Changes of respiratory chain activity in mitochondrial and synaptosomal fractions isolated from the gerbil brain after graded ischaemia

In this study we have examined (1) the integrated function of the mitochondrial respiratory chain by polarographic measurements and (2) the activities of the respiratory chain complexes I, II-III, and IV as well as the ATP synthase (complex V) in free mitochondria and synaptosomes isolated from gerbil brain, after a 30-min period of graded cerebral ischaemia. These data have been correlated with cerebral blood flow (CBF) values as measured by the hydrogen clearance technique. Integrated functioning of the mitochondrial respiratory chain, using both NAD-linked and FAD-linked substrates, was initially affected at CBF values of approximately 35 ml 100 g-1 min-1, and declined further as the CBF was reduced. The individual mitochondrial respiratory chain complexes, however, showed differences in sensitivity to graded cerebral ischaemia. Complex I activities decreased sharply at blood flows below approximately 30 ml 100 g-1 min-1 (mitochondria and synaptosomes) and complex II-III activities decreased at blood flows below 20 ml 100 g-1 min-1 (mitochondria) and 35-30 ml 100 g-1 min-1 (synaptosomes). Activities declined further as CBF was reduced below these levels. Complex V activity was significantly affected only when the blood flow was reduced below 15-10 ml 100 g-1 min-1 (mitochondria and synaptosomes). In contrast, complex IV activity was unaffected by graded cerebral ischaemia, even at very low CBF levels.     

Sulfur-related air pollutants induce the generation of platelet-activating factor, 5-lipoxygenase- and cyclooxygenase-products in canine alveolar macrophages via activation of phospholipases A2

Recent studies have shown that long-term in vivo exposure of dogs to neutral sulfur(IV)/sulfite aerosols induces mild inflammatory reactions, whereas the combination of neutral sulfite with acidic sulfur(VI)/sulfate aerosols evokes less pronounced effects. To understand underlying mechanisms, we studied in vitro the role of lipid mediators in the responses of alveolar macrophages (AMs) to sulfur-related compounds under neutral (pH 7) or moderate acidic (pH 6) conditions. Canine AMs incubated with sulfite at pH 7 released threefold higher amounts of platelet-activating factor than control (P < 0.005). Generation of arachidonic acid, leukotriene B4, 5-hydroxy-eicosatetraenoic acid, prostaglandin E2, thromboxane B2 and 12-hydroxyheptadecatrienoic acid increased twofold (P < 0.0005). However, these metabolites remained unchanged following incubation of AMs with sulfite at pH 6 or with sulfate at pH 7 or pH 6. Mediator release by sulfite-treated AMs at pH 7 stimulated respiratory burst activity of neutrophils. Inhibition of MAPK pathway by PD 98059, of cytosolic (cPLA2) and secretory phospholipases A2 by AACOCF3 and thioetheramide-PC, respectively, reduced sulfite-induced eicosanoid formation in AMs. Sulfite activated cPLA2 activity twofold at pH 7. This mechanism of sulfite-stimulated responses in phospholipid metabolism predicts that chronic exposure to sulfur(IV)/sulfite is associated with a considerable health risk.     

A microdialysis study of the effects of the nicotinic agonist RJR-2403 on cortical release of acetylcholine and biogenic amines

Transcortical dialysis was employed to investigate the effects of subcutaneous (s.c.) injections of RJR-2403 (1.2-7.2 mumol/kg) on extracellular levels of acetylcholine (ACh), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat. Systemic administration of RJR-2403 produced a 90% increase of cortical extracellular ACh levels that persisted for up to 90 minutes after injection. Norepinephrine and DA release were increased 124% and 131% above basal values, respectively. Serotonin (5-HT) levels in the dialysate were also significantly elevated by RJR-2403 (3.6 mumol/kg, s.c.) 70% above baseline at 90 minutes post-injection. Comparison of these responses to those of (-)nicotine from a previous study reveals little difference between the two compounds in their ability to influence cortical neurotransmitter release following systemic administration.     

