Variation of nitric oxide concentration during inspiration

OBJECTIVE: To evaluate the pattern of inspiratory nitric oxide concentration in a simple, constant flow delivery system during the use of two phasic-flow ventilatory modes. DESIGN: Laboratory study in a lung model. SETTING: University experimental laboratory. SUBJECT: Nitric oxide (800 ppm in nitrogen) was administered continuously into the inspiratory circuit to deliver a nitric oxide concentration of 10 and 40 ppm to a test lung during volume-controlled (constant flow) and pressure-controlled (decelerating flow) ventilation, with an FIO2 of 1.0. INTERVENTIONS: In each mode, minute ventilation of 7, 14, and 21 L/min and installation of mixing chambers (none, 1-L, 2-L, and 3.2-L turbulence boxes) were studied, respectively. Nitric oxide and nitric dioxide were monitored by chemiluminescence. Since the nitric oxide/nitrogen gas is the only nitrogen source in the system during ventilation with an FIO2 of 1.0, we evaluated the fluctuation in the inspiratory nitric oxide (NOx) concentration by measuring nitrogen with a fast-response analyzer. To test the effect of the measurement site, we measured nitric oxide concentrations using chemiluminescence at different positions in the inspiratory and expiratory limbs, with and without the mixing chambers, with a minute ventilation of 14 L/min and a nitric oxide concentration of 40 ppm. MEASUREMENTS AND MAIN RESULTS: Nitrogen dioxide production was not influenced by the flow pattern. During a nitric oxide concentration of 10 ppm, nitrogen dioxide was always < 0.6 ppm. During a nitric oxide concentration of 40 ppm, the highest nitrogen dioxide (4.47 ppm) concentration was found at the lowest minute ventilation and the largest inspiratory circuit volume. Nitric oxide values displayed by chemiluminescence indicated stable concentrations at all settings. However, without mixing chambers, NOx concentration calculated from nitrogen measurements demonstrated marked inspiratory fluctuations and was highest with a minute ventilation of 21 L/min and higher during pressure-controlled ventilation compared with volume-controlled ventilation (nitric oxide concentration of 40 ppm, pressure-controlled ventilation: 14.5 to 130.5 ppm; volume-controlled ventilation: 21.6 to 104.7 ppm; nitric oxide concentration of 10 ppm, pressure-controlled ventilation: 3.2 to 30.9 ppm; volume-controlled ventilation: 4.5 to 27.1 ppm). NOx concentration fluctuation decreased with an increasing mixing chamber, and was negligible at all settings with the 3.2-L turbulence box. Nitric oxide concentration fluctuation influenced chemiluminescence measurements. The displayed nitric oxide values varied, depending on the sampling site, and did not accurately reflect mean inspiratory nitric oxide concentration. Incorporation of a mixing chamber eradicated this sampling site influence. CONCLUSIONS: Continuous flow delivery of nitric oxide into the circuit of a phasic-flow ventilator results in marked inspiratory nitric oxide concentration fluctuation that is not detected by a slow-response chemiluminescence analyzer. Moreover, nitric oxide concentration fluctuation can influence the accuracy of the chemiluminescence measurements. These effects can be diminished by using additional mixing chambers to facilitate a stable gas concentration. As these mixing volumes increase the contact time of nitric oxide with oxygen, an increase of nitrogen dioxide has to be taken into account.     

Thymic alterations in feline GM1 gangliosidosis

GM1 gangliosidosis is an inherited metabolic disease characterized by progressive neurological deterioration with premature death seen in children and numerous animals, including cats. We have observed that thymuses from affected cats greater than seven months of age (GM1 mutant cats) show marked thymic reduction compared to age-matched normal cats. The studies reported here were done to describe alterations in the thymus prior to (less then 90 days of age) and during the development of mild (90 to 210 days of age) to severe (greater than 210 days of age) progressive neurologic disease and to explore the pathogenesis of the thymic abnormality. Although histologic examination of the thymus from GM1 affected cats less than 210 days of age showed no significant differences from age-matched control cats, thymuses from GM1 mutant cats greater than 210 days of age were significantly reduced in size (approximately 3-fold). Histologic sections of lymph nodes, adrenal glands, and spleens from GM1 gangliosidosis-affected cats showed no significant differences. Flow cytometric analyses showed a marked decrease in the percentage of immature CD4+CD8+ thymocytes (p < 0.001) and significantly increased CD4-CD8+ cells (p < 0.01) in GM1 mutant cats greater than 210 days of age when compared to normal age matched cats. Co-labelling with CD4, CD8, and CD5 indicated an increase in the percentage of GM1 mutant cat thymocytes at this age which were CD5high, suggesting the presence of more mature cells. Cytometric analyses of subpopulations of peripheral lymphocytes indicated an increase in CD4-CD8+ cells (p < 0.05) with concurrent decreases in CD4+CD8- and CD4-CD8- cells (which were not significant). Similar analyses of thymocyte and lymphocyte subpopulations from cats < 210 days of age showed no significant differences between GM1 mutant and normal cells. GM1 mutant cats at all ages had increased surface binding of Cholera toxin B on thymocytes, indicating increased surface GM1 ganglioside expression. Increases were highly significant in GM1 mutant cats greater than 210 days of age. In situ labelling for apoptosis was increased in GM1 mutant cats between 90 to 200 days of age when thymic masses were within normal limits. In GM1 mutant cats over 200 days of age, decreased labelling was observed when thymic mass was reduced and the CD4+CD8+ subpopulation, known to be very susceptible to apoptosis, was significantly decreased. These data describe premature thymic involution in feline GM1 gangliosidosis and suggest that increased surface GM1 gangliosides alters thymocyte development in these cats.     

