Dopamine involved in the nociceptive modulation in the parafascicular nucleus of morphine-dependent rat.

Dopamine regulates pain perception in some areas of the central nervous system. Previously, we have confirmed that dopamine potentiated the electric activities of the evoked discharges of pain-excited neurons (PENs) and inhibited those of pain-inhibited neurons (PINs) in the parafascicular nucleus (Pfn) of normal rats. The mechanism of action of dopamine on pain-related neurons in the Pfn of morphine-dependent rat is still unknown. The present study aimed to determine the effects of dopamine and its receptor antagonist droperidol on the pain-evoked responses of the PEN and PIN in the Pfn of morphine-dependent rats, and to compare the effects between the morphine-dependent rat and the normal rat. The trains of electric impulses applied to the sciatic nerve were used as noxious stimulation. The discharges of PEN or PIN in the Pfn were recorded by using a glass microelectrode. The results showed that intra-Pfn microinjection of dopamine decreased the frequency of noxious stimulation-induced discharges of PEN and increased the frequency of PIN. The intra-Pfn administration of droperidol produced an opposite effect. These results demonstrated that dopamine is involved in nociceptive modulation in the morphine-dependent rat, the responses to noxious stimulation between normal rat and morphine-dependent rat are completely opposite. The effect of dopamine is through the dopamine D(2) receptor of PENs and PINs in Pfn. The results suggest that the dopamine system of the Pfn may become a therapeutic target for analgesia and the treatment of morphine dependence.

L-364,718 potentiates electroacupuncture analgesia through cck-a receptor of pain-related neurons in the nucleus parafascicularis.

Electroacupuncture (EA) has been successfully used to alleviate pain produced by various noxious stimulus. Cholecystokinin-8 (CCK-8) is a neuropeptide involved in the mediation of pain. We have previously shown that CCK-8 could antagonize the analgesic effects of EA on pain-excited neurons (PENs) and pain-inhibited neurons (PINs) in the nucleus parafascicularis (nPf). However, its mechanism of action is not clear. In the present study, we applied behavioral and neuroelectrophysiological methods to determine whether the mechanisms of CCK-8 antagonism to EA analgesia are mediated through the CCK-A receptors of PENs and PINs in the nPf of rats. We found that focusing radiant heat on the tail of rats caused a simultaneous increase in the evoked discharge of PENs or a decrease in the evoked discharge of PINs in the nPf and the tail-flick reflex. This showed that radiant heat could induce pain. EA stimulation at the bilateral ST 36 acupoints in rats for 15 min resulted in an inhibition of the electrical activity of PEN, potentiation of the electrical activity of PIN, and prolongation in tail-flick latency (TFL), i.e. EA stimulation produced an analgesic effect. The analgesic effect of EA was antagonized when CCK-8 was injected into the intracerebral ventricle of rats. The antagonistic effect of CCK-8 on EA analgesia was reversed by an injection of CCK-A receptor antagonist L-364,718 (100 ng/µl) into the nPf of rats. Our results suggest that the pain-related neurons in the nPf have an important role in mediating EA analgesia. L-364,718 potentiates EA analgesia through the CCK-A receptor of PENs and PINs in the nPf.

The effect of dopamine on pain-related neurons in the parafascicular nucleus of rats.

Dopamine (DA) regulates pain perception in the central nervous system (CNS). However, the mechanism of the action of DA in pain-related neurons of the parafascicular nucleus (Pf) is not clear. The present study aimed to determine the effect of DA and its receptor antagonist, droperidol on the pain-evoked responses of the pain-excited neurons (PEN) and pain-inhibited neurons (PIN) in the Pf of rats and to analyze the mechanisms underlying this effect. The trains of electric impulses applied to the sciatic nerve were used as noxious stimulation. The discharges of PEN and PIN in the Pf were recorded by using a glass microelectrode. The results showed that, in the Pf, intra-Pf microinjection of DA (5 microg/0.5 microl) increased the frequency of noxious stimulation-induced discharges of the PEN and decreased the frequency of those of the PIN, while the intra-Pf administration of droperidol (0.15 microg/0.5 microl) produced an opposite effect. On the basis of the above-mentioned findings, we could conclude that DA and its receptors in the Pf are involved in the modulation of the nociceptive response by regulating the discharges of PEN and PIN.

Studies on glutamine synthetase from Streptomyces hygroscopicus var. jinggangensis.

