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Biomedical subjects

E Davidson

Publications and source records attributed to E Davidson.

At least 19 recordsLinked to original sources
PubMed1992

Mutagenesis of methionine-183 drastically affects the physicochemical properties of cytochrome c1 of the bc1 complex of Rhodobacter capsulatus.

Site-directed mutagenesis was used to investigate which of the highly conserved methionine residues (M183 and M205) provides the sixth axial ligand to the heme Fe in the cyt c1 subunit of the bc1 complex from the bacterium Rhodobacter capsulatus. These residues were changed to leucine (cM183L) and valine (cM205V). Two additional mutants were constructed, 1 in which a stop codon was inserted at M205 (cM205*) and the second in which 127 amino acids were deleted between the signal sequence and the putative C-terminal transmembrane alpha-helix (c delta SfuI). Only cM205V grew photosynthetically, and membranes isolated from this strain catalyzed quinol-dependent reduction of cyt c in amounts similar to that in a wild-type strain. Even though cM183L could not grow photosynthetically, it contained all the appropriate polypeptides and cofactors of the bc1 complex, as shown by SDS-PAGE and optical difference spectroscopy of intact membrane particles. Neither of the two deletion mutants contained a stable complex. Flash absorption spectroscopy using chromatophores showed no cytochrome c rereduction after oxidation by the reaction center in cM183L. The bc1 complex from each strain was isolated and characterized. Oxidation reduction midpoint potential titrations revealed that cyt c1 from cM183L had a dramatically shifted Em value (delta Em = -390 mV) compared with wild type and cM205V. While the optical absorption spectrum of cyt c1 from cM183L suggested that the c-type heme was low-spin, nonetheless it was able to react with the exogenous ligand carbon monoxide. The overall data support that M183, and not M205, is the sixth ligand to the heme Fe of cyt c1 of the bc1 complex.

Amino Acid Sequence↗
PubMed1992

Circulating aggregated platelets, number of platelets per aggregate, and platelet size during acute myocardial infarction.

Circulating aggregated platelets (total, reversibly and irreversibly aggregated), the number of platelets per aggregate and "big" platelets were measured by a modification of the Wu and Hoak method in 42 patients on the first, second and fifth day of acute myocardial infarction (AMI). Among them, 30 had an uncomplicated course and 12 patients had complications that occurred between the 5th and 10th day of hospitalization (7 patients had reinfarction and 5 died). In all patients the measured parameters were elevated compared with those of control subjects. There was a significant increase, especially after the first observation day, in the values of total aggregated platelets (37 +/- 11% vs 26 +/- 12%, p < 0.001), reversibly aggregated platelets (27 +/- 9% vs 17 +/- 8%, p < 0.001) and the average platelets per aggregate (8.6 +/- 0.3 vs. 2.3 +/- 0.4) in patients with versus without complications. In considering the role of platelets in the development of AMI, these findings may add information to the role of platelets in determining the course of AMI.

Blood Platelets↗
PubMed1992

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu, C.-A., Yu, L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Qo) site of the bc1 complex.

Amino Acid Sequence↗
PubMed1992

Rhodobacter capsulatus mutants lacking the Rieske FeS protein form a stable cytochrome bc1 subcomplex with an intact quinone reduction site.

The ubiquinol-cytochrome c oxidoreductase (or bc1 complex) of Rhodobacter capsulatus consists of three subunits: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, encoded by the fbcF, fbcB, and fbcC genes, respectively. In the preceding paper [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry (preceding paper in this issue)], we have observed that the apoproteins for cytochromes b and c1 are fully present in the intracytoplasmic membrane of R. capsulatus mutants containing low amounts of, or no, Rieske apoprotein. Here we present evidence that the redox midpoint potentials of cytochromes b and c1, as well as their ability to bind antimycin and stabilize a semiquinone at the Qi site, are unaffected by the absence of the Rieske subunit. This is the first report describing a mutant containing a stable bc1 subcomplex with an intact Qi site in the chromatophore membranes, and provides further evidence that a functional quinone reduction site can be formed in the absence of a quinol oxidation (Qo) site. Additional mutants carrying fbc deletions expressing the remaining subunits of the cytochrome bc1 complex were constructed to investigate the relationship among these subunits for their stability in vivo. Western blot analysis of these mutants indicated that cytochromes b and c1 protect each other against degradation, suggesting that they form a two-protein subcomplex in the absence of the Rieske protein subunit.

