Functional significance of the activities of glutaminase and ornithine-ω-aminotransferase in rat brain

The activities of glutaminase, glutamine synthetase (GS), arginase and ornithine amino transferase (orn-T) were studied in three regions of rat brain in heightened neuronal activity by producing convulsions by leptazol. These enzymes were studied in preconvulsive, convulsive and postconvulsive phases. Glutaminase activity was found to increase in all the three regions in the preconvulsive and convulsive phases. GS activity decreased in the preconvulsive phase but rose gradually to the control level when the postconvulsive phase was reached. The activity of arginase decreased in the cerebellum in preconvulsive and convulsive phases. However, in the cerebral cortex there was a decrease in the activity of this enzyme only in the convulsive phase. The results suggest that glutamine acts more likely as a precursor for the neurotransmitter pool of glutamate, while ornithine serves more as a precursor for the neurotransmitter pool of GABA.     

Opioid-specific recognition sites of the mu- and the delta-type in rat striatum after lesions with kainic acid

The binding of 3H-dihydromorphine (3H-DHM) and of 3H-D-ala2, D-leu5-enkephalin (3H-DADL), which are regarded as relatively selective ligands for mu- or delta-type opioid receptors, respectively, was estimated in total particulate fraction of the striatum of rats in vitro, either in tissue of rats after striatal chemolesions with kainic acid or in control rats (not operated or saline injected into the striatum). Kainate lesions reduced the Bmax values of 3H-DHM by about 78 - 88% depending on the method of calculation, and of 3H-DADL by greater than 90%. Furthermore they lowered the Kd-values, suggesting an increase in affinity. The results are discussed with regard to recent hypotheses on the structure and function of opioid receptors.     

Binding equations for interacting systems comprising multivalent acceptor and bivalent ligand: application to antigen-antibody systems

Consideration is given to the reversible interaction of a bivalent ligand, B, with a multivalent acceptor, A (possessing f reactive sites) which leads to the formation of a series of complexes, A_iB_j, comprising networks of alternating acceptor and ligand molecules. A binding equation is derived on the basis of a site association constant, k, defined in terms of reacted site probability functions. This equation, which relates the binding function, r (the moles of ligand bound per mole of acceptor) to the concentration of unbound ligand, m_b, is used to show that plots of r vs. 2km_B constructed with fixed but different values of $\text{k}\text{m}{}_{\mathrm{A}}$ intersect at the point ($\text{m}{}_{\mathrm{B}}\text{=}\text{1}\text{2k}\text{, r =}\text{f}\text{2}$) where the extent of reaction and the concentrations of those complexes for which $\text{j}\text{i}\text{=}\text{f}\text{2}$ attain maximal values. Corresponding Scatchard plots are shown by numerical example to be non-linear, their second derivative being positive for all r. It follows that such deviations from linearity cannot be taken alone as evidence for site heterogeneity in cross-linking systems. The binding equation obtained directly is shown to be identical with that obtained with f = 2 by summation procedures involving the general expression for concentrations of complexes, m_AiBj, formulated in terms of appropriate statistical factors. In this way, previous findings on precipitation and gel formation in cross-linking systems are correlated with the present development of binding theory.     

Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae

The escape of several enzymes from “ammonia catabolite repression” in gdhA^? (NADP-linked glutamate-dehydrogenase-less) mutants, as well as in gdhCR mutants of Saccharomyces cerevisiae, does not involve glutamine synthetase, either as a positive or as a negative control element. A glutamine-synthetase-less mutant (gln^?) was used in this demonstration.In addition to its derepressing effect on the NAD-linked glutamate dehydrogenase, the gdhCR mutation releases “nitrogen catabolite repression” on arginase and allatoinase, as well as glutamine repression on glutamine synthetase. A gdhCS mutation was used to demonstrate that these effects are not mediated through the NAD-linked glutamate dehydrogenase.     

