Stimulation of Free Fatty Acid and Diacylglycerol Accumulation in Cerebrum and Cerebellum During Bicuculline-Induced Status Epilepticus. Effect of Pretreatment with α-Methyl-p-Tyrosine and p-Chlorophenylalanine

The pool size and composition of free fatty acids (FFA) and diglycerides (DG) from the cerebrum and cerebellum of rats undergoing bicuculline-induced seizures were studied. A fourfold increase in cerebral FFA occurred 3-4 min after bicuculline injection; arachidonic and stearic acids were the principal fatty acids accumulated. Cerebellar FFA also increased, but to a lesser extent. An increased production of arachidonic acid took place in the cerebrum as a function of time after bicuculline injection. Other fatty acids produced were oleic, palmitic, and docosahexaenoic acids. A twofold increase in cerebral arachidonic acid was seen at the time of the first generalized tonic-clonic convulsion. However, a 13- to 17-fold increase in arachidonic acid was seen approximately 5-6 min after bicuculline injection. The rise in other FFA was much smaller. Stearoyl- and arachidonoyl-DG were also accumulated. The drug alpha-methyl-p-tyrosine was found to (a) potentiate the bicuculline-stimulated release of cerebellar FFA, and (b) inhibit by 70% the production of stearoyl- and arachidonoyl-DG in the cerebrum and cerebellum. Basal production of FFA was stimulated by p-chlorophenylalanine, but the drug had no effect on the bicuculline-induced changes. Hydrolysis of phospholipids enriched in stearoyl-arachidonoyl groups, such as phosphatidylinositol of excitable membranes, may be stimulated during seizures.     

The effects of bisulfite on growth and macromolecular synthesis in Escherichia coli

Bisulfite reversibly inhibits the growth of a variety of microorganisms and has been used as a preservative in foods and beverages for that reason. We have now measured macromolecule synthesis in Escherichia coli K12 after bisulfite treatment. RNA synthesis, the synthesis of total protein, and of an inducible enzyme, beta-galactosidase, stopped almost immediately upon addition of 2 mM (or higher concentrations) of bisulfite. These functions resumed after a lag whose duration depended on the concentration of bisulfite added. The synthesis of DNA was slowed upon bisulfite addition, but did not stop entirely. The inhibition of RNA synthesis by bisulfite took place in both stringent and relaxed strains of E. coli and was not relieved upon addition of chloramphenicol. Stringent control was therefore not involved in this effect. No effect on protein synthesis was observed in the cell-free system of E. coli (using poly(U) or MS2 RNA as messenger) at bisulfite concentrations up to 10 mM. Protein synthesis inhibition in vivo was apparently not due to a reaction of bisulfite with a component of this system. In additional experiments, RNA polymerase was not impaired by bisulfite, and the growth inhibition effect was shown to proceed in the presence of inhibitors of free radical chain reactions.     

Organic Acids of Ditylenchus triformis and Turbatrix aceti

Pyruvic acid, lactic acid and several tricarboxylic acid cycle acids were extracted from Ditylenchus triformis and Turbatrix aceti and identified. Fumaric acid was predominant in both nematodes. Small amounts o f malic and α-ketoglutaric acids and intermediate quantities o f lactic, citric, succinic, and pyruvic acids occurred in D. triformis. In T. aceti citric, lactic, and α-ketoglutaric acids were less abundant than succinic, malic and pyruvic acids. Only traces of aconitic and oxalacetic acids occurred in both nematodes. All the organic acids detected accounted for only about one per cent of the dry weight of nematodes o f the two species.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Growth of epithelium from a preneoplastic mammary outgrowth in response to mammary adipose tissue

