Local Activation of Metabotropic Glutamate Receptors Inhibits the Handling-Induced Increased Release of Dopamine in the Nucleus Accumbens but Not that of Dopamine or Noradrenaline in the Prefrontal Cortex: Comparison with Inhibition of Ionotropic Receptors

Abstract: On-line in vivo microdialysis was used to determine the effects of a 16-min handling period on release of dopamine (DA) in the nucleus accumbens and of DA and noradrenaline (NA) in the medial prefrontal cortex of awake, freely moving rats. DA and NA were determined in one HPLC run. Handling resulted in an immediate and strong increase of both catecholamines in the prefrontal cortex. Maximal values for DA were 295%, and for NA 225%, of controls. DA in the nucleus accumbens was also increased (to 135% of controls) but only after a short delay. Local inhibition of ionotropic glutamate receptors by continuous reversed dialysis of the drugs 6-cyano-7-nitroquinoxaline, d -2-amino-5-phosphonopentanoic acid, or dizocilpine did not significantly affect handling-induced increases in cortical DA and NA release. Neither did the agonist of metabotropic glutamate receptors, trans -(1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), or the GABA-B agonist baclofen. Reversed dialysis of dizocilpine in the nucleus accumbens was equally ineffective, but ACPD inhibited the increase in DA release in this area. Stimulation of metabotropic glutamate receptors in the nucleus accumbens was previously reported to inhibit activation of DA release in that area after stimulation of glutamatergic or dopaminergic afferents. It is concluded that metabotropic receptors in the nucleus accumbens are important for the control of activation of DA release in the accumbens by physiological stimuli but that a similar mechanism is lacking in the prefrontal cortex.     

Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty acid fructose esters

Lipase-catalyzed synthesis of fatty acid sugar esters through direct esterification was performed in 2-methyl 2-butanol as solvent. Fructose and saturated fatty acids were used as substrates and the reaction was catalyzed by immobilized Candida antarctica lipase. The effect of the initial fructose/acyl donor molar ratio and the carbon-chain length of the acyl donor as well as their reciprocal interactions on the reaction performance were investigated. For this purpose, an experimental design taking into account variations of the molar ratio (from 1:1 to 1:5) and the carbon-chain length of the fatty acid (from C8 to C18) was employed. Statistical analysis of the data indicated that the two factors as well as their interactions had significant effects on the sugar esters synthesis. The obtained results showed that whatever the molar ratio used, the highest concentration (73 g l⁻¹), fructose and fatty acid conversion yields (100% and 80%, respectively) and initial reaction rate (40 g l⁻¹ h⁻¹) were reached when using the C18 fatty acid as acyl donor. Low molar ratios gave the best fatty acid conversion yields and initial reaction rates, whereas the best total sugar ester concentrations and fructose conversion yields were obtained for high molar ratios.     

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.     

Regional Amino Acid Distribution in Relation to Function in Insulin Hypoglycaemia

The amino acids glutamate, aspartate, gamma-aminobutyric acid (GABA), and glutamine were measured as their dansyl derivatives in whole brain and specific brain regions by a sensitive double-labelling technique at various times during the development of hypoglycaemic encephalopathy. Hypoglycaemia was induced by administration of insulin (100 i.u./kg) to 24-h fasted rats. No significant changes in glutamate, GABA, or glutamine were detected in whole brain at any time up to and including the onset of hypoglycaemic convulsions. In cerebral cortex, however, GABA levels were reduced to 65% or normal prior to the appearance of neurological symptoms of hypoglycaemia. Onset of symptoms (severe catalepsy and loss of righting reflex, but before the onset of convulsions) was accompanied by marked decreases of glutamate and glutamine in striatum and hippocampus. These regions, in addition to cerebral cortex, show the greatest vulnerability to hypoglycaemic insult, according to previous anatomical studies. Aspartate levels were significantly increased (p less than 0.01) in the cerebral cortex of convulsing animals, confirming a previous report. No changes were detectable in any of the amino acids studied in medulla-pons at any time during the progression of hypoglycaemia. Cerebral cortex and striatum showed a selective net loss of amino acids (2.2 and 3.5 mumol/g. respectively) prior to the onset of insulin-hypoglycaemic convulsions.     

Carbon metabolism of Frankia spp. in root nodules of Alnus glutinosa and Hippophaë rhamnoides

The occurrence and localization of enzymes involved in glycolysis, tricarboxylic acid cycle and glyoxylate cycle in root nodules of Alnus glutinosa (L.) Vill. and Hippophaë rhamnoides L. ssp. rhamnoides were studied. The following enzymes, catalyzing reversible steps in the glycolysis, were found in both the endophyte Frankia spp. and the plant cytosol of Alnus nodules: fructose-1,6-diphosphate aldolase, glyceralde-hyde-3-phosphate dehydrogenase, phosphoglycerate kinase and enolase. The enzymes catalyzing irreversible steps in glycolysis, viz. hexokinase and pyruvate kinase, were detectable only in the plant cytosol. Similar results were obtained with nodule homogenates of Hippophaë. This indicates the absence of a complete glycolysis in the endophyte. Vesicle clusters of the nodule endophyte of Alnus contained various dehydrogenases of the tricarboxylic acid cycle and showed activity of glutamate oxaloacetate transaminase. Respiration studies showed that vesicle clusters take up oxygen when supplied with NAD, glutamate and malate together. No oxygen uptake was found when any of these compounds was omitted. Vesicle clusters from both Alnus and Hippophaë nodules showed no detectable activity of the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Since these enzymes are known to be present in Frankia Avcll, when grown in a medium with Tween 80 as carbon source, it is suggested that the glyoxylate cycle enzymes are repressed in the root-nodule symbioses.     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Purification of glycopeptides of human ceruloplasmin and immunoglobulin D by high-pressure liquid chromatography