The potent induction of intestinal heme oxygenase by the organotin compound, bis(tri-n-butyltin)oxide

Oral administration of bis(tri-n-butyltin)oxide, an important organotin biocidal agent, produces a substantial elevation in heme oxygenase activity when measured at 16 hours in rat small intestine. An apparent Km for hemin of 100 microM is the same in both control and the organotin-induced 9,000 X g supernatant preparations. Concomitant with elevated heme oxygenase activity there occurs a substantial reduction in benzo(a)pyrene hydroxylase activity (approximately 20% of controls) and cytochrome P-450 concentration (approximately 60% of controls). These perturbations of heme metabolism in intestinal epithelium of the rat define an important new toxicological effect of organotins and raise the possibility that concurrent oral ingestion of environmental pollutants can directly affect the cytochrome P-450-dependent metabolism of other chemicals in the intestine.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. II. ACTH-induced changes in subcellular corticosterone

Zona fasciculata-reticularis subcellular structures were implicated in corticosterone transport and secretion by noting changes in subcellular corticosterone during a 30-min period following ACTH stimulation. Six decapsulated adrenal homogenate subcellular fractions separated by gradient centrifugation were characterized cytochemically and morphologically. Predominant components in each of six fractions were: floating lipid droplets, 0.125 M sucrose (no organelles), cytosol (0.25 M sucrose supernatant with 0.25-1.2 micron electron dense granules), microsomes (interface between 0.5 M and 1.1 M sucrose layers), mitochondria (boundary between 1.1 M and 2.2 M sucrose layers) and nuclei (centrifuge pellet). Whole glands and most subcellular fractions showed peak corticosterone levels 10 to 15, and 30 min after stimulation. Sucrose and cytosolic fractions contained about 75% of the total corticosterone, responded to stimulation most significantly, and were rich in protein. In these two fractions only cytosol contained structures; these consisted of 0.15-1.2 micron electron dense granules.     

An examination of the forms of scatchard plots of binding results outside domains of sigmoidality

General expressions are formulated for the first and second derivatives of the Scatchard function, r/[S], with respect to the binding function, r, from an equation that describes the binding of a ligand to a two-state acceptor system (either isomerizing or polymerizing). The expressions are utilized to determine the sign of the second derivative for particular systems under conditions where the first derivative is negative for all r. The work therefore correlates with previous studies, which stressed conditions of existence of critical points in Scatchard plots, by examining more fully possible forms of binding curves outside such domains of sigmoidality. Particular attention is given to the condition, d(r/[S])/dr < 0 and d²(r/[S])/dr² > 0 for all r (which defines a Scatchard plot convex to the r-axis). In agreement with previous findings it is proven that the isomerizing acceptor model cannot give rise to this form of plot and is therefore distinguished from negatively co-operative allosteric models. On the other hand, the polymerizing acceptor model may yield such a Scatchard plot, a feature demonstrated by formulating explicit conditions for its manifestation when ligand binding is exclusive to the polymeric state, and illustrated numerically for a system in which ligand binds to both oligomeric states. Distinction between such systems and those exhibiting negative co-operativity is possible on the basis of the Scatchard plots, which exhibit dependence on acceptor concentration in the case of a polymerizing acceptor; indeed, an example is provided where variation of acceptor concentration for a system characterized by fixed interaction parameters effects a conversion from sigmoidal binding behaviour to that typified by a Scatchard plot convex to the r-axis.     

Effect of thyroxine and thiourea on cholesterol total lipid and glycogen contents of brain of Singi fish (Heteropneustes fossilis bloch)

Three consecutive days injections of thyroxine of different doses (1, 2 and 4 μg/g of body weight) caused significant increase in cholesterol content of cerebrum of Singi fish at 25°C in comparison to the control. The cholesterol content of cerebellum, midbrain and medulla oblongata was enhanced significantly with higher doses of 2 and 4 μg of thyroxine per g of body weight. The lipid and glycogen contents of whole brain were also found to increase with different doses of thyroxine after three consecutive days injections. These cellular constituents decreased with hypothyroid condition induced by thiourea treatment.The results indicate the thyroid hormonal regulation of lipid and carbohydrate metabolism in brain of Singi fish.     

