On the mechanism of biosynthesis of leukotrienes and related compounds

[10D-³H; 3-¹⁴C]- and [10L-³H; 3-¹⁴C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B₄ and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the ³H/¹⁴C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A₄ whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.     

Free Fatty Acids in the Rat Brain in Moderate and Severe Hypoxia

Abstract: The effects of mild, moderate, and severe hypoxia on cerebral cortical concentrations of free fatty acids (FFAs) were investigated in artificially ventilated rats under nitrous oxide anaesthesia. No change occurred during either mild (arterial Po₂ 35–40 mm Hg) or moderate (Po₂ 25–30 mm Hg) hypoxia. The effects of severe hypoxia (Po₂ about 20 mm Hg) combined with hypotension (mean arterial blood pressure 80–85 mm Hg) varied with the EEG pattern and the tissue energy state. Thus, a major increase in total as well as in individual FFAs occurred first when EEG was severely depressed (almost isoelectric) and energy homeostasis disrupted. On a relative basis the greatest change occurred in free arachidonic acid. It is concluded that hypoxia is associated with an increase in the concentrations of FFAs in brain tissue, provided that tissue oxygen deficiency is severe enough to cause tissue energy failure. However, an increase in FFAs does not invariably accompany minor reductions in the adenylate energy charge (EC) of the tissue.     

Cholinergic receptors in human hippocampus-- regional distribution and variance with age

The anterio-posterior distribution of cholinergic receptor binding sites in human hippocampus (five parts) as well as the effect of age (age range 3 days - 85 years) on receptor properties has been studied. Muscarinic binding sites was measured using labelled quinuclidinyl benzilate (³H-QNB) as ligand and labelled tubocurarine (³H-TC) was used for measurement of nicotine-like binding sites.The highest number of ³H-QNB binding sites in human hippocampus was measured at 3 days and 3 weeks of age and the lowest at 82 years of age. The proportion of high and low affinity muscarinic binding sites respectively was about the same at all ages investigated.A decrease in ³H-QNB binding sites with age was found in the anterior parts of the hippocampus (age range 55–84 years). When individual data for number of ³H-TC binding sites were plotted against corresponding number of ³H-QNB binding sites a strong correlation was observed in most of the different regions of the hippocampus.     

Regeneration of NADH with immobilized systems of alanine dehydrogenase and hydrogen dehydrogenase

Summary An alternative approach to the regeneration of coenzymes using immobilized hydrogen dehydrogenase (hydrogenase) is described. Hydrogenase isolated from Alcaligenes Eutrophus was immobilized to porous glass particles and used in combination with alanine dehydrogenase for formation of alanine, while the NADH consumed was regenerated by molecular hydrogen. Different physical arrangements of the two enzymes were compared. Alanine was conveniently assayed with a specially designed enzyme thermistor method.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

Structural studies of the capsular antigen of haemophilus infuenzae type c

The structure of the capsular antigen from Haemophilus influenza type c has been investigated, n.m.r. spectroscopy being the principal method used. It is concluded that the antigen is composed of repeating-units having the following structure:

O-Acetyl groups are present in ～90% of the repeating-units.     

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

Structural studies of the polysaccharide antigen of Eubacterium saburreum, Strain L 32

The structure of the polysaccharide antigen produced by Eubacterium saburreum, strain L 32, has been investigated. The principal methods used were methylation analysis, graded hydrolysis with acid, and n.m.r. spectroscopy. The polysaccharide, which contains the unusual sugar 3,6-dideoxy-D-arabino-hexose (tyvelose, Tyv), is composed of trisaccharide repeating-units having the following structure: