The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: evolutionary implications

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba(3) and caa(3)) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) under reducing anaerobic conditions. The rate of NO consumption and N(2)O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 +/- 0.7 mol NO/mol ba(3) x min and 32 +/- 8 mol NO/mol caa(3) x min at [NO] approximately 50 microM and 20 degrees C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme x min), is definitely significant. We also show that for ba(3) oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar Cu(B)(+) coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb(3) terminal oxidases. Our findings represent functional evidence in support of this hypothesis.     

Pulmonary lymphangitic spread of carcinoma: appearance on CT scans

Chest computed tomography (CT), including high-resolution CT with thin (1.5-mm) sections was used to evaluate proved (pathologically or clinically) lymphangitic spread (LS) of tumor in 12 patients. These appearances were compared with thin-section scans obtained in 11 healthy subjects. Thin-section CT demonstrated findings consistent with thickening of the normal lung interstitium. In all patients, thin sections showed an increase in the number of peripheral lines (1-2 cm in length) that were diffuse in generalized disease and localized in focal disease. Normal peripheral arcades were not increased in number, but the limbs forming the arcades were thickened in all patients. A diffuse increase in linear and curvilinear structures (reticular pattern) was seen toward the center of the lung. Polygonal structures 1-2 cm in diameter were seen in seven patients with LS but not in healthy subjects. Fissures were thickened in nine patients. Selected 1.5-mm-thick CT sections are recommended through abnormal areas (seen at CT or on chest radiographs) or if these are normal at three levels (midapex, hilus, and 3 cm above the diaphragm) when scanning patients with tumors known to cause LS.     

Characterization of the human jumonji gene

While constructing a cDNA library of human embryos, we have isolated a clone homologous to jumonji, a mouse gene required for neural tube formation. We have determined the complete coding sequence of the human homologue (JMJ) and deduced the amino acid sequence of the putative protein. We show here that human and mouse jumonji putative proteins are homologous and present 90% identity. During human embryogenesis, JMJ mRNAs are predominantly expressed in neurons and particularly in dorsal root ganglion cells. They are also expressed in neurons of human adult cerebral cortex. In view of these observations, we propose JMJ as a candidate gene for developmental defects of the central nervous system in the human. The human JMJ gene maps at position 6p24-6p23.      

Comparison of the fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm injection

A prospective study was carried out to compare the fertilizing capability and pregnancy outcome following intracytoplasmic sperm injection (ICSI) using spermatozoa obtained from ejaculates, or surgically from epididymis or seminiferous tubules. A total of 77 ICSI cycles (one per patient) was included. In all, 28 patients had severe oligoasthenoteratozoospermia, 19 patients had obstructive azoospermia and 30 patients had non-obstructive azoospermia. The main outcome measures were fertilization rate per injected metaphase II oocyte and the clinical pregnancy rate per embryo transferred back to the female recipients. In patients with severe oligoasthenoteratozoospermia, the fertilization and pregnancy rates were 79 and 25 %. In patients with obstructive azoospermia, for whom epididymal spermatozoa were used, these were 75 and 28%, and in the non-obstructive group for which testicular spermatozoa were used for injection, they were 69 and 21% respectively. These rates were not significantly different in the three groups (P = 0.85 and P = 0.14 respectively), suggesting that spermatozoa from the ejaculates and epididymal or testicular biopsies are able to fertilize equally by using ICSI. Live birth per embryo transfer was significantly reduced in patients with non-obstructive azoospermia compared to the other two groups. The high abortion rate (50%) in the group in which testicular spermatozoa were used raises doubts about the developmental competence of such embryos.      

Aid coordination for health sector reform: a conceptual framework for analysis and assessment

Recent widespread interest in health sector policy and institutional reform in lower income countries has coincided with heightened concern for aid coordination. Because the health budgets of many low income countries are highly aid dependent, donors are strongly placed to make aid conditional on health care reforms. However, given the growing number and heterogeneity of multilateral, bilateral and international non-governmental donors operating in many of these countries, there is concern that if external efforts are not coordinated, the aims of health care reform--namely improving efficiency, effectiveness and equity--will not be met. Evidence is mounting that without effective coordination arrangements, donors may weaken rather than improve fragile health systems, undermining attempts to reform those systems. This paper traces the factors fuelling current interest in coordination, in particular with reference to its contribution to the goals of health sector reform. Aid coordination is defined and its principles elaborated. A framework is developed by which to assess the variety of coordination mechanisms which are evolving at the county level. In light of this framework, a case is made for greater and more critical analysis of aid coordination arrangements. The paper concludes that if health sector reform is to be successful in low income countries, current enthusiasm for coordination needs to be harnessed. The framework offered here provides a way of assessing the variety of coordination mechanisms currently proliferating, which could be used to enhance health sector reform.     

Arg-Gly-Asp-dependent occupancy of GPIIb/IIIa by applaggin: evidence for internalization and cycling of a platelet integrin