The expression of alpha-fetoprotein and albumin genes in rat liver during chemical carcinogenesis

The effect of the hepatocarcinogen 3′-methyl-4-dimethylaminoazobenzene on α-fetoprotein (AFP) and albumin gene expression in rat liver was studied. Serum concentrations of AFP and albumin were measured. Amounts of AFP mRNA and albumin mRNA in rat livers were determined by hybridization of total cytoplasmic RNAs to their cDNAs. Dramatic increases in serum AFP concentrations coincided with increases in AFP biosynthesis and amount of AFP mRNA in livers of carcinogen-treated rats. In contrast, no or little change in albumin mRNA concentration was found in livers of rats treated with 3′-methyl-4-dimethylaminoazobenzene. Concomitantly, there was little change in liver albumin biosynthesis or serum albumin concentrations during hepatocarcinogenesis.     

The role of superoxide and hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and by ascorbic acid

Purified commercial hyaluronic acid contains significant amounts of iron. Addition of Fe2+ to solutions of it causes depolymerization, which is inhibited by catalase and scavengers of the hydroxyl radical (. OH) but not by superoxide dismutase. Fe3+ is ineffective. Ascorbic acid also depolymerizes hyaluronic acid, apparently because it can reduce Fe3+ in the reaction mixtures to Fe2+. Ascorbate-induced depolymerization is inhibited by the specific iron chelator desferrioxamine, by catalase, and by scavengers of the hydroxyl radical. The relevance of these observations to rheumatoid arthritis and inflammatory joint diseases is discussed.     

An approach to the identification of adenosine''s inhibitory site on adenylate cyclase

The separate and combined effects of molybdate and dithiothreitol on the stability of human uterine 9 S estrogen receptor were studied. Maximal, short-term, protection of the 9 S estrogen receptor was achieved by the joint inclusion of both stabilizing agents in cytosol buffers. This molybdate—dithiothreitol-mediated stability was dependent on reducing agent concentration inferring sulphydryl involvement in 9 S receptor protection by molybdate. The study also showed that molybdate—dithiothreitol could not prevent the gradual decay of the 9 S estrogen receptor to the 4 S form in cytosols stored at 4°C over prolonged periods.     

Ultrastructure-function correlative studies for cardiac cryopreservation. V. Absence of a correlation between electrolyte toxicity and cryoinjury in the slowly frozen,cryoprotected rat heart

Hearts were frozen to ?17 °C in the initial presence of 2.1 m DMSO. Attempts were made to prevent or minimize the consequences of an osmotic shock based on Lovelock's classical hypothesis of freezing injury. Substitution of mannitol or potassium for NaCl before freezing did not improve the results, nor did perfusion of thawed hearts with hyperosmotic perfusate. It was found that freezing and thawing resulted in a significant attenuation of coronary flow and that, as a result of this, DMSO was apparently retained within the heart after thawing. DMSO was also difficult to remove at 30 °C in the absence of prior freezing and caused a significant drop in coronary flow upon institution of DMSO washout with balanced salt solution. The blanching of freezing and thawing was also seen, in milder form, in nonfrozen hearts. For both frozen-thawed and nonfrozen hearts, the blanching was associated with DMSO washout with balanced salt solution. Flow was improved by perfusion with hyperosmotic perfusate in both nonfrozen and in frozen-thawed hearts, but the improvement was largely temporary. Evidence from earlier studies indicates that electrolyte concentrations during freezing cannot be correlated with cardiac cryoinjury, in support of the present findings. It is suggested instead that cryoprotectant toxicity may be the chief agent of injury under the conditions studied.     

Developmental profile of three enolase isozymes in rat brain determination from one-cell embryo to adult brain

Levels of three enolase isozymes (αα, αγ and γγ) were determined in rat tissues from one-cell embryo to adult brain with a sensitive enzyme immunoassay system. Each embryo of the early stage (gestational age, 0–3 days) contained about 5 × 10^?17 mol of αα enolase. The nervous system-specific αγ and γγ enolases would be detected in the embryos of 6–8 days, which contain no histologically recognizable neurones. The 8-day embryos contained 4.3 × 10^?17 and 3.4 × 10^?16 mol of αγ and γγ enolases. Amounts of all the three enolases were increased with growth of the embryo. The nervous system-specific enolases (αγ and γγ) in the brain kept increasing until 1–2 months of postnatal age, whereas the αα enolase level in the brain was relatively constant after the 15-day embryo through the adult rat.     