Summary We investigated the effects of conditioned media derived from mouse mammary fat pads on the proliferation of CL-S1 cells, an epithelial cell line originally isolated from a preneoplastic mammary outgrowth line. Cell proliferation in vitro in serum-free defined medium was compared to that in this medium conditioned using intact mammary fat pad pieces or isolated fat pad adipocytes. Culture medium was conditioned by incubating the conditioning material in defined culture medium for 24 h at 37°C. Conditioned medium induced CL-S1 proliferation as much as 10- to 20-fold above the minimal levels of growth in control cultures after 13 d of culture. The growth-stimulatory factor(s) had an apparent molecular weight of greater than 10 kDa. This growth-stimulatory activity was both heat and trypsin stable. Because the role of adipose tissue is to store and release lipids, we next tested whether lipids are released during medium conditioning. The lipid composition of the fat pad conditioned medium was characterized using both thin layer and gas liquid chromatography. These lipid analyses indicated that the fat pad pieces released significant amounts of fatty acids and phospholipids into the medium during the conditioning period. The free fatty acid composition included both saturated and unsaturated molecules, and about 80% of the total fatty acids consisted of palmitate, stearate, oleate, and linoleate. These same fatty acids were a structural component of the majority of phospholipid found in the medium. The addition of palmitate or stearate to defined medium had no effect or was inhibitory for CL-S1 proliferation, depending on the concentration used. Defined medium supplemented with oleate, arachidonate, or linoleate induced CL-S1 proliferation, and the inhibitory effects of palmitate and stearate were overcome by addition of oleate and linoleate. These data indicate that both unsaturated and saturated fatty acids are released from intact adipose cells of the mouse mammary fat pad and that fatty acids can influence the growth of prenoplastic mouse mammary epithelium. Thus, unsaturated fatty acids, perhaps in conjunction with other substances released simultaneously, are candidate molecules for the substances that mediate the effect of adipose tissue on growth of epithelium. This work was supported in part by a grant from the American Institute for Cancer Research; grant CA 46885 from the National Institutes of Health, Bethesda, MD; and by State of Washington initiative 171.     

Metabolic Interactions Between Glucose, Glycerol, Alanine and Acetate in Leishmania braziliensis panamensis Promastigotes

¹³C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of ¹⁴CO₂ production from ¹⁴C-labeIed substrate. Cells incubated with [2-¹³C]glycerol released acetate, succinate and D-lactate in addition to CO₂. Cells incubated with acetate released only CO₂. More succinate C-2/C-3 than C-l/C-4 was released from both [2-¹³C]glycerol and [2-¹³C]glucose, indicating that succinate was formed predominantly by CO₂ fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of ¹⁴CO₂ production from [l,(3)-¹⁴C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased ¹⁴CO₂ production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of ¹⁴CO₂ production from [U-¹⁴C]- and [l-¹⁴C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool.     

In Vivo Substitution of Choline for Sodium Evokes a Selective Osmoinsensitive Increase of Extracellular Taurine in the Rat Hippocampus

Recent investigations have demonstrated that taurine and phosphoethanolamine (PEA) are the amino acids most sensitive to microdialysis-perfusion with reduced concentrations of NaCl. The aim of the present work was to assess the importance of Na+ deficiency in evoking this response. Further, the previously described selectivity of replacement of Cl- with acetate with respect to amino acid release was reinvestigated. The hippocampus of urethane-anesthetized rats was dialyzed with Krebs-Ringer bicarbonate buffer, and amino acid concentrations of the perfusate were determined. Choline chloride was then stepwise substituted for NaCl, and, in some cases, mannitol (122 mM) was included in low sodium-containing media. In other experiments, NaCl was replaced with sodium acetate. The dialysate levels of taurine increased selectively in response to Na+ substitution. The elevation of taurine was linearly related to the increase in choline chloride, and maximal levels amounted to 335% of basal levels. The increase in extracellular taurine was not inhibited by perfusion with medium made hyperosmotic with mannitol. Replacement of Cl- with acetate stimulated the release of taurine to 652% of resting levels. In addition, PEA levels increased to 250% of control concentration. Other amino acids were unaffected by Cl- substitution. The results show that taurine transport is considerably more sensitive to Na+ depletion than glutamate transport, which also is known to be Na+ dependent. The taurine increase evoked by low Na+ is not caused by cellular swelling as it was unaffected by hyperosmolar medium. Finally, substitution of acetate for Cl- causes a specific elevation of extracellular taurine and PEA, possibly as a result of cytotoxic edema.     

