The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.

Alcaligenes↗

PubMed1992

Overexpression, purification and characterization of the Escherichia coli MelR transcription activator protein.

The gene encoding Escherichia coli MelR protein has been cloned in the expression vector pJLA502. MelR has been overexpressed, substantially purified and shown to bind to DNA fragments carrying the melAB promoter. A truncated version of the melR gene, encoding the C-terminal half of MelR, was also cloned into pJLA502; the protein product of this truncated gene binds to the melAB promoter but was not overproduced. A number of amino acid substitutions were made in the recognition helices of two putative helix-turn-helix motifs in the C-terminal part of MelR, and the effects of these mutations on MelR-dependent transcription initiation at the melAB promoter have been measured.

Amino Acid Sequence↗

PubMed1992

Regulation of low affinity IgE receptor (CD23) expression on mononuclear phagocytes in normal and asthmatic subjects.

Mononuclear phagocytic cells contain low affinity receptors for IgE (Fc epsilon RII or CD23) which induce cellular activation in the presence of specific allergen. These studies were performed to quantify the expression by monocytes and alveolar macrophages of Fc epsilon RII in asthma and to determine biologic response modifiers that regulate Fc epsilon RII. Whereas 2.5 +/- 1.0% of the monocytes obtained from normal volunteers were Fc epsilon RII positive, this increased to 16.7 +/- 2.4% in asthma (p < 0.001). Stimulation of Fc epsilon RII expression on monocytes was shown to be an activity of IL-4 (24.5 +/- 5.9%), granulocyte-macrophage-CSF (28.1 +/- 5.2%), IFN-alpha (15.8 +/- 5.3%), IFN-gamma (10.4 +/- 3.7%), and macrophage-CSF (7.3 +/- 0.7%) but not of IL-2, IL-6, or TNF-alpha. Expression of Fc epsilon RII by these cytokines was associated with the induction of specific mRNA transcripts. Using Fc epsilon RII subtype specific primers in the polymerase chain reaction expansion of cDNA, cytokine-induced receptors were shown to be Fc epsilon RIIb. Alveolar macrophages from nonasthmatic subjects displayed minimal expression of Fc epsilon RII (3.2 +/- 1.2%); however, these receptors were present on 69.2 +/- 6.3% of asthmatic volunteers (p < 0.001). Induction of Fc epsilon RII appears specific for allergic asthma insofar as these receptors are also not expressed in subjects with interstitial lung disease (1.3 +/- 1.3%). As assessed by shift in mean fluorescence, instillation of allergen in the asthmatic's airway further up-regulated Fc epsilon RII on alveolar macrophages by 151 +/- 7%. Up-regulation of Fc epsilon RII in atopic individuals may therefore reflect allergen-induced exposure of mononuclear phagocytes to one or more of these cytokines. These studies suggest a mechanism by which an immunologic stimulus that leads to the production of these cytokines (e.g., allergen or viral infection) would contribute to the development or exacerbation of allergic disease.

Allergens↗

PubMed1992

'The comparative strengths of commercial glass ionomer cements'.

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Glass Ionomer Cements↗

PubMed1992

Prenatal screening for Down's syndrome with use of maternal serum markers.