Immunolocalization of natriuretic peptide receptor B in the rat kidney

The natriuretic peptide receptor (NPR) family consists of three receptor subtypes: two transmembrane forms that contain a guanylyl cyclase intracellular domain (NPR-A and NPR-B), and one truncated form (NPR-C). Because of the lack of specific agonists and antagonists for each receptor subtype and to the difficulty to detect the presence of small quantities of NPR-B by ligand binding studies, polyclonal antibodies against a peptide whose sequence was chosen from a region of the extracellular domain of rat NPR-B that is not homologous to sequences in NPR-A and NPR-C were developed. Western blotting with affinity-purified anti-NPR-B (413-426)-Tyr revealed a polypeptide of approximately 120 kD on COS-1 cell membranes transfected with rat NPR-B cDNA. The antibody recognized a second polypeptide, approximately 5 to 10 kD smaller, which probably represents the unglycosylated receptor. Anti-NPR-B (413-426)-Tyr did not show crossreactivity to any other NPR. Western blotting analysis with anti-NPR-B (413-426)-Tyr also identified a protein of appropriate size in renal vascular membranes. These results were supported by immunohistochemistry findings that demonstrated staining for NPR-B on papillary and medullary capillaries, glomeruli, and renal arteries. This study concludes that NPR-B is present in the rat kidney, although it was only detected in vascular structures.     

Mannolipid donor specificity of glycosylphosphatidylinositol mannosyltransferase-I (GPIMT-I) determined with an assay system utilizing mutant CHO-K1 cells

The microsomal enzyme glycosylphosphatidylinositol mannosyltransferase I (GPIMT-I) catalyses the transfer of a mannosyl residue from beta-mannosylphosphoryldolichol (beta-Man-P-Dol) to glucosamine-alpha(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Man alpha(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis. While the transfer of [3H]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [3H]Man-P-Dol, GPIMT-I activity could be characterized with an in vitro enzyme assay system employing membrane fractions from Lec15 or Lec35 cells. These CHO cell mutants apparently contain elevated levels of endogenous GlcN-aPI due to the inability to synthesize (Lec15) or utilize (Lec35) beta-Man-P-Dol in vivo. The presence of a saturated alpha-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since beta-mannosylphosphorylpolyprenol (beta-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as beta-[3H]Man-P-Dol as a mannosyl donor. When beta-[3H]-Man-P-Dol and alpha-[3H]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the beta-mannosyl-phosphoryl linkage. beta-[3H]Man-P-dolichols containing 11 or 19 isoprenyl units were equally effective substrates for GPIMT-I. Membrane fractions from Lec 9, a CHO mutant that apparently lacks polyprenol reductase activity and synthesizes very little beta-Man-P-Dol, but accumulates beta-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with beta-[3H]Man-P-Dol in vitro. This indirect assay suggests that GlcN-aPI does not accumulate in Lec 9 cells, possibly because it is mannosylated via beta-Man-P-Poly, or perhaps the small amount of Man-P-Dol formed by the mutant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

The BsmI vitamin D receptor restriction fragment length polymorphism (bb) influences the effect of calcium intake on bone mineral density

Previous studies of the vitamin D receptor (VDR) polymorphisms and bone mineral density (BMD) have suggested that there may be differences in calcium absorption among groups of women with different VDR genotypes, and that the association may be stronger in younger women. To investigate the association between the VDR polymorphisms and BMD, this study was undertaken in the Framingham Study Cohort and a group of younger volunteers. Subjects from the Framingham Study (ages 69-90 years) included those who underwent BMD testing and who had genotyping for the VDR alleles (n = 328) using polymerase chain reaction methods and restriction fragment length polymorphisms with BsmI (B absence, b presence of cut site). A group of younger volunteer subjects (ages 18-68) also underwent BMD testing and VDR genotyping (n = 94). In Framingham Cohort subjects with the bb genotype, but not the Bb or BB genotypes, there were significant associations between calcium intake and BMD at five of six skeletal sites, such that BMD was 7-12% higher in those with dietary calcium intakes greater than 800 mg/day compared with those with intakes < 500 mg/day. The data also suggested that BMD was higher in persons with the bb genotype only in the group with calcium intakes above 800 mg/day. No significant differences were found in the Framingham Cohort for age-, sex-, and weight-adjusted BMD at any skeletal site between those with the BB genotype and those with the bb genotype regardless of 25-hydroxyvitamin D levels or country of origin. In the younger volunteers, BMD of the femoral neck was 5.4% higher (p < 0.05) in the bb genotype group compared with the BB group and 11% higher (p < 0.05) in males with the bb genotype compared with the BB group. There were no significant differences at the lumbar spine. In this study, the association between calcium intake and BMD appeared to be dependent upon VDR genotype. The findings of an association between dietary calcium intake and BMD only in the bb genotype group suggests that the VDR genotype may play a role in the absorption of dietary calcium. Studies that do not consider calcium intake may not detect associations between VDR genotype and BMD. In addition, the association between VDR alleles and BMD may become less evident in older subjects.     