Antimycin A↗
PubMed1992

Cytochrome c2 mutants of Rhodobacter capsulatus.

Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These findings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.

Amino Acid Sequence↗
PubMed1992

Big platelets in hyperlipidemic patients.

Both "big" platelets and hyperlipidemia are associated with increased coronary risk. This study was undertaken to search for a possible effect of various hypolipidemic drugs on big platelets. The percentage of big platelets, assessed microscopically, was measured in 66 patients who had hyperlipidemia of various types. Twenty-seven patients with hypertriglyceridemia were randomly selected to receive either fish oil or placebo in a crossover study. Another group of 39 patients with hypercholesterolemia, among them 13 with heterozygous familial hypercholesterolemia (FH), received lovastatin. The pretreatment level of big platelets was elevated, and similar in all groups: 23.3 +/- 12% versus 22 +/- 9%, in the fish oil versus placebo group, 19.1 +/- 6.3% versus 24 +/- 11% in the FH versus non-FH primary hypercholesterolemia group (reference value, 6.8 +/- 3.5%). After treatment, despite the improvement in lipoprotein profile, the percentage of big platelets did not change. The relationship between lipid reduction and big platelets is thus questionable, and necessitates further study.

Aged↗
PubMed1992

The effect of dementia on acute care in a geriatric medical unit.

Treatment of dementia costs billions of dollars in long-term care and community services every year. Dementia also burdens the acute care system and may contribute to financial problems for hospitals serving large numbers of demented elderly. In a specialized geriatric medical unit devoted to acute care of the frail elderly, Alzheimer's disease and vascular and mixed dementias afflicted 63% of inpatients and were associated with excess consumption of nursing resources, complications of treatment, nosocomial infections, lengthy hospitalizations, and financial losses to the hospital. Due in part to the effects of dementia on mobility, continence, and nutrition, demented patients suffered more frequently from life-threatening infections, sepsis, iatrogenic disease, and prolonged hospital stays. Hospital losses were 75% higher for demented patients than for nondemented patients. Dementia affected the majority of acute care patients in this study. However, it was rarely coded as an admitting diagnosis, even though it may have been the proximate cause of the medical morbidity which led to the acute hospitalization. In addition, despite the significant impact of dementia on the hospital course and costs, it was a factor in hospital reimbursement in less than one third of cases. The results indicate that dementia was not considered to be an acute diagnosis, nor was it recognized as a complex medical illness. The impact of dementia on acute hospitalization, including the mechanisms by which dementia prolongs the hospital stay, requires further investigation.

Activities of Daily Living↗
PubMed1991

Cholesterol does not remove the gel-liquid crystalline phase transition of phosphatidylcholines containing two polyenoic acyl chains.

Homoacid (single-acid) phosphatidylcholines containing two linoleate (18:2), arachidonate (20:4), or docosahexaenoate (22:6) chains were dispersed in water to form multilamellar vesicles. The influence of cholesterol on the gel to liquid phase transitions was studied by differential scanning calorimetry (DSC). The pure dipolyenoic phosphatidylcholines exhibited very broad endothermic transitions (widths of 28 to 38 C degrees at scanning rates of 5 C degrees/min) in the temperature range of about -80 degrees C to -30 degrees C. The mixing of cholesterol into the phospholipids in proportions up to 50 mol% had little effect on the temperatures, widths or enthalpy changes associated with the phase transitions. The data suggest that cholesterol does not interfere with the packing of these lipids in the gel state, possibly because the packing is already irregular or because the solubility of cholesterol in these lipids is low, or both.

