Circular dichroism detection in high-performance liquid chromatography: Evaluation of the anisotropy factor

The application of a circular dichroism (c.d.) detection system in HPLC using a chiral stationary phase is presented. The simultaneous measurement of the absorbance and c.d. signal allows the evaluation of the anisotropy factor (g = Δ/) and thus the determination of the enantiomeric excess (e.e.) of the eluates. When this detection system is used in preparative chiral chromatography the collection of the enantiomeric fractions can be readily optimized.     

Conjugation pathways in liver disease.

1. The activities of microsomal glucuronyltransferase and thiomethyltransferase, and those of cytosolic sulphotransferase, acetyltransferase, glutathione transferase and thiomethyltransferase were measured in abnormal (cirrhosis and chronic hepatitis) and normal livers. 2. Glucuronyltransferase and sulphotransferase were investigated with 2-naphthol and ethinyloestradiol as substrates. p-Aminobenzoic acid, benzo(a)pyrene-4,5-epoxide and 2-mercaptoethanol were the substrates of acetyltransferase, glutathione transferase and thiomethyltransferase, respectively. 3. Enzyme activities are expressed as nmol min-1 incubation mg-1 protein and the averages (+/- s.d.) are given. With 2-naphthol as substrate, the glucuronyltransferase activity was 6.55 +/- 4.10 (abnormal liver, n = 33) and 7.81 +/- 4.02 (normal liver, n = 26) (NS); whereas sulphotransferase activity was 0.28 +/- 0.18 (abnormal liver, n = 35) and 0.68 +/- 0.43 (normal liver, n = 26) (P less than 0.01). Glucuronyltransferase activity towards ethinyloestradiol was 102.5 +/- 56.9 (abnormal liver, n = 30) and 107 +/- 59.9 (normal liver, n = 26) (NS), whereas sulphotransferase activity was 57.2 +/- 36.0 (abnormal liver, n = 35) and 122 +/- 67.6 (normal liver, n = 28) (P less than 0.01). Acetyltransferase activity was 0.84 +/- 0.83 (abnormal liver, n = 35) and 3.84 +/- 1.65 (normal liver, n = 26) (P less than 0.01). Glutathione transferase activity was 0.83 +/- 0.68 (abnormal liver, n = 35) and 2.90 +/- 1.59 (normal liver, n = 25) (P less than 0.01) and thiomethyltransferase activity was 1.00 +/- 0.69 (abnormal liver, n = 34) and 3.99 +/- 1.49 (normal liver, n = 25) (P less than 0.01). 4. Liver disease lowers the activities towards the substrates studied of sulphotransferase, acetyltransferase, glutathionetransferase and thiomethyltransferase but not that of glucuronyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methaemoglobin formation in the koala and the possum

Measurements were made of glutathione (GSH) levels, catalase activity and the oxidant sensitivity of the erythrocytes from the koala (Phascolarctos cinereus) and the common brushtail possum (Trichosurus vulpecula). The oxidant sensitivity was tested by treating the haemolysates with either 0.55 him H₂O₂ or 1.4mm NaNO₂. The erythrocytes of the koala had greater levels of GSH and catalase and yet were found to be more susceptible to oxidation induced by both these oxidants.     

Bradykinin-induced release of PGI2 from aortic endothelial cell lines: responses mediated selectively by Ca2+ ions or a staurosporine-sensitive kinase.

