Failure of multitarget detection of Borrelia burgdorferi-associated DNA sequences in synovial fluids of patients with juvenile rheumatoid arthritis: a cautionary note.
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Biomedical subjects
Publications and source records attributed to T J White.
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Establishing the phylogeny of fungi and protists often has proved difficult owing to the simple morphologies and convergent characters in these organisms. We used DNA sequences of nuclear small-subunit ribosomal RNA genes to determine phylogenetic relationships among three major classes of organisms considered to be fungi--Basidiomycetes, Ascomycetes and Chytridiomycetes--and to assess the taxonomic position of Neocallimastix, an economically important anaerobic rumen microorganism whose classification is controversial. The Basidiomycetes and Ascomycetes, two classes of nonflagellated fungi, are the most closely related taxa. Chytridiomycetes, though bearing flagella, group with these higher fungi rather than with the protists. Neocallimastix, a eukaryote lacking mitochondria and variously classified as a protist or as a fungus, shows closest molecular affinities with the Chytridiomycete fungi in the order Spizellomycetales.
The morphological, ecological, and clinical diversity among ascomycete fungi that are pathogenic to humans suggest that the potential for pathogenicity may have arisen multiple times within these higher fungi. We have obtained 18S ribosomal DNA sequences from a diverse group of human pathogenic fungi in order to determine their evolutionary origins. The fungi studied include a skin pathogen that is confined to humans (Trichophyton rubrum) and three systemic, facultative parasites that cause histoplasmosis (Histoplasma capsulatum), blastomycosis (Blastomyces dermatitidis) and coccidioidomycosis (Coccidioides immitis) in humans and other higher animals. Also included in our analysis are representatives of non-pathogenic fungi, as well as two opportunistic pathogens, Pneumocystis carinii and Candida albicans, that cause severe disease in immunocompromised individuals, especially those with AIDS. Two of the fungi we sequenced, T. rubrum and C. immitis, are limited to asexual modes of reproduction and therefore lack the sexual structures that are most useful for evolutionary comparison as well as being essential for classification among the higher fungi. Coccidioides immitis is particularly problematic owing to its contradictory and confusing asexual morphologies, which have caused it to be placed in three fungal classes and the protista. Our analysis shows that the specialized, superficial parasite and the systemic, facultative parasites, including C. immitis, are closely related ascomycetes, which clearly demonstrates the power of molecular characters to compensate for missing or confusing reproductive morphology. Analysis also shows that the opportunistic pathogens are more distantly related, with the likely explanation that pathogenicity has arisen more than once within the Ascomycetes.
1. Thiophene and its two monobromo derivatives were administered to rats and the amounts of thioether excreted in urine were measured by an assay based on Ellman's reagent. This assay, which involves extraction and hydrolysis, was validated by determining extraction and hydrolysis efficiencies for several authentic thioethers including N-acetyl-S-(2-thienyl)-L-cysteine, a previously reported metabolite of thiophene and 2-bromothiophene. 2. The thioethers present in urine of animals dosed with thiophenes have been examined chromatographically. Contrary to previous reports, the present work indicates that S-substituted, N-acetyl-L-cysteines (mercapturic acids) are not important thioether metabolites of thiophene in rats, and the small quantity of such compounds formed does not include either of the two simple S-thienyl derivatives. 3. The two monobromo thiophenes form higher proportions of thioethers than does thiophene, and one of these thioethers, arising from 3-bromothiophene, was identified, chromatographically, as N-acetyl-S-(3-thienyl)-L-cysteine.
Nucleotide sequences obtained from regions of the ribosomal DNA repeat were compared by phylogenetic methods and combined with a statistical evaluation to clarify the relationships among the genera Cystofilobasidium, Filobasidium (F.) and Filobasidiella (Fl.), to assess the affinity of Filobasidiella neoformans and Filobasidiella depauperata, and to compare the varieties of Fl. neoformans. With appropriate primers, the nuclear 18S, 5.8S and internal transcribed spacer (ITS) regions of the ribosomal RNA genes (rDNA) of 10 strains were amplified with the polymerase chain reaction. The resulting DNA products were compared by digestion with endonucleases and analysis of restriction fragments. Single strands of the 5.8S rDNA and ITS regions were subsequently sequenced by the dideoxy method. Statistical support for the phylogeny inferred from parsimony analysis of aligned 5.8S rDNA sequences was determined by bootstrapping. There were no nucleotide substitutions in this region, nor in the ITS, among strains of Fl. neoformans that differ in variety and serotype. There was strong support for retaining Fl. depauperata and Fl. neoformans in the same genus, but nucleotide substitutions can be used to distinguish the two species. There was no support for combining the genera Filobasidium, Filobasidiella or Cystofilobasidium.
