analgesia) for renal colic.

STUDY OBJECTIVE: To compare the analgesic efficacy and safety of IV ketorolac, the only nonsteroidal antiinflammatory drug indicated for parenteral use in acute pain in the United States, with IV meperidine and with a combination of the two agents in renal colic. METHODS: We carried out a double-blind, randomized, multicenter clinical trial in the emergency departments of four urban tertiary care teaching hospitals. Our study subjects were 154 patients with suspected renal colic. Each subject received an initial IV dose of ketorolac 60 mg, meperidine 50 mg, or both supplemented as needed beyond 30 minutes with additional doses of meperidine. RESULTS: The main outcome measures were changes in pain-intensity and pain-relief scores, amount of supplemental meperidine required, end-of-study drug tolerability, and adverse events. Analyses of 106 subjects with confirmed renal colic indicated that ketorolac and the combination were significantly better than meperidine alone by all efficacy measures, including pain relief and time elapsed before the need for supplemental meperidine. By 30 minutes, 75% of the ketorolac group and 74% of the combination group had a 50% reduction in pain scores, compared with 23% of the meperidine group (P < .001). The ketorolac and combination groups did not differ significantly in any of the efficacy measures. CONCLUSION: IV ketorolac, alone or in combination with meperidine, was superior to IV meperidine alone in moderate and severe renal colic. Because many subjects in all three treatment groups received supplemental meperidine and because response to ketorolac alone cannot be predicted, clinicians may choose to initiate treatment with a ketorolac-meperidine combination.

Adult↗
PubMed1996

Modeling indoor air concentrations near emission sources in imperfectly mixed rooms.

Assessments of exposure to indoor air pollutants usually employ spatially well-mixed models which assume homogeneous concentrations throughout a building or room. However, practical experience and experimental data indicate that concentrations are not uniform in rooms containing point sources of emissions; concentrations tend to be greater in close proximity to the source than they are further from it. This phenomenon could account for the observation that "personal air" monitors frequently yield higher concentrations than nearby microenvironmental monitors (i.e., the so-called "personal cloud" effect). In this project, we systematically studied the concentrations of a tracer gas at various distances from its emission source in a controlled-environment, room-size chamber under a variety of ventilation conditions. Measured concentrations in the proximity of the source deviated significantly above the predictions of a conventional well-mixed single-compartment mass balance model. The deviation was found to be a function of distance from the source and total room air flow rate. At typical air flow rates, the average concentration at arm's length (approximately 0.4 meters) from the source exceeds the theoretical well-mixed concentration by a ratio of about 2:1. However, this ratio is not constant; the monitored concentration appears to vary randomly from near the theoretical value to several times above it. Concentration data were fitted to a two-compartment model with the source located in a small virtual compartment within the room compartment. These two compartments were linked with a stochastic air transfer rate parameter. The resulting model provides a more realistic simulation of exposure concentrations than does the well-mixed model for assessing exposure to emissions from active sources. Parameter values are presented for using the enhanced model in a variety of typical situations.

Air↗
PubMed1996

P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.

We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes. This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), representing "cytochrome P450', be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen is no longer recommended in mouse gene nomenclature. "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates.

Alleles↗
PubMed1996

Effects of dietary fat with or without nicotinic acid on nutrient flow to the duodenum of dairy cows.

Four Holstein cows, fitted with ruminal and duodenal cannulas, were utilized in a 4 x 4 Latin square design to investigate the effects of supplementing nicotinic acid to diets that contained 35% alfalfa haylage, 15% corn silage, and either 50% of a low fat concentrate or 10% whole raw soybeans and 40% of a high fat concentrate containing tallow. Treatments in a 2 x 2 arrangement were 1) low fat, no supplemental nicotinic acid; 2) low fat, 12 g/d of nicotinic acid; 3) high fat, no supplemental nicotinic acid; and 4) high fat, 12 g/d of nicotinic acid. The DMI and OM apparently and truly digested in the rumen and apparently digested postruminally were not different among treatments. Addition of fat to the diet decreased the concentration of total VFA in ruminal fluid but did not alter the molar proportions of any of the VFA; supplementation of nicotinic acid tended to decrease the molar proportion of acetate. Amounts of NAN, microbial N, nonammonia nonmicrobial N, and AA that flowed to the duodenum were similar among diets. The concentration of urea N in plasma decreased, and concentrations of cholesterol and triglycerides increased, when cows were fed supplemental fat. Milk composition and production of milk, 4% FCM, and milk components were not altered by addition of fat or nicotinic acid to the diet. Supplementation of fat or nicotinic acid to diets of dairy cows was not beneficial in this experiment.

Animal Nutritional Physiological Phenomena↗
PubMed1996

Pharmacokinetics of penicillin G in plasma and interstitial fluid collected with dialysis fiber bundles in sheep.