BACKGROUND: Approximately 35 percent of all cases of Down's syndrome in fetuses can be detected by measuring maternal serum alpha-fetoprotein during the second trimester in the general population of pregnant women. Recent case-control studies indicate that this detection rate could be approximately doubled by measuring serum levels of unconjugated estriol and chorionic gonadotropin, which are abnormally low and abnormally high, respectively, in women carrying fetuses affected by Down's syndrome. METHODS: We prospectively screened 25,207 women and adolescents in the second trimester of pregnancy and assigned each a risk of fetal Down's syndrome with an algorithm that took into account measurements of all three serum markers in combination with maternal age. On this basis, 1661 subjects (6.6 percent) were initially assigned a second-trimester risk of fetal Down's syndrome of at least 1 in 190, and 962 (3.8 percent) were offered amniocentesis for chromosomal analysis after verification of gestational age. Gestational age was determined on the basis of the first day of the last menstrual period or, when available, by ultrasonography. RESULTS: Among the 760 women and adolescents who chose amniocentesis, 20 cases of fetal Down's syndrome were detected, along with 7 other chromosomal disorders. There was 1 additional case of fetal Down's syndrome among the 202 women who chose not to have amniocentesis. The rate of detection of Down's syndrome was thus 58 percent (21 of 36 expected cases), and the frequency of identifying a fetus with Down's syndrome in women undergoing amniocentesis was 1 per 38 amniocenteses (95 percent confidence interval, 1 in 25 to 1 in 62). CONCLUSIONS: Measuring serum alpha-fetoprotein, chorionic gonadotropin, and estriol is more effective in screening for fetal Down's syndrome than measuring maternal serum alpha-fetoprotein alone. Such an expanded protocol can readily be incorporated into existing prenatal screening programs.

Adolescent↗

PubMed1992

Elevation of interleukin-6 in response to a chronic inflammatory stimulus in mice: inhibition by indomethacin.

Intraperitoneal (i.p.) injection of a mineral oil such as pristane induces a chronic inflammatory response in mice. This is characterized by a large influx of macrophages and other inflammatory cells into the peritoneal cavity for months after injection of the oil. By using the B9 cell bioassay, it was found that injection of pristane caused a marked and prolonged elevation of interleukin-6 (IL-6) levels in the peritoneal cavities of the mice. IL-6 was undetectable (less than 15 U/mL) in the peritoneal fluids of unprimed mice and during the first week after injecting pristane. From 4 to 20 weeks, the concentration of IL-6 increased to an apparent plateau with concentrations ranging from 200 to 2,000 U/mL. Increasing the dose of pristane did not substantially increase the peritoneal levels of IL-6 established at 20 weeks after pristane treatment. At later times (by day 250), the level decreased to 263 +/- 217 U/mL. However, mice that developed plasma cell tumors around day 300 showed high levels of IL-6 in the ascites fluid (650 to 2,400 U/mL). Serum levels of IL-6 were also elevated in pristane-primed mice but were substantially lower than those found in the peritoneal cavity. Chronic administration of the nonsteroidal anti-inflammatory drug indomethacin decreased the levels of IL-6 by 75% to 80%. Experiments performed in vitro showed that pristane-elicited macrophages secreted low levels of IL-6 constitutively and high levels of IL-6 in the presence of lipopolysaccharide. Both IL-6 and prostaglandin E2 production were inhibited by addition of indomethacin to macrophage cultures in vitro. Treatment of mice with pristane may provide a model system for studying the inflammatory pathways that control IL-6 levels in vivo. The relevance of these results to elucidation of the role of IL-6 in plasma cell tumorigenesis is discussed.

Animals↗

PubMed1992

Extracellular (axenic) development in vitro of the erythrocytic cycle of Plasmodium falciparum.

Merozoites of the erythrocytic stage of Plasmodium falciparum were suspended in erythrocyte sonicate medium with ATP and pyruvate and mixed with Matrigel to form a soft gel. The gel was overlaid with complete medium; this was replaced with fresh medium at 12, 24, and 36 hr. At these times and also at 45 hr rhodamine 123 was added to some cultures and gels were sampled. Viable extracellular forms showing rhodamine fluorescence were seen: rings at 12 hr, trophozoites and early schizonts with pigment at 36 hr, and late schizonts with developing merozoites at 45 hr. These merozoites were shown to be infective to erythrocytes added to the cultures at 45 hr. Electron micrographs of 36-hr trophozoites show the organisms to have only their single plasma membrane; no parasitophorous membrane is evident. We conclude that the complex process of entry, the intactness of the host erythrocyte, and the parasitophorous membrane are not essential to the development of a merozoite through its complete asexual cycle.

Adenosine Triphosphate↗

PubMed1992

An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syndrome.