Combinatorial manipulation of three key active site residues in glycinamide ribonucleotide transformylase

The enzyme glycinamide ribonucleotide transformylase (EC 2.1.2.2) has previously been shown to have three key polar active site residues important for catalysis: N106, H108 and D144. Mutations of any of these three residues lead to substantially decreased catalytic activity, although none of them are completely irreplaceable. In order to determine whether any alternative arrangement of amino acids at these three positions could lead to an active protein, all three of these residues were simultaneously subjected to saturation site-directed mutagenesis. The resulting combinatorial library of mutant genes was screened for those encoding active proteins using functional complementation. Glycinamide ribonucleotide transformylase was found to be capable of tolerating no more than one mutation amongst these key residues, since the only proteins found to be sufficiently active to allow growth of auxotrophic cells on selective media were the wild-type and enzymes containing a single mutation to one of these residues. It seems likely that no enzymes containing two or more mutations of these three residues possess significant catalytic activity. The combinatorial approach used could prove to be quite useful in protein engineering and protein evolution experiments.      

Cytokines in human breast milk

Out of the total number at 200 suspected cases of otomycoses consisting of 40 malnourished and 160 apparently healthy children examined in this study between the months of July and August in Edo State, 64 Cases (32%) were identified to be of fungal aetiology on the basis of positive culture and careful microscopic examination. The state at protein energy malnourishment was deterwined using physicians' comments in their case files. The fungal agents isolated were Aspergillus niger 28 (43.8%); A. fumigatus 4 (25%); Fusarium solari 4 (6.3%); Candida albicans 8 (12.5%); and Hendersonula teruloidea types torn B 5 (6.3%). Of these isolates, A. niger having an solation rate of (43.8%) was found to be the most predominant fungal species associated with otomycosis.     

Marked decrease of neuropeptide Y Y2 receptor binding sites in the hippocampus in murine prion disease

Using autoradiographic binding methodology with monoiodinated peptide YY together with the agonists neuropeptide Y (NPY) and NPY (13-36), as well as in situ hybridization with oligonucleotide probes complementary to the NPY Y2 receptor (Y2-R) mRNA, we have studied whether or not intracerebral prion inoculation affects Y2-Rs in male CD-1 mice. Monoiodinated peptide YY binding, mainly representing Y2-Rs, was down-regulated by 85% in the CA1 strata oriens and radiatum and by 50-65% in the CA3 stratum oriens 110-140 days postinoculation. In the CA3 stratum radiatum, where the mossy fibers from the dentate granule cells project, there was a significant decrease in PYY binding at 110-120 days. Y2-R mRNA, moderately expressed both in the CA1 and CA3 pyramidal cell layers and the granule cell layer in the dentate gyrus, showed a slight, but not significant, decrease in CA3 neurons 130 days postinoculation. The results indicate that the accumulation of the scrapie prion protein in the CA1-3 region strongly inhibits NPY binding at the Y2-Rs, which, however, is only marginally due to reduced Y2-R mRNA expression. The loss of the ability of NPY to bind to inhibitory Y2-Rs may cause dysfunction of hippocampal circuits and may contribute to the clinical symptoms in mouse scrapie.     

Acquired inhibitors to factors V and X after exposure to topical thrombin: interference with monitoring of low molecular weight heparin and warfarin

Repeated surgical exposure to topical bovine thrombin is known to be associated with the development of antibodies to bovine and human thrombin and factor V. This is demonstrated by abnormalities of in vitro coagulation assays and, rarely, postoperative bleeding. We describe a 4-year-old child in whom an antibody to bovine factor X developed after cardiac surgery; this antibody interfered with the heparin anti-Xa assay, thereby complicating the monitoring of heparin therapy.     

Mitochondrial aldehyde dehydrogenase polymorphism in Asian and American Indian populations: detection of new ALDH2 alleles

Genetic deficiency of the mitochondrial aldehyde dehydrogenase (ALDH2) is frequent in Asian peoples where it is an important factor negatively regulating drinking behavior. To obtain additional information on gene geography of known ALDH2 alleles, and look for new variants, ALDH2 genes were evaluated in a Chinese population from Taiwan, a Yakut population of Siberia, and in five North American Indian populations. A novel approach based on a single-strand conformation polymorphism assay, and polymerase chain reaction-directed mutagenesis was developed for genotyping. In the Taiwan Chinese population, the ALDH2(2) allele frequency was 0.319 +/- 0.025, and this allele was not detected in the Yakut population nor in the five North American Indian populations. However, a new allele, ALDH2(3), was detected in Pima Indians at a frequency of 0.044 +/- 0.022, and this allele was also observed in 1 of 49 Pueblo samples. ALDH2(3) is a silent transition 1464 G-->A, and it possibly has a wide distribution among North American Indians. A new subtype of the ALDH2(2) allele, designated as ALDH2(2Taiwan), was found in 1 of 174 Chinese from Taiwan. ALDH2(2Taiwan) is characterized by two G-->A transitions at bases 1486 and 1510, resulting in Glu-->Lys substitutions at both the 479 and 487 positions. Thus, this second nonconservative ALDH2 substitution occurs within the sequence of the already inactive ALDH2(2) allele.