Simultaneous measurement of O2 radicals and pulmonary vascular reactivity in rat lung

The role of endogenous radicals in the regulation of pulmonary vascular tone was evaluated by simultaneous measurement of pulmonary artery pressure and lung radical levels during exposure of isolated rat lungs to varying inspired O2 concentrations (0-95%) and angiotensin II. Lung radical levels, measured "on-line" using luminol and lucigenin-enhanced chemiluminescence, decreased in proportion to the degree of alveolar hypoxia. Radical levels fell during hypoxia before the onset of pulmonary vasoconstriction and promptly returned to basal levels with restoration of normoxic ventilation. Mild alveolar hypoxia (10% O2), which failed to decrease chemiluminescence, did not trigger pulmonary vasoconstriction. Although chemiluminescence tended to decrease more as the hypoxic response strengthened, there was not a simple correlation between the magnitude of the change in chemiluminescence induced by hypoxia and the strength of the hypoxic pressor response. Normoxic chemiluminescence was largely inhibited by superoxide dismutase but not catalase. Superoxide dismutase also increased normoxic pulmonary vascular tone and the strength of the pressor response to hypoxia and angiotensin II. Thus the predominant activated O2 species in the lung, during normoxia, was the superoxide anion or a closely related substance. Alteration of endogenous radical levels can result in changes in vascular tone. It remains uncertain whether the decrease in lung radical production during hypoxia caused pulmonary vasoconstriction or was merely associated with hypoxic ventilation.     

Non-secretion of blood group antigens and susceptibility to infection by Candida species

One of the innate defences against superficial infections by Candida species appears to be the ability of an individual to secrete the water-soluble form of his ABO blood group antigens into body fluids. There was a significantly higher number of non-secretors (48.9%) among 174 patients with either oral or vaginal candida infections compared with the proportion of non-secretors in the local population (26.6%). The protective effect afforded by the secretor gene might be due to the ability of glycocompounds in the body fluids of secretors to inhibit adhesins on the surface of the yeast. In attachment studies, preincubation of blastospores with boiled secretor saliva significantly reduced their ability to bind to epithelial cells. Non-secretor saliva did not reduce the binding and often enhanced the numbers of attached yeasts. Possible host-parasite interactions underlying the susceptibility of non-secretors to candida and other infections are discussed.     

Standard specimens for stain calibration: application to Romanowsky-Giemsa staining.

Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.     

The quantitative analysis of endogenous folate catabolites in human urine.

In man folates are catabolized and excreted as inactive cleaved degradation products, a mixture of pteridines and p-aminobenzoylglutamate (pABGlu) or its acetamido derivative (apABGlu). The daily rate of excretion represents the inescapable use of the vitamin in metabolic activity and thus has implications for determining the recommended dietary allowance for the vitamin. Furthermore, the rate of catabolism has been suggested to rise during pregnancy and in certain disease states. A method is described for the quantitative extraction and assay of the folate catabolites pABGlu and apABGlu in human urine. Aliquots of 24-h urine collections are acidified and applied to columns of Dowex 50W cation-exchange resin. The catabolites are selectively batch-eluted with increasing concentrations of HCl. The fraction containing pABGlu is diazotized and then applied to a C18 Sep Pak column for further purification and concentration. The fraction containing apABGlu was deacetylated and reapplied to the Dowex column and then treated identically to the pABGlu fraction. The methanolic concentrates of both extracts were evaporated to dryness and reconstituted with water and pABGlu was regenerated by reductive cleavage of the diazotized material with Zn/HCl. The extracts of the two catabolites were separated by reverse-phase HPLC using a Radial Pak C18 column. Recovery of isolated material was monitored by the addition of high specific activity tritiated labels of both compounds added as internal standards to all urine aliquots prior to purification and analysis.     