A new method of collection of interstitial fluid (ISF) (the site of most bacterial infections) was developed for the determination of free (unbound) penicillin G concentrations in sheep. Dialysis fiber bundles for the collection of ISF were first characterized in vitro and subsequently implanted in the subcutaneous fascia of the dorsal thorax parallel to the vertebral column in sheep. The sheep were then dosed intravenously with 26.4 and 52.9 mg/kg of sodium penicillin G using a crossover experimental design. Plasma and ISF dialysate were collected after dosing for determination of penicillin G concentrations using high pressure liquid chromatography (HPLC). The concentration of penicillin G in the ISF dialysate was calculated with the recovery ratio determined for each fiber bundle. The decline of penicillin G concentrations in ISF dialysate paralleled the disappearance of the drug from plasma providing evidence for the rapid diffusion of penicillin G into the fiber bundles. Pharmacokinetic analysis determined that the disposition of penicillin G was best described by a two-compartment open model with penicillin concentrations in plasma (Cp) defined by two biexponential equations, Cp = 170.64e-7.16t + 31.04e-1.56t for the low dose and Cp = 418.19e-1.56t for the high dose.

Animals↗
PubMed1995

Vascular tissue plasminogen activator and the development of coronary artery disease in heart-transplant recipients.

BACKGROUND: An aggressive and potentially fatal form of coronary artery disease may develop after cardiac transplantation. We studied the role of vascular tissue plasminogen activator (t-PA), the primary mediator of fibrinolysis, in the development of this problem. METHODS: We studied 78 consecutive recipients of cardiac allografts over a five-year period, and we collected follow-up data over a mean (+/- SE) of 32.5 +/- 2.0 months. The patients were studied with ventricular function tests, serial endomyocardial biopsies (16.6 +/- 0.5 per patient), and annual coronary angiography. Measurements of t-PA and its inhibitor were performed immunocytochemically on unfixed cryostat sections of endomyocardial-biopsy specimens with the use of monoclonal antibodies to t-PA and its inhibitor. RESULTS: In biopsy specimens obtained during the first three months of follow-up, 38 allografts had a normal distribution of t-PA in arteriolar smooth-muscle cells, whereas in 40 allografts there was depletion of t-PA that persisted in subsequent follow-up. Coronary artery disease developed during follow-up in 31 of 40 allografts (78 percent) with depletion of t-PA, but the disease developed in only 9 of the 38 allografts (24 percent) with normal t-PA levels (P < 0.001). Allografts with depletion of t-PA also had the t-PA inhibitor and were at greater risk for earlier and more severe disease than were allografts with normal arteriolar t-PA levels. Twelve patients whose allografts were depleted of t-PA either received a second transplant or died, whereas only one of the patients whose allografts had persistently normal t-PA levels died (P < 0.001). CONCLUSIONS: These findings reveal an association between the depletion of t-PA from arteriolar smooth-muscle cells and the subsequent development of coronary artery disease and decreased graft survival. Although we cannot be certain about a cause-and-effect relation, our data suggest a possible role for deficient fibrinolysis in the development of coronary artery disease in transplanted human hearts.

Coronary Angiography↗
PubMed1995

Pain and tissue-interface pressures during spine-board immobilization.

STUDY OBJECTIVES: Although spine boards are one of the main EMS means of immobilization and transportation, few studies have addressed the discomfort and potential harmful consequences of using this common EMS tool. We compared the levels of pain and tissue-interface (contact) pressures in volunteers immobilized on spine boards with and without interposed air mattresses. DESIGN: Prospective crossover study. SETTING: Emergency department of Methodist Hospital of Indiana, Indianapolis, Indiana. PARTICIPANTS: Twenty healthy volunteers who had not taken any analgesic drugs in the preceding 24 hours, were not experiencing any pain at the time of the study, and did not have history of chronic back pain. INTERVENTIONS: To simulate prehospital transport conditions, we immobilized volunteers with hard cervical collars and single-buckle chest straps on wooden spine boards with or without commercially available medical air mattresses. The crossover order was randomized. After 80 minutes, immobilization measures were discontinued and the subjects were allowed to get off the boards for a recovery period of 60 minutes. Subjects were then studied for a second 80-minute period with the opposite intervention. At baseline and at 20-minute intervals, the level of pain was rated with a 100-mm visual analog scale. Tissue-interface pressures were measured at the occiput, sacrum, and left heel. RESULTS: Mean pain on the visual analog scale was 9.7 mm at the end of the mattress period and 37.5 mm at the end of the no-mattress period (P = .0001). Although there were no significant differences in pain between the two groups at time 0, volunteers reported significantly more pain during the no-mattress period at 20 (P = .003), 40 (P = .0001), and 60 minutes (P = .0001). All 20 subjects reported that immobilization on the spine board with the mattress was "much better" (five-point scale) than that without the mattress. Interface pressure levels were significantly less in the mattress period than in the no-mattress period measured at occiput (P = .0001), sacrum (P = .0001), and heel (P = .0001). CONCLUSION: In a simulated immobilization experiment, healthy volunteers reported significantly less pain during immobilization on a spine board with an interposed air mattress than during that on a spine board without a mattress. Tissue-interface pressures were significantly higher on spine boards without air mattresses. This and previous studies suggest that immobilization on rigid spine boards is painful and may produce tissue-interface pressure high enough to result in the development of pressure necrosis ("bedsores"). Emergency care providers should consider the use of interposed air mattresses to reduce the pain and potential tissue injury associated with immobilization on rigid spine boards.