In a double-blind randomized study, we evaluated the effects of 25 mg vs 50 mg of hydrochlorothiazide in 51 elderly patients (aged 68.9 +/- 7.0 years) with isolated systolic hypertension (blood pressure, 160 to 239 mm Hg systolic and less than 90 mm Hg diastolic). Dose levels could be increased to twice daily to control blood pressure. The reductions in blood pressure (25.4/6.8 mm Hg and 28.9/7.4 mm Hg) and proportion of patients in whom blood pressure was controlled (78% and 89%) were similar in the lower- and higher-dose groups during the titration phase. However, serum potassium level was reduced more in the higher-dosage (0.57 mmol/L) than the lower-dosage (0.17 mmol/L) group. There were no significant changes in blood pressure during a 24-week maintenance phase. No patient required withdrawal from the study because of adverse effects, and cognitive-behavioral function was well preserved. We conclude that hydrochlorothiazide is effective and well tolerated in older patients with isolated systolic hypertension, many of whom may be effectively treated with 25 mg of hydrochlorothiazide once daily.
The human lung fibroblast cell line MRC-5 constitutively produces a megakaryocyte potentiator activity, identified in murine bone marrow liquid culture assays for acetylcholinesterase and megakaryocyte colony assays in the presence of low concentrations of IL-3. The production levels of this activity were increased after stimulation with the phorbol ester analog, mezerein, and the calcium ionophore, A23187. Complete purification of a protein having this activity from conditioned media of induced MRC-5 cells was achieved using gel filtration and ion exchange chromatography. The first 14 residues of the purified protein identified by amino-terminal sequencing were identical to the first 14 residues of IL-6. Recombinant human IL-6 was tested and found to promote megakaryocyte growth. IL-1 beta, another component detected in MRC-5 conditioned media, was unable to promote megakaryocyte colony formation but did reduce the concentration of IL-6 necessary to support megakaryocyte colony formation. Immunoprecipitation using rabbit antiserum prepared against IL-6 removed the megakaryocyte growth activity found in MRC-5 conditioned media. Thus, connective tissue cells such as fibroblasts in the bone marrow may co-stimulate thrombocytopoiesis via IL-6 and, possibly, via IL-1 production.
We compared the efficacy and adverse effects of antihypertensive drug regimens in 690 men past age 60 with diastolic blood pressure 90-114 mm Hg and systolic blood pressure less than 240 mm Hg. They received either a low (25-50 mg) or high (50-100 mg) dose of hydrochlorothiazide daily. Of 644 patients who completed the hydrochlorothiazide titration, 375 (58.2%) were responders (diastolic blood pressure less than 90 and less than or equal to 5 mm Hg below baseline) and 92.8% of these completed a 6-month maintenance period. Blood pressure was reduced from 157.6/98.5 mm Hg by 18.3/9.5 mm Hg with low dose hydrochlorothiazide and by 20.4/9.6 mm Hg with high dose hydrochlorothiazide; more patients achieved goal blood pressure with the high dose. Whites and blacks responded equally. Serum potassium less than 3.5 mmol/l occurred in 104 of 321 (32.3%) of the high dose versus 62 of 333 (18.6%) of the low dose hydrochlorothiazide patients. The 269 nonresponders to hydrochlorothiazide were randomly assigned in a double-blind study to receive hydralazine, methyldopa, metoprolol, or reserpine in addition to hydrochlorothiazide; 79.2% responded to the addition of the second drug and 87.3% of these completed a 6-month maintenance phase. Overall, there were no significant efficacy differences among the step 2 regimens. We conclude that the lower dose of hydrochlorothiazide was nearly as effective as the higher dose, and the addition of a second drug was effective and generally well tolerated in elderly patients.