BACKGROUND: Since 1990 several clusters of multidrug-resistant tuberculosis have been identified among hospitalized patients with the acquired immunodeficiency syndrome (AIDS). We investigated one such cluster in a voluntary hospital in New York. METHODS: We compared exposures among 18 patients with AIDS in whom tuberculosis resistant to isoniazid and streptomycin was diagnosed from January 1989 through April 1990 (the case patients) with exposures among 30 control patients who had AIDS and tuberculosis susceptible to isoniazid, streptomycin, or both. We also compared exposures among the 14 case patients hospitalized during the six months before the diagnosis of tuberculosis (the exposure period) with those among 44 control patients with AIDS matched for duration of hospitalization. Mycobacterium tuberculosis isolates were typed with analysis of restriction-fragment-length polymorphism (RFLP). RESULTS: Case patients with drug-resistant tuberculosis were significantly more likely than controls with drug-susceptible tuberculosis to have been hospitalized during their exposure periods (14 of 18 vs. 10 of 30) (odds ratio, 7.0; 95 percent confidence interval, 1.6 to 36; P = 0.006). Case patients hospitalized during their exposure periods were significantly more likely to have been hospitalized on the same ward as a patient with infectious drug-resistant tuberculosis than were either controls with drug-susceptible tuberculosis hospitalized during their exposure periods or controls matched for duration of hospitalization (13 of 14 vs. 2 of 10 and 23 of 44) (odds ratio, 52; 95 percent confidence interval, 3.1 to 2474; P less than 0.001; and odds ratio, infinity; 95 percent confidence interval, 2.4 to infinity; P = 0.005, respectively). Among those hospitalized on the same ward, the rooms of case patients were closer to that of the nearest patient with infectious tuberculosis than were the rooms of controls matched for duration of hospitalization. M. tuberculosis isolates from 15 of 16 case patients had identical patterns on RFLP analysis. Of 16 patients' rooms tested with air-flow studies, only 1 had the recommended negative-pressure ventilation. CONCLUSIONS: Multidrug-resistant tuberculosis is readily transmitted among hospitalized patients with AIDS. Physicians must be alert to this danger and must enforce adherence to the measures recommended to prevent nosocomial transmission of tuberculosis.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Limiting access and patient selection in liver transplantation.

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Ethics, Medical↗

PubMed1992

An unsuccessful cotinine-assisted intervention strategy to reduce environmental tobacco smoke exposure during infancy.

OBJECTIVE: To test a low-intensity physician's office-based intervention strategy using infant urine cotinine measurements, aimed at reducing infant exposure to environmental tobacco smoke. DESIGN: A randomized intervention trial. SETTING: Offices of 28 physicians (pediatricians and family practitioners) and two hospital-based clinics in the Greater Portland, Me, area. PARTICIPANTS: Infants brought to the physician by a parent (usually the mother) for an initial well-child visit. A total of 518 of these infants and their mothers consented to provide information about household smoking habits and to provide a sample of the infant's urine for biochemical analysis. SELECTION PROCEDURE: From among the consecutive sample of 518 enrolled mother/infant pairs, 103 mothers reported that they smoked 10 or more cigarettes per day, and these were randomized on an individual basis for intervention (52 pairs) or control (51 pairs). Randomization took place at the study center when the infant's urine sample and information about household smoking habits were received. INTERVENTION: The physician telephoned the mother to report the urine cotinine result and to explain its meaning. The physician then signed and sent an individualized form letter to the mother, providing specific recommendations for changing household smoking habits. RESULTS: Follow-up urine cotinine measurements were obtained in 27 (52%) of 52 infants from the intervention group and in 29 (57%) of the 51 controls 2 months later. The mean log ratio of the follow-up to initial urine cotinine measurements was 6% lower in the intervention group than in the control group. This difference was not statistically significant. CONCLUSION: The low-intensity intervention strategy did not significantly influence infant exposure to environmental tobacco smoke in the household.

Adult↗