Release of cholecystokinin-like immunoreactivity from slices of dorsal and ventral striatum of rat brain

Dorsal and ventral striatum may differ in their neuronal organisation and function. In a comparative in vitro study, we investigated the release of cholecystokinin-like immunoreactivity from slices of dorsal and ventral striatum, respectively. Release of immunoreactivity was induced by veratridine. The dopamine D₂-receptor agonist RU 24926 enhanced, while substance P reduced the release from slices of dorsal striatum. The two agents had no effect on the release of immunoreactivity from slices of ventral striatum. It is concluded that the discrepancy in the modulation of the release of cholecystokinin-like immunoreactivity reflects differences in neuronal organisation in both functionally important areas.     

Comparison of endogenous phosphorylation of hen and rat spinal cord proteins and partial characterization of optimal phosphorylation conditions for hen spinal cord

The optimal conditions for the endogenous phosphorylation of hen spinal cord cytosolic and membrane proteins with 5 μM [γ-³²P]ATP, 10 mM MgCl₂, were determined by 10% SDS-polyacrylamide gel electrophoresis, autoradiography, and microdensitometry. Phosphate incorporation increased linearly with concentrations ranging from 35–75 μg/100 μl for cytosolic proteins and 21–125 μg/200 μl for membrane proteins. Optimal incubation times, temperatures, and pH values were 60 s, 30°C, and 6.0, respectively, for spinal cord cytosolic proteins and 15 s, 45°C, and 8.0, respectively, for spinal cord membranes. Prominent species differences in protein phosphorylation between these fractions in hens and similarly prepared fractions in rats, co-electrophoresed, include 80K and 30K protein phosphate acceptors unique to rat spinal cord cytosol, 60K and 16K protein phosphate acceptors characteristic of rat spinal cord membranes, a 50K protein phosphate acceptor present only in hen spinal cord membranes, and greater phosphorylation of a more abundant 20K protein in both hen spinal cord fractions. The functional significance of these differences is presently unclear. However, their characterization provides a basis from which to launch future investigations of the biochemistry, pharmacology, and toxicology of spinal cord protein phosphorylation and indicates that caution should be exercised in the choice of an animal model with characteristics appropriate to those of the system it is representing.     

Stimulation of canine pancreatic polypeptide, gastrin, and gastric acid secretion by ranatensin, litorin, bombesin nonapeptide and substance P

Four dogs with chronic gastric fistulas were give intravenous bombesin nonapeptide (B9), ranatensin, and litorin by constant infusion for 90 min at 1.2 micrograms x kg-1 on separate days. A dose response study with substance P (1.5, 3.0, 60, 18 and 54 micrograms x kg-1 x h-1) was also carried out and all tests compared to a standard protein meal (10g x kg-1). Plasma gastrin and PP were measured by radioimmunoassay and gastric acid by autobiuret titration. Substance P failed to stimulate gastric acid secretion or release either pancreatic polypeptide (PP) or gastrin. Basal gastrin levels were 8 +/-2 fmol/ml. The peak increment of gastrin released by bombesin was 95 +/- 16, ranatensin 22 +/- 6, litorin 18 +/- 4, and meal 39 +/- 5 fmol/ml. Bombesin caused significantly greater release of gastrin than a meal, litorin or ranatensin (P less than 0.01). Basal gastric secretion was 23 +/- 4 microequiv./min. B9 produced a peak acid secretion of 356 +/- 124 muequiv./min. There was no significant difference between the bombesin-like peptides (P less than 0.01). Basal plasma PP was 38 +/- 12 fmol/ml. B9 produced a peak PP increment of 600 +/- 50, litorin 137 +/- 36, ranatensin 98 +/- 11, and a meal 305 +/- 58 fmol/ml. B9 released significantly more PP than either litorin of ranatensin (P less than 0.01). The different amino acid sequences of the peptides are probably responsible for their potency. The substitution of a penultimate phenylalanine residue in litorin and ranatensin for leucine in bombesin does not prevent PP or gastrin release by bombesin-like peptides. Since bombesin-like peptides are widely distributed in the gastrointestinal tract of man and stimulate both acid and gut hormone secretion, it is possible that they might play a physiological role in the modulation of gastrointestinal function.     