Adolescent↗
PubMed1995

alpha-Glutathione S-transferase as a marker of hepatocellular damage in chronic hepatitis C virus infection.

alpha-Glutathione S-transferase (alpha GST) may be a good serologic marker of hepatocellular damage because of its low molecular weight, uniform hepatic distribution, high cytosolic concentration, and short half-life. To determine the clinical utility of alpha GST in patients with chronic hepatitis C virus (HCV) infection, serum alpha GST levels were measured in 96 patients with chronic HCV infection, of whom 47 subsequently underwent interferon-alpha therapy. Patients were simultaneously evaluated with conventional liver biochemistry, serum HCV RNA levels, and liver histology. Different methods of serum collection did not affect alpha GST values, indicating that this was a stable serum marker. In 93% of patients with chronic HCV infection, alpha GST was elevated and showed an excellent correlation with serum aminotransferases. Histologic analysis revealed a correlation of alpha GST with both lobular inflammation and bile duct lesions. There was no correlation between serum alpha GST levels and the demographic features, mode of transmission, virologic, other histologic parameters, or subsequent response to interferon-alpha. During serial monitoring in patients undergoing interferon-alpha therapy, elevation of serum alpha GST correlated with biochemical relapse and in some patients virologic relapse in the presence of normal liver biochemistry. alpha GST was persistently elevated in all nonresponders. Four of six of those patients who responded completely followed by early relapse had elevated alpha GST intermittently or continuously during therapy despite normalization of serum aminotransferases. Two of five of those with a complete and sustained response had elevated alpha GST during treatment and follow-up, and both were also seropositive for HCV RNA during follow-up. These data demonstrate that alpha GST is a stable marker, has similar diagnostic utility as serum aminotransferases, and may have a role in the monitoring of patients undergoing interferon-alpha therapy.

Adult↗
PubMed1995

Variations of hepatitis C virus NS5B sequence (nucleotides 8261-8566) do not correlate with response to interferon-alpha therapy.

Hepatitis C virus (HCV) is an RNA virus with the NS5B gene encoding an RNA-dependent RNA polymerase. Interferon-alpha (IFN-alpha) is effective against HCV and its effect is believed to be related to its antiviral activity. To determine whether sequence variations of the HCV NS5B region correlate with response to IFN therapy, pretreatment serum samples from 40 patients with chronic HCV infection who were subsequently treated with IFN (> or = 3 MU thrice weekly for 24 weeks) and had well-characterized biochemical responses were studied. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to generate an approximately 365-bp fragment from which nucleotide sequence and genotypes were determined. By comparing the nucleotide sequences and the encoded amino acid sequences of samples from each group, no response group-specific nucleotide or encoded amino acid substitution was identified. Most of the substitutions identified were synonymous (usually by changes at the third position of the codon). These data suggest that these substitutions were selective rather than spontaneous events. Of the few non-synonymous substitutions identified, none was correlated with subsequent response to IFN, either within or across genotypes.

Adult↗
PubMed1995

Electromyography of the reticulum, abomasum and duodenum in dairy cows with left displacement of the abomasum.

Electromyographic (EMG) recordings of the reticulum, abomasal corpus, pyloric antrum and duodenum of six dairy cows with left displacement of the abomasum (LDA) were made in order to substantiate abomasal atony as a prerequisite to abomasal displacement. EMG recordings were made when LDA was present as well as when absent. Mean values were determined in five of six cows for the maximum peak or amplitude, mean peak values, peak-to-peak interval and count of the electrical response activity (ERA) for each 15 min segment of the waveform recordings. Segments containing phase III migrating myoelectric activity were not analysed. LDA positive periods were compared to LDA negative periods in each cow. The 6 h period (transition period) prior to the diagnosis of LDA was analysed separately. Paired t-tests were applied to group values with statistical significance established at the P = 0.05 level. There was a significant decrease in the ERA count during the LDA positive periods in the abomasal corpus (-1.40% to -7.88%, P = 0.0217) and in the pyloric antrum (-2.05% to -11.98%, P = 0.0430). A corresponding significant increase occurred in the peak-to-peak interval. During the transition period spike activity in the duodenum increased 0.5% to 48.31% (P = 0.0474) and the peak-to-peak interval was significantly decreased. No extended periods of atony were observed in the abomasum during this study.

Abomasum↗