Using indirect immunofluorescence microscopy we examined the distribution and cycling of GPIIb/IIIa after binding to applaggin, a high-affinity Arg-Gly-Asp (RGD)--containing ligand. Resting, unfixed platelets were incubated with applaggin for 30 minutes at 37 degrees C, and bound applaggin was detected by an affinity-purified rabbit anti- applaggin antibody. Examination of intact cells showed a rim pattern for applaggin, consistent with its binding to the platelet surface. Staining of Triton X-100--permeabilized cells showed an intracellular pool of applaggin. Competition of applaggin binding by either AP-2, an anti-GPIIb/IIIa monoclonal antibody (MoAb) that blocks fibrinogen binding, or the synthetic peptide RGDW eliminated both surface and intracellular staining, indicating that applaggin is binding to GPIIb/IIIa in an RGD-dependent manner. Inhibition of platelet activation by PGE1 and theophylline had no effect on the observed staining patterns, indicating that cellular activation is not required for surface binding and subsequent internalization. To evaluate whether occupancy of functional binding sites on GPIIb/IIIa is required for internalization, we used mAb15, an anti-GPIIIa antibody that neither blocks fibrinogen binding nor induces the expression of ligand-induced binding sites on GPIIb/IIIa. In these studies mAb15 was internalized in a manner analogous to both AP-2 and applaggin, showing that occupancy of the RGD binding site is not required to initiate receptor internalization. To estimate the size of the newly internalized pool of applaggin, 125I-applaggin--binding studies were performed. Displacement of bound 125I-applaggin by excess unlabeled applaggin or EDTA showed that at least 17% of bound applaggin was nondisplaceable when binding was performed under conditions permitting membrane flow and internalization. These data indicate that GPIIb/IIIa is internalized in unstimulated platelets independent of cellular activation or occupancy of the functional binding site(s) of GPIIb/IIIa by RGD-containing ligands. Thus, internalization of GPIIb/IIIa may represent a mechanism by which the surface expression of this adhesion receptor is regulated.     

Behavioural Effects of the Methanolic Root Bark Extract of Securinega Virosa in Rodents

Securinega virosa is used traditionally as sedative in children and in mental illnesses. In this study, the behavioral effects of methanolic root bark extract of S. virosa were investigated in mice. The results revealed that the extract significantly (P<0.05) and dose-dependently reduced the onset and prolonged the duration of sleep. The extract significantly (P<0.05) decreased exploratory activity and reduced the rate of apomorphine-induced stereotyped climbing at the doses tested (6.25–25mg/kg). It also produced a significant and dose-dependent motor coordination deficit in mice at the doses tested (P<0.01). The intraperitoneal median lethal dose in mice was 774.6mg/kg while the preliminary phytochemical screening revealed the presence of alkaloids, tannins, saponins and flavonoids. These results suggest that methanolic root bark extract of S. virosa contains biologically active principles that are sedative in nature and lend pharmacological credence to the ethnomedical use of the plant.     

Chronic daily headache with medication overuse: a randomized follow-up by neurologist or PCP

Several studies have shown the benefit of withdrawal therapy when medication overuse headache (MOH) is suspected. Our aim was to compare the effect of withdrawal therapy in patients followed by a neurologist (group A, n  = 42) and a primary care physician (PCP) (group B, n  = 38). Patients were randomized to A or B, and follow-up was at 3, 6 and 12 months. Calculated mean headache (MH at 6 months + MH at 12 months)/2 (primary end-point) was similar; A 1.04 (0.87, 1.21) and B 1.02 (0.82, 1.21) ( P  = 0.87). The number of patients with 50% improvement of headache days was also similar; 14/42 in group A vs. 12/34 in B ( P  = 0.86) at 3 months, 15/42 vs. 11/33 ( P  = 0.83) at 6 months and 15/42 vs. 14/38 ( P  = 0.92) at 12 months. Days without headache during the last 9 months of follow-up were 123 (96, 150) in group A and 137 (112, 161) in B ( P  = 0.62). After 3 months one-third were classified as MOH. Patients with MOH improved similarly in group A and B, and so did patients without MOH. Within 1 year 7/42 in A and 9/38 in B had recurrent medication overuse ( P  = 0.43). In summary, there were no significant differences in follow-up results between the two groups.     

Mespirenone and other 15,16-methylene-17-spirolactones, a new type of steroidal aldosterone antagonists

The ability of a series of 15,16-methylene-spirolactones in comparison to known antimineralocorticoids to inhibit the renal actions of aldosterone was tested in adrenalectomized, glucocorticoid-treated rats. The standard procedure involved continuous i.v. infusion with an isotonic solution of low sodium content (0.05% NaCl + 5.2% glucose, 3 ml/rat/h) supplemented with d-aldosterone [1 micrograms/(kg X h)] resulting in a long-lasting reduction of renal sodium excretion, increase of renal potassium excretion and hence decrease of the urinary Na/K-ratio. In some experiments sodium input was increased (0.2% NaCl + 4.3% glucose or 0.9% NaCl, respectively). The test drugs either were administered orally 1 h before start of the infusion or were added to the infused solution. With the exception of two steroids which could only be tested at single doses, all compounds were administered at three doses ranging from 2.2 to 40 mg/kg (p.o.) or from 0.83 to 6.7 mg/kg/h (i.v.). Spironolactone or spirorenone (oral administration) and potassium canrenoate (i.v. infusion) served as reference compounds. The antimineralocorticoid activity of the steroids was judged by the increase in the aldosterone-lowered Na/K-ratio in urine which was collected at hourly intervals for 15 or 21 h, respectively. Adrenalectomized, glucocorticoid-treated rats receiving an i.v. infusion without aldosterone were used as controls. To obtain preliminary information on potential antiandrogenic and progestogenic (side-)effects, binding of the test-compounds to androgen receptors (rat prostate cytosol) and progestogen receptors (rabbit uterus cytosol) was measured in vitro using 3H-dihydrotestosterone (DHT) and 3H-progesterone (prog.) as tracer and unlabelled DHT and prog. as references. All steroids tested exhibited antimineralocorticoid activity. For compounds tested at three doses levels the potency relative to the standard used was evaluated using regression analysis based on the Na/K-ratio or the log (Na X 100)/K-ratio. The relative potency of the other compounds was estimated by comparing the biological effect of single doses of test drug and standard drug, respectively, using nonparametric statistical tests.(ABSTRACT TRUNCATED AT 400 WORDS)