Streptomyces hygroscopicus var. jinggangensis produces validamycin, an important antibiotic used in agriculture. It was found that there was a positive correlation between the specific activity of mycelial glutamine synthetase (GS) and validamycin biosynthesis in our laboratory. So, in this paper, the purification, characteristics and regulatory properties of GS are reported. The native enzyme had a molecular weight of approximately 530,000 and was composed of 12 identical subunits, each 43,000. Electronic microscopic examination of preparations negatively stained disclosed that the subunits were arranged in two hexagonal rings that lay one on top of the other in a face-to-face fashion. Mg2+ or Mn2+ was absolutely needed as cofactor for GS activity. The enzyme activity was regulated by feedback inhibition and cumulative feedback inhibition. In addition, it was also regulated through a covalent modification, adenylylation and deadenylylation, suggesting that the covalent modification of GS exists not only in Gram-negative bacteria but also in some Gram-positive bacteria.

Purification and properties of glutamate synthase from Nocardia mediterranei.

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.

[Permeabilization of yeast Trigonopsis variabilis FA10 cells for enhancing apparent activity of D-amino acid oxidase].

D-amino acid oxidase(DAO) is an intracellular enzyme in Trigonopsis variabilis FA10. The whole cells of yeast exhibited very low DAO activity. Various treatment with physical and chemical agents, such as, freezing and thawing, acetone, butanol and cetyltrimethylammoinium bromide altered membrane permeability and increased cellular DAO activity. It was demonstrated that the performance of acetone was dependent on the concentration of the solvent, the incubation time and the temperature. Maximum enzyme activity of the cells was achieved with 30-35% acetone, between 4 degrees C and 28 degrees C. The process was very quick and permeabilization occurred in 5 minutes or less. On the other hand, the thermostability of DAO in permeabilized cells was higher than in cell extract. It was proved that Cephalosporin C could be effectively converted to glutaryl-7ACA by permeabilized Trigonopsis variabilis cells with 83% yield.

[Enzymatic conversion of cephalosporin C to glutaryl-7-aminocephalosporanic acid using whole cells of the yeast Trigonopsis variabilis FA10].

A process for the production of glutaryl-7-aminocephalosporanic acid (GL-7ACA) from cephalosporin C(CPC) using permeabilized cells of yeast Trigonopsis variabilis FA10 containing D-amino acid oxidase (DAO) is described. It was found that the bioconversion of CPC to GL-7ACA was interfered by the catalase activity presented in the cells that hydrolyzed the hydrogen peroxide and resulted in the accumulation of alpha-keto-adipyl-7-ACA (AKA-7ACA) and decrease of GL-7ACA yield. the methods to overcome this problem including the addition of extra H2O2 and use of catalase inhibitor, NaN3, were developed and the rate of GL-7ACA from CPC were 73% and 70.1%, respectively. Another alternative method was to incubate the permeabilized FA10 cells at pH10.5-11.0 for 30 minutes at 20 degrees C which served to selectively inactivate the catalase. In the bioconversion of CPC to GL-7ACA using pH10.5-treated cells without catalase activity, the high reaction yield of GL-7ACA(84%) was achieved.

The route of incorporation of nitrogen atom into rifamycin during its biosynthesis.

In this paper, it was first shown that under various conditions of nitrogen supply the rifamycin yield was positively correlated with the mycelial specific activity of glutamine synthetase (GS), then the enhancing effect of glutamine, the product of GS, on rifamycin biosynthesis was demonstrated with resting cell system. The stimulatory effect of glutamine was more pronounced than that of glutamate, and not reduced by a GS specific inhibitor, DON. However, the increase in yield brought about by glutamate, and by asparagine was strongly inhibited by this inhibitor. Glutamine-CO15NH2 amd glutamate-alpha-15NH2 were synthesized and compared for the incorporation of 15N into rifamycin. It was found that the amide nitrogen was incorporated to a much greater extent than the alpha-amino nitrogen, showing that glutamine was a direct precursor of the nitrogen atom in rifamycin. In addition, synthesis of A-32 (C7N), an intermediate secreted by an inactive mutant rif 1, was also greatly stimulated by glutamine, and the synthetic C7N was found to be able to stimulate the biosynthesis of rifamycin. Based upon the above results, the route of incorporation of nitrogen atom into rifamycin is summarized as follows: (Formula: see text).