Life cycle of Ixodes minor (Acari: Ixodidae) in the laboratory

OBJECTIVE: It is commonly believed that the full moon exerts an influence on violence and aggression in psychiatric settings. The literature to date is contentious. This study used a robust methodology to examine the hypothesis that there was an increased frequency of violent and aggressive behaviour among hospitalised psychiatric clients at the time of the full moon. METHOD: Prospective data were collected in five inpatient psychiatric settings across the Northern Sydney Area Health Service. Morrison's hierarchy of violence and aggression was used to rate behaviour. Lunar phases were clearly defined and Poisson regression used to examine relationships between lunar phase and violence. Extraneous temporal variation was considered. RESULTS: No significant relationship was found between total violence and aggression or level of violence and aggression and any phase of the moon. CONCLUSION: Future research could profitably examine the implications of a belief in the lunar effect among health workers in the face of evidence that no relationship exists between violence, aggression and the lunar cycle.     

The efficacy and cost effectiveness of vaccination against influenza among elderly persons living in the community

BACKGROUND: Despite recommendations for annual vaccination against influenza, more than half of elderly Americans do not receive this vaccine. In a serial cohort study, we assessed the efficacy and cost effectiveness of influenza vaccine administered to older persons living in the community. METHODS: Using administrative data bases, we studied men and women over 64 years of age who were enrolled in a large health maintenance organization in the Minneapolis-St. Paul area. We examined the rate of vaccination and the occurrence of influenza and its complications in each of three seasons: 1990-1991, 1991-1992, and 1992-1993. Outcomes were adjusted for age, sex, diagnoses indicating a high risk, use of medications, and previous use of health care services. RESULTS: Each cohort included more than 25,000 persons 65 years of age or older. Immunization rates ranged from 45 percent to 58 percent. Although the vaccine recipients had more coexisting illnesses at base line than those who did not receive the vaccine, during each influenza season vaccination was associated with a reduction in the rate of hospitalization for pneumonia and influenza (by 48 to 57 percent, P < or = 0.002) and for all acute and chronic respiratory conditions (by 27 to 39 percent, P < or = 0.01). Vaccination was also associated with a 37 percent reduction (P = 0.04) in the rate of hospitalization for congestive heart failure during the 1991-1992 season, when influenza A was epidemic. The costs of hospitalization for all types of illness studied were lower in the vaccinated group during 1991-1992 (range of reduction, 47 to 66 percent; P < 0.005) and for acute and chronic respiratory conditions and congestive heart failure in 1990-1991 (reductions of 37 percent and 43 percent, respectively; P < or = 0.05). Direct savings per year averaged $117 per person vaccinated (range, $21 to $235), with cumulative savings of nearly $5 million. Vaccination was also associated with reductions of 39 to 54 percent in mortality from all causes during the three influenza seasons (P < 0.001). CONCLUSIONS: For elderly citizens living in the community, vaccination against influenza is associated with reductions in the rate of hospitalization and in deaths from influenza and its complications, as compared with the rates in unvaccinated elderly persons, and vaccination produces direct dollar savings.     

Genetic transformation of Vitreoscilla sp

Of all the methods customarily used to transform E. coli we found only electroporation to be effective for transformation of the Gram-negative bacterium Vitreoscilla, yielding 5.10(5) transformants/microgram of plasmid DNA. The conditions used were close to those described for E. coli E. coli plasmids are stably maintained in Vitreoscilla. This is the first report of exogenous DNA transfer in Vitreoscilla which opens the way for the application of recombinant-DNA techniques to study this unique group of organisms.