Trace metal levels in commercially prepared tissue culture media

Four trace elements, lead, copper, tin and zinc, in addition to certain electrolytes, were measured in 11 commercially prepared tissue culture media. Glass media bottles and plastic tissue culture dishes and flasks were treated with a HCl acid solution to determine the amounts of trace metals leached from their surfaces. Zinc, lead and copper were detected in all media. Tin was detected only in RPMI Medium 1640, fetal bovine serum, minimum essential medium and penicillin-streptomycin. It is possible that a major cause of variability in tissue culture experimental results may be due to effects on growth caused by fluctuation in trace element contamination from batch to batch. Variability in establishing primary cultures of corneal endothelial cells was traced to high lead levels in commercially prepared tissue culture media. A strong case is made for continued diligent efforts to expand analytical horizons and our definition of substances in culture media.     

Swainsonine, a potent mannosidase inhibitor, elevates rat liver and brain lysosomal alpha-D-mannosidase, decreases Golgi alpha-D-mannosidase II, and increases the plasma levels of several acid hydrolases

Swainsonine, a toxic plant alkaloid reported to be the agent that induces in animals a neurological condition very similar to the hereditary lysosomal storage disease mannosidosis, and to inhibit the formation of complex glycoproteins of the asparagine-linked class, was recently shown [D.R.P. Tulsiani, T.M. Harris, and O. Touster, (1982) J. Biol. Chem. 257, 7936-7939] to be a highly potent and specific inhibitor of Golgi mannosidase II in addition to being a strong inhibitor of lysosomal mannosidase. In the present study the effect of administered swainsonine on tissue enzyme levels was investigated. The activity of Golgi mannosidase II was markedly decreased (22% of control) without changes occurring in the activities of several other Golgi enzymes. However, the effects of swainsonine on lysosomal enzymes was unexpected. In liver, acid mannosidase increased markedly, instead of decreasing as would be expected from a compound reported to induce a mannosidosis-like condition. Similarly, the principal change in brain was a substantial increase in lysosomal mannosidase levels. In plasma, most lysosomal enzymes increased. These results indicate that the pathological effects of swainsonine are not solely attributable to its being an inhibitor of lysosomal alpha-D-mannosidase and are probably a consequence of abnormal processing of glycoproteins.     

Determination of gamma-aminobutyric and glutamic acids in rat brain by liquid chromatography with electrochemical detection

The determination of glutamic and gamma-aminobutyric acids in rat brain regions by derivatization with o-phthaldialdehyde-thiol, isocratic separation by liquid chromatography, and quantification by electrochemical detection provides a simple and precise method for assessing changes in glutamatergic and GABAergic neuronal systems. Gamma-aminobutyric acid was eluted in 30-35 minutes followed by a washout step with 90% methanol to remove all amino acid derivatives with longer retention times. Homoserine was used as an internal standard. Significant increases in gamma-aminobutyric acid content in nucleus accumbens and substantia nigra could be detected 20 minutes after injection of 400 mg/kg valproic acid.     

Stimulation of gastrin secretion from the perfused rat stomach by somatostatin antiserum

The effect of a high capacity somatostatin antiserum on antral gastrin secretion was examined in an isolated vascularly perfused rat stomach preparation. Infusion of somatostatin antiserum diluted 1:1 and 1:9 with Krebs buffer solution produced significant increases in gastrin secretion throughout the period of infusion. Neither infusion of somatostatin antiserum diluted 1:99 nor infusion of control rabbit serum had any effect on gastrin secretion. The data indicate that antral somatostatin excercises a continous restraint on gastrin secretion in the basal state.