1. Bradykinin (100 nM) triggers release of nitric oxide and prostacyclin from both AG07680A and AG04762 bovine cultured aortic endothelial cells. The exposure of these cells to bradykinin is in each case associated with a striking rise in intracellular calcium ion concentration. 2. Exposure of AG07680A cells to 250 nM ionomycin was followed also by a significant release of prostacyclin, whereas 250 nM ionomycin had no capacity to stimulate release of prostacyclin from AG04762 cells. 3. There was a similar concentration-dependent increase in intracellular calcium ion concentration on exposure of AG07680A and AG04762 cells to ionomycin. 4. Exposure of AG04762 cells for 10 min to staurosporine produced a concentration-dependent inhibition (IC50 = 107 +/- 14 nM) in bradykinin-stimulated prostacyclin release. There was no similar inhibitory effect of staurosporine in AG07680A cells. 5. Bradykinin (10 nM) triggered release of nitric oxide from both AG07680A and AG04762 cells, and the effect was not inhibited by 500 nM staurosporine. There was a similar ionomycin-dependent release of nitric oxide from both cell types. 6. These results identify a common pathway for bradykinin-dependent nitric oxide release from both AG07680A and AG04762 cells, involving increases in intracellular calcium ion concentration. In contrast, the bradykinin-dependent release of prostacyclin may involve one of two pathways (involving an increase in intracellular calcium or activation of a staurosporine-sensitive kinase), and the two pathways are selectively exploited in AG07680A and AG04762 cells, respectively.     

Rational diagnosis and treatment in unclassified arthritis: how clinical data may guide requests for Lyme serology and antibiotic treatment.

To improve the appropriateness and efficiency of diagnostic serological tests and subsequent antibiotic treatment, clinical data from 102 patients with unclassified arthritis were analysed to investigate whether the presence of positive IgG antibodies to Borrelia burgdorferi could be predicted. The clinical data were blindly ranked from 1 to 4 (1, Lyme arthritis unlikely; 4, Lyme arthritis very likely). Antibodies to B burgdorferi were positive in nine of 102 patients (9%). Six of 15 (40%) patients with rank numbers 3 and 4 were positive for antibodies to B burgdorferi, in contrast with only three of 87 (3%) patients with rank numbers 1 and 2. The likelihood ratio of positive Lyme serology for patients ranked 3 and 4 was 12.0, for patients ranked 2 to 4, 4.5, and for patients with arthritis of the knee, 3.0. These likelihood ratios were associated with a post-test probability of 55, 30, and 20% respectively. The clinical history in patients with unclassified arthritis can largely predict the presence of antibodies to B burgdorferi. The absolute value of a likelihood ratio can be a contributing factor in deciding to request tests for antibodies to B burgdorferi in patients with unclassified arthritis.     

Differential distribution of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat forebrain

Summary Contradictory immunohistochemical data have been reported on the localization of N-acetylaspartylglutamate in the rat forebrain, using different carbodiimide fixation protocols and antibody purification methods. In one case, N-acetylaspartylglutamate immunoreactivity was observed in apparent interneurons throughout all allocortical and isocortical regions, suggesting possible colocalization with GABA. In another case, strong immunoreactivity was observed in numerous pyramidal cells in neocortex and hippocampus, suggesting colocalization with glutamate or aspartate. Reconciling these disparate findings is crucial to understanding the role of N-acetylaspartylglutamate in nervous system function. Antibodies to N-acetylaspartylglutamate and a structurally related molecule, N-acetylaspartate, were purified in stages, and their cross-reactivities with protein conjugates of N-acetylaspartylglutamate and N-acetylaspartate were monitored at each stage by solidphase immunoassay. Reduction of the cross-reactivity of the anti-N-acetylaspartylglutamate antibodies to N-acetylaspartateprotein conjugates to about 1% eliminated significant staining of most pyramidal neurons in the rat forebrain. Utilizing highly purified antibodies, the distributions of N-acetylaspartylglutamate and N-acetylaspartate were examined in several major telencephalic and diencephalic regions of the rat, and were found to be distinct. N-acetylaspartylglutamate-immunoreactivity was observed in specific neuronal populations, including many groups thought to use GABA as a neurotransmitter. Among these were the globus pallidus, ventral pallidum, entopeducular nucleus, thalamic reticular nucleus, and scattered non-pyramidal neurons in all layers of isocortex and allocortex. N-acetylaspartate-immunoreactivity was more broadly distributed than N-acetylaspartylglutamate-immunoreactivity in the rat forebrain, appearing strongest in many pyramidal neurons. Although N-acetylaspartate-immunoreactivity was found in most neurons, it exhibited a great range of intensities between different neuronal types.