The effects of hydrocortisone on lung structure in fetal lambs

The effect of cortisol infusion on fetal lung development was studied in lambs. Changes were compared with those of control groups of saline-infused fetuses of the same age (day 132) and normal late gestation fetuses (142 +/- 4.6 days). Cortisol was infused into five fetal lambs at 129 days of gestation at a rate of 17.0 mg/day. Four fetuses were delivered by hysterotomy at the onset of labour-like uterine activity (58 +/- 3 h). In cortisol-infused fetuses the concentration of cortisol in fetal plasma and tracheal fluid rose to levels similar to those in normal fetuses during the last week of gestation. Progesterone concentration in maternal plasma declined at about 48 h after the start of treatment. Cortisol-infused lambs showed increases in fixed lung volume, specific lung volume, absolute volume of both parenchyma and non-parenchyma and the proportion of the parenchyma which was potential airspace and a decrease in the proportion of parenchyma. For cortisol-infused lambs Type II cell size and the abundance of lamellar bodies, and the volume fraction of cell occupied by the nucleus were similar to the 142 day group, whereas Golgi apparatus and RER were closer to age matched saline-infused (day 132) controls. Glycogen content was midway between the two control groups. We conclude that infusion of cortisol for about 60 h at physiological levels, beginning at 0.85 of gestation, accelerates many, but not all aspects of pulmonary parenchymal maturation, expressed in terms either of morphogenesis of the gas exchange area or differentiation of Type II alveolar cells.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Entry kinetics and mouse virulence of Ross River virus mutants altered in neutralization epitopes.

Previously we identified the locations of three neutralization epitopes (a, b1 and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were found to have single amino acid substitutions in the E2 protein (S. Vrati, C.A. Fernon, L. Dalgarno, and R.C. Weir, Virology 162:346-353, 1988). We have now studied the biological properties of these variants in BHK cells and their virulence in mice. While variants altered in epitopes a and/or b1 showed no difference, variants altered in epitope b2, including a triple variant altered in epitopes a, b1, and b2, showed rapid penetration but retarded kinetics of growth and RNA and protein synthesis in BHK cells compared with RRV T48, the parent virus. Variants altered in epitopes a and/or b1 showed no change in mouse virulence. However, two of the six epitope b2 variants examined had attenuated mouse virulence. They had a four- to fivefold-higher 50% lethal dose (LD50), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues similar to those of RRV T48. The ID50 of the triple variant was unchanged, but infected mice had an increased average survival time. This variant produced lower levels of viremia in infected mice. On the basis of these findings we propose that both the receptor binding site and neutralization epitopes of RRV are nearby or in the same domain of the E2 protein.     

Diallel analysis for sex-linked and maternal effects

Genetic models including sex-linked and maternal effects as well as autosomal gene effects are described. Monte Carlo simulations were conducted to compare efficiencies of estimation by minimum norm quadratic unbiased estimation (MINQUE) and restricted maximum likelihood (REML) methods. MINQUE(1), which has 1 for all prior values, has a similar efficiency to MINQUE(), which requires prior estimates of parameter values. MINQUE(1) has the advantage over REML of unbiased estimation and convenient computation. An adjusted unbiased prediction (AUP) method is developed for predicting random genetic effects. AUP is desirable for its easy computation and unbiasedness of both mean and variance of predictors. The jackknife procedure is appropriate for estimating the sampling variances of estimated variances (or covariances) and of predicted genetic effects. A t-test based on jackknife variances is applicable for detecting significance of variation. Worked examples from mice and silkworm data are given in order to demonstrate variance and covariance estimation and genetic effect prediction.     

Establishing the robustness of short-tandem-repeat statistics for forensic applications.

Before the introduction of a four-locus multiplex short-tandem-repeat (STR) system into casework, an extensive series of tests were carried out to determine robust procedures for assessing the evidential value of a match between crime and suspect samples. Twelve databases were analyzed from the three main ethnic groups encountered in casework in the United Kingdom: Caucasians, Afro-Caribbeans, and Asians from the Indian subcontinent. Independence tests resulted in a number of significant results, and the impact that these might have on forensic casework was investigated. It is demonstrated that previously published methods provide a simple procedure for correcting allele frequencies--and that this leads to conservative casework estimates of evidential value.