Retinal degeneration induced by taurine deficiency in light-deprived cats.

The effect of light deprivation on the retinal degeneration induced by taurine deficiency in cats was examined in this study. After 25 weeks of taurine-free diet, taurine levels in plasma, retina, heart, cerebellar and cerebral cortex decrease to 16-25% of normal. The ERG a-wave and b-amplitudes also decreased as a consequence of the taurine-free diet. After 15 weeks of taurine-free diet the b-wave amplitude decreased to 36-41% of normal, and after 25 weeks both the a-wave and the b-wave were undetectable. The structure of the outer segments of photoreceptors, examined by electron microscopy appeared disturbed in the taurine-deficient cats, showing disorientation of the disk membranes, vesiculation and swelling. The time course of taurine deficiency and the retinal degeneration pattern were observed in all cats fed the taurine-free diet, irrespective of the conditions of lighting. These results indicate that the morphological and functional disturbances induced by taurine deficiency in cats are independent of the physiological stimulation of the retina by light.

Pharmacological identification of retinal cells releasing taurine by light stimulation.

The effect of drugs blocking synaptic activity at different retinal levels was examined in this study, in an attempt to identify the origin of the light-stimulated release of 3H-taurine from the chick retina. It was determined by autoradiography that the chick retina accumulates taurine in photoreceptors, in cells from the inner nuclear layer, and in processes of the inner plexiform layer. All these are possible sites for the release of taurine upon illumination. To discriminate among these possibilities, the effects of aspartate, tetrodotoxin, strychnine, picrotoxin, chlorpromazine, tubocurarine, atropine, glutamate diethyl esther, alpha-amino adipate and 2-amino-4-phosphonobutyrate were studied. Aspartate (10 mM), which is known to eliminate the light response of cells postsynaptic to photoreceptors, induced a marked increase of 150% in the resting efflux of 3H-taurine but did not decrease significantly the light-stimulated release. Tetrodotoxin, which blocks amacrine cell responses, decreased 3H-taurine release stimulated by light by less than 20%. The efflux of taurine was unaffected by strychnine, picrotoxin, tubocurarine, atropine, chlorpromazine, and 2-amino-4-phosphonobutyrate, whereas it was increased by glutamate diethyl esther and alpha-amino adipate. These results, all together, point to photoreceptors as the cells releasing 3H-taurine in response to light.

Physiology of ammonium assimilation in Neurospora crassa.

In Neurospora crassa the assimilation of high and low concentrations of ammonium occurs by two different pathways. When the fungi are growing exponentially on ammonium excess, this compound is fixed by a glutamic dehydrogenase and an octameric glutamine synthetase (GS). The synthesis of this GS polypeptide (beta) is regulated by the nitrogen source present in excess; being higher on glutamate, intermediate on ammonium, and lower on glutamine. When N. crassa is growing in fed-batch ammonium-limited cultures a different polypeptide of GS (alpha), arranged as a tetramer, is synthesized. In both conditions synthesis in vivo correlates with the data obtained with an in vitro translation system primed with N. crassa RNA. This different expression of alpha and beta GS polypeptides was also observed when the cultures were shifted from excess to low nitrogen, and vice versa. By agarose gel electrophoresis in the presence of methylmercury hydroxide, some separation of different mRNAs that direct the in vitro synthesis of alpha and beta GS polypeptides has been accomplished. Data are presented that establish the operation of the tetrameric alpha GS and of glutamate synthase in the assimilation of ammonium in low concentration.

Heterogeneity of glutamine synthetase polypeptides in Neurospora crassa.

Purified preparations of Neurospora crassa glutamine synthetase contain two nonidentical polypeptides that can be separated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 7 M urea. These polypeptides are synthesized both in vivo and in a heterologous cell-free protein-synthesizing system. The data presented indicate that both polypeptides contain an active site for glutamine synthetase activity and suggest that there is not a precursor-product relationship between them.

Nitrogen source regulates glutamine synthetase mRNA levels in Neurospora crassa.

Neurospora crassa glutamine synthetase mRNA was measured by its capacity to direct the synthesis of the specific protein in a cell-free system derived from rabbit reticulocytes. N. crassa cultures grown on glutamate as the sole nitrogen source had higher mRNA activities than did those grown on glutamine. The differences were about 10-fold when polysomal RNA was used for translation and about 5-fold when either total cellular RNA or polyadenylic acid-enriched cellular RNA was used. These data indicate that in exponentially growing N. crassa, the nitrogen source regulates glutamine synthetase by adjusting specific mRNA levels.

Neurospora crassa glutamine synthetase. Translation of specific messenger ribonucleic acid in a cell-free system derived from rabbit reticulocytes.

The total reticulocyte lysate cell-free protein-synthesizing system was incubated in the presence of Neurospora crassa RNA. With the aid of an antibody directed against purified N. crassa glutamine synthetase, the synthesis of a specific protein was detected. This protein precipitates with antiglutamine synthetase using both direct and indirect procedures, migrates with the same molecular weight as the monomer of N. crassa glutamine synthetase when subjected to acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and chromatographs as N. crassa glutamine synthetase on anthranilate-bound Sepharose. These data indicate the translation of the mRNA that codes for N. crassa glutamine synthetase. This RNA behaves as poly(A)-containing material when fractionated on oly(U)-Sepha-rose.