This study was designed to determine whether blood pressure reduction, per se, causes adverse effects on cognitive and behavioral function in elderly hypertensive patients. Men with mild-to-moderate diastolic hypertension who had passed their 60th birthday were entered into the trial. After a placebo washout period, they were assigned in a randomized, double-blind manner to one of two groups receiving hydrochlorothiazide (either 25 mg once or twice daily or 50 mg once or twice daily). Responders entered a 1-year maintenance period. Nonresponders were randomly assigned to double-blind treatment with hydralazine, methyldopa, metoprolol, or reserpine added to the diuretic therapy. During the placebo and treatment periods, patients underwent a battery of psychometric tests designed to assess cognitive function, motor skills, memory, and affect. A separate questionnaire assessed the patient's ability to perform activities of daily living. A subset of patients blindly being treated with placebo received the same battery of tests as a control for practice effect. The results showed that there was similar improvement on the psychometric tests between those patients whose blood pressure was successfully reduced and the placebo-treated control group. Therefore, the practice effect did not obscure a true deterioration in function. There were no substantive differences between the lower and higher doses of diuretic or among the four drugs added to the diuretic, although there were qualitative differences in side effects. We conclude that blood pressure reduction, per se, does not adversely affect cognitive and behavioral function in elderly hypertensive patients and that antihypertensive treatment is safe and effective in these patients.
To examine the effect of amino acid substitutions in lysozyme on the binding of antibodies to lysozyme, we purified lysozyme from the egg whites of California quail and Gambel quail. Tryptic peptides were isolated from digests of the reduced and carboxymethylated lysozymes and subjected to quantitative analysis of their amino acid compositions. The two proteins were identical by this criterion. Each peptide from the California quail lysozyme was then sequenced by quantitative Edman degradation, and the peptides were ordered by homology with other bird lysozymes. California quail lysozyme is most similar in amino acid sequence to bobwhite quail lysozyme, from which it differs by two substitutions: arginine for lysine at position 68 and histidine for glutamine at position 121. California and bobwhite quail lysozymes were antigenically distinct from each other in quantitative microcomplement fixation tests, indicating that substitutions at one or both of these positions can alter the antigenic structure of lysozyme. Yet neither of these positions is among those claimed to account for the precise and entire antigenic structure of lysozyme [Atassi, M. Z., & Lee, C.-L. (1978) Biochem. J. 171, 429--434]. Two possible explanations for this discrepancy are discussed.
Rectally administered thiopental was evaluated for sedation in pediatric computed tomography and compared with "cardiac cocktail" sedation. The drug produced sedation as effective as the "cocktail" with easier administration, more rapid onset, and shorter duration of sedation. Although no complications were observed, careful observation for respiratory depression is suggested.
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For evolutionary reasons, we determined the primary structure of rat lysozyme. The chymotryptic peptides from the reduced and carboxymethylated protein were sequenced and aligned by homology with the sequence of human lysozyme. Overlaps were confirmed by partial structures of tryptic peptides and an automatic sequencer run on the whole protein. By comparing this lysozyme sequence with those of human and baboon and taking into account paleontological estimates of the times of divergence of these species from one another, an approximate estimate of the average rate of lysozyme evolution was made. This rate is not significantly different from the average rate of lactalbumin evolution in mammals--a finding which is at variance with Dickerson's [Dickerson, R.E. (1971), J. Mol. Evol. 1, 26] and Dayhoff's [Dayhoff, m.o., ed. (1972), Atlas of Protein Structure and Sequence, Vol. 5, Silver Spring, Md., The National Biomedical Research Foundation] conclusion that lactalbumin evolution has been faster than lysozyme evolution. Our finding raises the possibility that the gene duplication event responsible for the origin of lactalbumin from lysozyme was more ancient than is generally supposed. Furthermore, from comparison of the rates of lysozyme evolution in rodents and primates, it is suggested that generation time is not a key factor in lysozyme evolution.
A preliminary model was prepared for the development of superficial burns which can result from the interaction of faulty defibrillator and other electrical operating room equipment. A thermal model previously used to predict retinal coagulation was adapted to give temperature rise history of the skin. Skin resistance is assumed to decrease rapidly due to the instability of a small volume near the surface. Heating due to dissipation of the defibrillator capacitor energy in this skin volume is shown to be high enough to cause burns. A sensitivity analysis is included to isolate the dominant input parameters. Recommendations are made for future research.
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A new assay for chloramphenicol in biological fluids has been developed that offers sensitivity, specificity, precision, accuracy, economy, and ease of performance. The assay is based on the enzymological acetylation of chloramphenicol catalyzed by an R factor-mediated enzyme. [(14)C]acetyl coenzyme A serves as the donor of the labeled acetyl group, and the product, [(14)C]acetoxychloramphenicol, is separated from the labeled precursor by utilizing its preferential extraction into benzene. The product is then quantified by liquid scintillation counting. This assay measures chloramphenicol concentrations in both plasma and other biological specimens and in the presence of other antibiotics, hemolysis, or jaundice. Its rapidity and ease of performance are useful for clinical laboratories, and its sensitivity allows determinations on 10 mul of plasma.