To facilitate structural studies of glycoproteins, reverse-phase high-pressure liquid chromatography (HPLC) methods have been developed for preparative isolation of glycopeptides and have been applied to human ceruloplasmin as an example of glycopeptides containing glucosamine (GlcN) and to human immunoglobulin D (IgD) for glycopeptides containing galactosamine (GalN). The use of RP-P columns and of trifluoroacetic acid and heptafluorobutyric acid as counterions was investigated. Various elution systems (both isocratic and programmed gradient) were used with n-propanol to assess the relative hydrophilicity of the peptides. The procedure developed for the GlcN glycopeptides of ceruloplasmin enabled purification of nine major chymotryptic peptides (ranging in size from 15 to 29 residues) and also of many minor peaks. These were characterized by amino acid and endgroup analysis, and the complete sequence of five was determined. These represent three different sites of GlcN attachment in the amino-terminal half of the ceruloplasmin chain. The procedures developed have enabled isolation of glycopeptides from ceruloplasmin having a single GlcN oligosaccharide attached; the latter are valuable for study of the structure and function of the carbohydrate groups. Separation of GalN glycopeptides from IgD was more difficult because of the high content of GalN in the hinge. Purification and sequence analysis was aided by partial removal of sugar by treatment with HF and by other methods. Four (or five) GalN oligosaccharides are attached to serine or threonine residues in the IgD hinge region, and all but one are in close proximity in the repeating sequence Ala-Thr-Thr-Ala-Pro-Ala-Thr-Thr.     

Isolation and characterization of plasma membrane from lactating bovine mammary gland

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5′-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg²⁺-ATPase, (Na⁺ + K⁺)-ATPase, γ-glutamyl transpeptidase, phosphodiesterase I, akaline phosphatase and xanthine oxidase were also high (12–18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide compositions in both light and heavy membranes were similar upon SDS-polyacrylamide gel electrophoresis.     

Specific drug sensitive transport pathways for chloride and potassium ions in steady-state ehrlich mouse ascites tumor cells

A major aim of this investigation was to determine whether, in steady-state ascites cells, Cl^? transport can be partitioned into a furosemide-sensitive cotransport with K⁺ and a separate 4,4′-isothiocyanostilbene-2,2′-disulfonic acid (DIDS) sensitive self-exchange. Both Cl^? and K⁺ fluxes were studied. The furosemide- and Cl^? sensitive K⁺ fluxes were equivalent, both in normal ionic media and when the external K⁺ concentration, [K⁺]_o, was varied from 4 to 30 mM. The stoichiometry of the furosemide-sensitive Cl^? and K⁺ fluxes was 2 Cl^?: 1 K⁺ at 0.1 and 0.5 mM drug levels but increased to 3 Cl^? : 1 K⁺ at 1.0 mM furosemide. DIDS at 0.1 mM had no effect on the K⁺ exchange rate but inhibited Cl^? exchange by $\text{39\% ± 2}$ (S.E.). The effects of DIDS and 0.5 mM furosemide on Cl^? transport were additive but 1.0 mM furosemide and DIDS had overlapping inhibitory actions. Thus furosemide acts on components of K⁺ and Cl^? transport which are linked to each other, but the drug also inhibits an additional DIDS-sensitive Cl^? pathway, when present at higher concentrations. The dependence of the furosemide-sensitive K⁺ and Cl^? transport on [K⁺]_o was also studied; both fluxes fell as the [K⁺]_o increased. The latter results recall those in an earlier study by Hempling (Hempling, H.G. (1962) J. Cell. Comp. Physiol. 60, 181–198).     

A ginger derivative,zingerone—a phenolic compound—induces ROS‐mediated apoptosis in colon cancer cells (HCT‐116)

Zingerone (ZO), an active phenolic agent derived from Zingiber officinale (Ginger), has many pharmacological properties such as antioxidant, antiangiogenic, and antitumor. However, its potential value in cancer and the mechanism by which ZO wields its therapeutic effects remain obscure. Therefore, in this current study, we explored the effects of ZO on suppressing cell proliferation and enhancing apoptosis in colon cancer cells (HCT116). Our results indicated that ZO significantly enhances the production of reactive oxygen species, lipid peroxidation (thiobarbituric acid reactive substance [TBARS]), and loss of cell viability; and reduces mitochondrial membrane potential and antioxidant levels (SOD, CAT, and GSH) in ZO‐treated HCT116 cells in a dose‐dependent (2.5, 5, and 10 µM) manner. Furthermore, ZO induces oxidative stress‐mediated apoptosis as evidenced by apoptotic morphological changes predicted by AO/EtBr, Hoechst staining and further confirmed by comet assay. Moreover, immunoblotting techniques showed that ZO treatment effectively enhances Bax, caspase‐9, and caspase‐3 expressions and decreases the expression of Bcl‐2 in colon cancer cells. Together, our results evidenced that the antitumor effects of ZO reduce cell proliferation and stimulate apoptosis through modulating pro‐ and antiapoptotic molecular events in HCT116 colon cancer cells. Therefore, based on our findings, ZO may be used as a therapeutic agent for the treatment of colon cancer.