The Influence of water salinity on the free amino acid concentration in muscle and hepatopancreas of adult shrimps, Penaeus japonicus

Variations of the total free amino acid (FAA) pool and the content of specific amino acids have been measured in the muscle and hepatopancreas of adult shrimps, Penaeus japonicus, acclimatized at five water salinities: 38, 32, 26, 20 and 14%‰ The FAA content is always higher in muscle than in hepatopancreas at all tested salinites. On the other hand, the hepatopancreas exhibits the highest concentrations of essential amino acids. Two steps in the evolution of FAA content can be observed, the first one regarding decrease in salinity from 38 to 20%‰ and the second one, when salinity goes below 20%_°. The first step can be characterized by a 16% decrease of total FAA content in the muscle and a 36% increase in the hepatopancreas. In muscle, the variations are mainly due to changes in non-essential FAA content, whereas in the hepatopancreas, they are linked to variations in essential FAA content. The other step is characterized by a drastic increase in moisture and decrease in FAA content in both studied organs when water salinity is 14%‰ The total FAA content is about 40% lower in shrimps at 14%_° compared to 38%‰ seawater salinity. During adaptation, the FAA pool (mainly NEFAAs) of muscle seems to be directly related to osmoregulation, whereas in the hepatopancreas, its evolution seems to be linked with energy expenditure and protein synthesis. The results are evaluated in order to elucidate the role of FAA in intracellular osmoregulation and in relation to animal ecology.     

Influence of cytosolic pH changes on the organisation of the endoplasmic reticulum in epidermal cells of onion bulb scales: Acidification by loading with weak organic acids

Summary The anastomosing ER system of epidermal cells of onion bulb scales is composed of three modifications: lamellar and tubular elements, located in the cell periphery, and long tubular stands located deeper in the cytoplasm. Cytoplasmic acidification of epidermal cells by loading with weak organic acids like acetic or propionic acid causes the decay of the lamellar elements and the disappearance of long tubular strands. Organelle movement is also inhibited. The effects depend on the pH of the incubation medium and on the administered acid concentration, and are characterized by a distinct lag phase of about 7 min. The induced ER changes are transient with adaptation starting after about 50min. Buffer components alone have little influence on the cellular ER organization within a pH-range of 4.0–8.0. However, the pH of the medium strongly affects the time course of the effects as well as recovery after omitting the administered acid. Both modulation and recovery occur more rapidly at neutral or slightly alkaline pH. Actin filaments, which play a major role in ER organization and organelle movement, are not affected by cytosolic acidification.Dedicated to the memory of Professor Oswald Kiermayer     

Actions of Excitatory Amino Acids on Somatostatin Release from Cortical Neurons in Primary Cultures

L-Glutamate, N-methyl-D-aspartic acid (NMDA), quisqualate, and kainate were found to increase endogenous somatostatin release from primary cultures of rat cortical neurons in a dose-dependent manner. The rank order of potency calculated from the dose-response curves was quisqualate greater than glutamate = NMDA greater than kainate, with EC50 values of 0.4, 20, and 40 microM, respectively. Alanine, glutamine, and glycine did not modify the release of somatostatin. The stimulation of somatostatin release elicited by L-glutamate was Ca2+ dependent, was decreased by Mg2+, and was blocked by DL-amino-5-phosphonovaleric acid (APV) and thienylphencyclidine (TCP), two specific antagonists of NMDA receptors. The NMDA stimulatory effect was strongly inhibited by APV in a competitive manner (IC50 = 50 microM) and by TCP in a noncompetitive manner (IC50 = 90 nM). The release of somatostatin induced by the excitatory amino acid agonists was not blocked by tetrodotoxin (1 microM), a result suggesting that tetrodotoxin-sensitive, sodium-dependent action potentials are not involved in the effect. Somatostatin release in response to NMDA was potentiated by glycine, but the inhibitory strychnine-sensitive glycine receptor did not appear to be involved. Our data suggest that glutamate exerts its stimulatory action on somatostatin release essentially through an NMDA receptor subtype.