Calorimetry, Differential Scanning↗
PubMed1991

Transient ischemic attack-related syncope.

The records of 483 patients admitted to the emergency room because of syncope were reviewed. Thirty seven patients (7.7%) were found to suffer from transient ischemic attack- (TIA) related syncope. This group is the subject of this report. Of these patients, 28 (76%) were men (mean age 71 years). Seven patients reported previous syncopal episodes. Past history revealed a high rate of ischemic heart disease (70%) and hypertension (68%). Concurrent neurologic symptoms, which led to the diagnosis of TIA-related syncope, included mainly vertebrobasilar symptoms: vertigo (in 55% of the patients), ataxia (46%), parasthesia (41%). Two patients most probably were presenting bilateral carotid artery disease. Various diagnostic tests (including electroencephalography, computed tomography, sonography, and cerebral angiography) were used to exclude other causes of syncope. During follow-up (mean 14.5 months) four patients (11%) had an additional episode of TIA and in three of them syncope reappeared. One patient had a complete stroke. We conclude that TIA is a much more frequent explanation for syncope than has been previously argued. These patients tend to be elderly males with high incidence of ischemic heart disease and hypertension. The concurrent neurologic symptoms, leading to the diagnosis, represent mainly vertebrobasilar territory ischemia.

Aged↗
PubMed1991

Comparison of basal and prandial insulin therapy in patients with secondary failure of sulphonylurea therapy.

A group of 35 normal weight patients with secondary failure of sulphonylurea therapy (fasting plasma glucose greater than 8.0 mmol l-1 on maximal dose of sulphonylurea) were randomly assigned to receive either a single injection of a basal insulin supplement (human ultralente insulin, n = 16) or three or four injections of a preprandial insulin supplement (human unmodified insulin, n = 19). Patients performed self-monitoring of capillary blood glucose and adjusted their insulin doses in an effort to achieve fasting and preprandial capillary glucose concentrations of less than 7.0 mmol l-1. Blood glucose control after 16 weeks of insulin therapy was improved to a similar extent by both regimens (HbA1 basal insulin group 12.5 +/- 1.2 (+/- SD) falling to 10.7 +/- 2.2%; preprandial group 12.0 +/- 1.6 falling to 9.5 +/- 1.6%). Preprandial insulin gave better control of daytime blood glucose levels but fasting plasma glucose did not differ between the two regimens (basal group 10.6 +/- 3.6, preprandial group 11.1 +/- 3.6 mmol l-1). Insulin dose was greater in the preprandial group (44.1 +/- 17.9 U day-1) than in the basal group (26.7 +/- 12.5 U day-1 (p less than 0.005), but there was no difference in the frequency or severity of hypoglycaemia between the two treatments. Only the preprandial therapy group showed significant weight gain (2.7 +/- 3.0 kg). While both regimens led to improvement of blood glucose control, these results suggest that neither basal nor preprandial insulin alone can achieve ideal blood glucose control through 24 h in patients with fairly severe failure of control on sulphonylurea therapy.

Blood Glucose↗
PubMed1990

Native valve infective endocarditis in the 1970s versus the 1980s: underlying cardiac lesions and infecting organisms.

A retrospective review of the records of 135 patients with proven or suspected endocarditis, seen between January 1970 and December 1987, is presented. Among the findings: (1) Mitral valve prolapse (MVP) as an underlying lesion was more common in the 1980s group of patients (22%) than in the 1970s group (6%, p less than 0.01); (2) no significant difference was found in the occurrence of pathogens between the 1970s and the 1980s groups of patients; (3) in most patients (17 of 19) with MVP, the organisms isolated were Streptococci viridans; (4) most patients (15 of 17) with MVP had undergone a dental procedure without prior antibiotic treatment in the two months prior to admission. Prophylactic antibiotic treatment is suggested in patients with MVP undergoing dental procedure, especially in a subset of patients with flail or redundant valve leaflet as 16 of our MVP patients (out of 17) had this pathology on echocardiogram.

