Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Lipid associated calcium ionophores in islet cell plasma membrane following glucose stimulation

The effect of glucose exposure on lipid associated calcium ionophoretic activity was measured in cultured neonatal rat pancreatic islet cells using two model systems. The first measured the ability of a lipid extract of islet cells to facilitate calcium transfer from an aqueous to organic phase and thus detected lipids which transfer calcium in the manner of authentic ionophores or which chelate the ion. In this system glucose stimulation was followed by an increase in total cell ionophoretic activity and a decrease in the activity associated with the plasma membrane. The second system measured the transfer of calcium across an artificial phospholipid membrane and detected authentic ionophoretic activity. In this model an increase in total ionophoretic activity was again seen following glucose but there was no change in the ionophoretic activity of a plasma membrane extract. The results indicate that the lipid modifications which accompany glucose-induced insulin release may alter cellular calcium stores by decreasing lipid bound calcium at the plasma membrane and increasing the capacity for calcium ionophoresis at intracellular sites.     

Cytogenetic studies in spontaneous abortuses

Summary In a series of 450 products of conception received for cytogenetic analysis, tissue culture was attempted on 309, and karyotypes were established using banding techniques in 154 singleton specimens. Abnormalities of karyotype were identified in 19%; of these abnormalities, 48% were autosomal trisomies. Gestational age was decreased in the abnormal specimens, and their developmental age was retarded by comparison with their gestational age. Factors contributing to the relatively low incidence of abnormality are examined. The major factor appears to be the clinical interest of collaborating staff, leading to selection, either intentional or unintentional, of particular phenotypes and hence a non-random series. A negative relationship is suggested between frequency of monsomy X and autosomal trisomy, both being associated with maternal age.     

Magnetic separation in biotechnology

New developments in magnetic labelling techniques for cells and microspheres have extended the useful range of magnetic separation, particularly high gradient magnetic separation, into biotechnical areas. The basic magnetic principles involved are reviewed and representative samples of labelling techniques and results drawn from the past three years are presented. Illustrative examples of large scale operation in other industries are also presented, demonstrating the potential of the biotechnological applications.     

Nitrogen catabolite repression in a glutamate auxotroph of Saccharomyces cerevisiae

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is subject to nitrogen catabolite repression. In the present study we examined the physiological effects of glutamate auxotrophy on cellular metabolism and on the nitrogen catabolite repression of asparaginase II. Glutamate auxotrophic cells, incubated without a glutamate supplement, had a diminished internal pool of alpha-ketoglutarate and a concomitant inability to equilibrate ammonium ion with alpha-amino nitrogen. In the glutamate auxotroph, asparaginase II biosynthesis exhibited a decreased sensitivity to nitrogen catabolite repression by ammonium ion but normal sensitivity to nitrogen catabolite repression by all amino acids tested.     

The regulation of K+ influx in excised barley roots

The electrochemical potential differences for potassium, between excised barley (Hordeum vulgare L.) roots and external media containing 0.05 mM KCl+0.5 mM CaSO₄, were determined over a 4-h period during which initially low-K⁺ roots accumulated K⁺ by pretreatment in 50 mM KCl plus 0.5 mM CaCl₂. This pretreatment resulted in increased internal [K⁺], decreased K⁺ influx (as measured from 0.05 mM KCl+0.5 mM CaSO₄) and decreased values of . These observations indicate that the decline of K⁺ influx associated with increased internal K⁺ concentration cannot be accounted for by passive adjustment to the electrochemical gradient for this ion.     

The influence of potassium content on the kinetics of potassium influx into excised ryegrass and barley roots

The influx of K⁺ into excised roots of barley (Hordeum vulgare L.) and ryegrass (Lolium multiflorum L.) previously grown with or without K⁺ was measured in K⁺ solutions ranging in concentration from 0.01 to 50 mM. In both species the K⁺ influx was lower in the roots with high K⁺ content. The extent of reduction by high internal [K⁺] decreased with external concentration above 1 mM. These results support the contention that at high external concentrations passive diffusion makes significant contributions to observed fluxes.     

L-Asparaginase of Saccharomyces cerevisiae: an extracellular Enzyme.

During recent studies conducted with suspensions of three strains of Saccharomyces cerevisiae, it was observed that ammonia was rapidly liberated when L-asparagine was added to the medium. Subsequent investigation has revealed that these strains of S. cerevisiae have an externally active asparaginase as well as an internally active one. The appearance of the external asparaginase is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The internal enzyme appears to be constitutive. The external activity is relatively insensitive to para-hydroxymercuribenzoate inhibition, whereas the internal activity is highly inhibited by this compound.