Adolescent↗
PubMed1990

Vitamin E uptake by human intestinal cells during lipolysis in vitro.

Vitamin E uptake by Caco-2 cells, a human intestinal cell line, was studied by incubating the cells with alpha-tocopherol/triglyceride emulsions with or without bile activated lipase or lipoprotein lipase. During a 1-h incubation, vitamin E was transferred to Caco-2 cells only in the presence of triglyceride hydrolysis by bile activated lipase and not by lipoprotein lipase. Incubation with either lipase resulted in hydrolysis of approximately 20% of the medium [3H]-triolein to free fatty acids and a 3-5-fold increase in cellular radioactivity. In the absence of lipases but the presence of taurocholate, addition of oleic acid in an amount equal to the molar concentration of triglyceride (5.7 mM) to triglyceride emulsions containing either alpha-tocopherol or cholesteryl ester resulted in an increase in cellular [3H]-triglyceride and alpha-tocopherol or cholesteryl ester. We suggest that the absorption of hydrophobic molecules such as vitamin E may occur in the presence of bile and amphipathic lipids via the uptake of micellar neutral lipids by the intestine.

Cell Line↗
PubMed1990

Plasma viscosity, fibrinogen and haematocrit in the course of unstable angina.

Plasma viscosity, fibrinogen and haematocrit were measured in 80 patients with unstable angina on the 1st, 2nd and 5th day of hospitalization. In the group of patients who developed acute myocardial infarction (AMI) during hospitalization (20 patients), plasma viscosity was elevated during the entire observation period (1.69 +/- 0.05 cp, normal range 1.38-1.48 cp), while in patients who did not develop AMI, plasma viscosity was only mildly elevated (1.59 +/- 0.05 cp, P less than 0.001 vs the group who developed AMI) and tended to normalize towards the 5th day of hospitalization. Fibrinogen and haematocrit showed similar variations between the two groups. In a group of 20 patients who received heparin, the studied parameters were similar to the group who did not develop AMI. The importance of haemorheological factors in the clinical course of unstable angina is thus emphasized.

Adult↗
PubMed1990

A method for the determination of circulating aggregated platelets and its application to patients in the course of unstable angina.

A method for the quantitative and qualitative determination of the number of aggregated platelets is described. One milliliter of venous blood was separated equally into two solutions. One solution composed of EDTA (ethylenediaminetetraacetic acid) and formaldehyde (solution F) contained reversibly and irreversibly aggregated platelets, and the second solution, composed of EDTA alone (solution E), contained irreversibly aggregated platelets. By microscopic readings, the percentage of platelets forming aggregates was determined. Reversibly aggregated platelets were estimated by subtracting the percentage of aggregated platelets in solution E from that in solution F. The average amount of platelets per aggregate was calculated by dividing the number of aggregated platelets in solution F by the number of aggregates per 1000 platelets counted. The reference ranges (means +/- SDs) established in 100 healthy persons were 5.8% +/- 2.4% (1% to 9%) for solution F, 3.9% +/- 1.8% (0% to 7%) for solution E, and 2.2 +/- 0.18 (2.0 to 2.5) for the average number of platelets per aggregate. Twenty hospitalized patients without heart disease had values similar to those of 100 normal subjects. In 50 patients with acute myocardial infarction, the percentage of aggregated platelets in solution F was 23.8% +/- 10.3%; in solution E, 4.0% +/- 3.0%; and the average number of platelets per aggregate, 2.9 +/- 0.7. The mean variance for five daily consecutive measurements was 0.52% for solution F, 0.63% for solution E, and 0.002 for the average number of platelets per aggregate. An even lesser mean variance was observed when the interobserver-vs-intraobserver and the intersmear-vs-intrasmear variations were tested. In patients with acute myocardial infarction, the interobserver-vs-intraobserver variance was 5.6% for solution F, 2.2% for solution E, and 0.005 for the average number of platelets per aggregate. The parameters studied were unaffected by different blood drawings, assay tubes, or venous stasis. In 80 patients with unstable angina, the studied parameters as well as the percentage of "big" platelets were measured on hospital days 1, 2, and 5. In 25 patients in whom acute myocardial infarction developed during hospitalization, the percentage of aggregated platelets was 28.1% +/- 8.3%. Most of them (71%) were reversibly aggregated and did not change during hospitalization. The average number of platelets per aggregate was 3.9 +/- 1.6, and the percentage of big platelets was 12.5% +/- 7.2%, both values not undergoing subsequent changes. In patients in whom acute myocardial infarction did not develop, the percentage of aggregated platelets decreased to 14.2% +/- 6.1% on day 5. Most aggregated platelets (58.8% to 90%) were irreversibly aggregated.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1989

Mutations conferring resistance to quinol oxidation (Qz) inhibitors of the cyt bc1 complex of Rhodobacter capsulatus.

Several spontaneous mutants of the photosynthetic bacterium Rhodobacter capsulatus resistant to myxothiazol, stigmatellin and mucidin--inhibitors of the ubiquinol: cytochrome c oxidoreductase (cyt bc1 complex)--were isolated. They were grouped into eight different classes based on their genetic location, growth properties and inhibitor cross-resistance. The petABC (fbcFBC) cluster that encodes the structural genes for the Rieske FeS protein, cyt b and cyt c1 subunits of the cyt bc1 complex was cloned out of the representative isolates and the molecular basis of inhibitor-resistance was determined by DNA sequencing. These data indicated that while one group of mutations was located outside the petABC(fbcFBC) cluster, the remainder were single base pair changes in codons corresponding to phylogenetically conserved amino acid residues of cyt b. Of these substitutions, F144S conferred resistance to myxothiazol, T163A and V333A to stigmatellin, L106P and G152S to myxothiazol + mucidin and M140I and F144L to myxothiazol + stigmatellin. In addition, a mutation (aer126) which specifically impairs the quinol oxidase (Qz) activity of the cyt bc1 complex of a non-photosynthetic mutant (R126) was identified to be a glycine to an aspartic acid replacement at position 158 of cyt b. Six of these mutations were found between amino acid residues 140 and 163, in a region linking the putative third and fourth transmembrane helices of cyt b. The non-random clustering of several inhibitor-resistance mutations around the non-functional aer126 mutation suggests that this region may be involved in the formation of the Qz inhibitor binding/quinol oxidation domain(s) of the cyt bc1 complex. Of the two remaining mutations, the V333A replacement conferred resistance to stigmatellin exclusively and was located in another region toward the C terminus of cyt b. The L106P substitution, on the other hand, was situated in the transmembrane helix II that carries two conserved histidine residues (positions 97 and 111 in R. capsulatus) considered to be the axial ligands for the heme groups of cyt b. The structural and functional roles of the amino acid residues involved in the acquisition of Qz inhibitor resistance are discussed in terms of the primary structure of cyt b and in relation to the natural inhibitor-resistance of various phylogenetically related cyt bc/bf complexes.

Alkenes↗
PubMed1989

Tumour-infiltrating lymphocytes and interleukin-2 in treatment of advanced cancer.

Tumour-infiltrating lymphocytes (TIL) were isolated and expanded from small tumour biopsy samples of twenty-eight patients (thirteen with malignant melanoma, seven with renal cell carcinoma, and eight with non-small-cell lung cancer). The patients were treated with autologous expanded TIL (about 10(10)) and continuous infusions of recombinant human interleukin-2(1-3 x 10(6) U/m2 per 24 h). 29% of the patients with renal cell cancer and 23% of those with melanoma achieved objective tumour responses lasting 3-14 months. Toxic side-effects were limited, and no patient required intensive-care monitoring. Adoptive immunotherapy with TIL and interleukin-2 may be an effective systemic approach to the treatment of some patients with malignant melanoma and renal cell carcinoma.

Carcinoma, Renal Cell↗