N-acetylaspartylglutamate acts as an agonist upon homomeric NMDA receptor (NMDAR1) expressed in Xenopus oocytes.

The electrophysiological effects of N-acetylaspartylglutamate (NAAG), an endogenous peptide restrictively distributed in the central nervous system, were studied using Xenopus oocytes injected with RNAs transcribed from cloned glutamate receptor cDNAs. NAAG induced an inward current, dose dependently, in oocytes injected with RNA for an N-methyl-D-aspartate receptor subunit (NMDAR1). In contrast, the oocytes injected with RNAs for AMPA-selective glutamate receptors (GluR1, GluR3, GluR1+GluR2 and GluR2+GluR3) scarcely responded to NAAG, and the oocytes injected with RNA for kainate receptor (GluR6) did not respond to NAAG. The half-maximal response (ED50) value of NAAG on expressed NMDAR1 was 185 microM, which shows that NAAG is about 115-times less potent than L-glutamate (Glu), the ED50 of which value was 1.6 microM. The maximal current amplitude induced by NAAG was about 70% of that by Glu. NAAG-induced current in NMDAR1-injected oocytes was potentiated by glycine, dose-dependently antagonized by DL-2-amino-5-phosphonovaleric acid, and blocked by magnesium ions in a voltage-dependent fashion. These results suggest that NAAG is one of the endogenous agonists selective for NMDAR1.     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Genetic studies on site-specific endodeoxyribonucleases in Bacillus subtilis: multiple modification and restriction systems in transformants of Bacillus subtilis 168

Summary We transformed B. subtilis 168 with DNA from B. subtilis IAM1231, IAM1192 and ATCC6633. When we examined the restriction activities of the transformants in vivo and in vitro using phage 105C we found the following: (1) Cells of either IAM1231 or IAM1192 have two modification and restriction systems (Bsu1231(1)-system and Bsu1231(II)-system in IAM1231, and Bsu1192(I)-system and Bsu1192(II)-systems in IAM1192), and cells of ATCC6633 have only one system (Bsu6633-system). (2) The restriction enzymes of all of these five systems are site-specific endonucleases. (3) The nucleotide sequence specificities of the enzymes involved in Bsu1231(I)-system, Bsu1192(I)-system and Bsu6633-system are the same; and those of Bsu1231(II)-system and Bsu1192(II)-system are the same. The sequence specificities of these two groups are different from each other and also different from those of the Bsu168-system of B. subtilis 168, the BsuR-system of B. subtilis R and the Bsu1247(I)-and Bsu1247(II)-systems which are systems of B. subtilis IAM1247. (4) Transformants possessing four different modification and restriction systems (Bsu1231(I)-, Bsu1247(I)-, BsuR- and Bsu168-systems) were constructed. (5) Transformation of two derivatives of 168 that were m _R ⁺ r _R ⁺ by DNA from IAM1231 produced 16 transformants that had the Bsu1231(II) restriction system, but had lost the BsuR system. Transformation of a derivative of 168 that was m _1247(II) ⁺ r _1247(II) ⁺ by DNA from m _1231(II) ⁺ r _1231(II) ⁺ -or m _R ⁺ r _R ⁺ -derivative of 168 produced about 100 each of transformants that had the Bsu1231(II)-restriction system or the BsuR-restriction system. But all these transformants lost the Bsu1247(II)-system.     

Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing

The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37 degrees C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group 'Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-PROTEOBACTERIA: Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group 'Desulfotomaculum lineage I', but it was only distantly related to other known species.     

Protein engineering for thermostability.

Studies with small, monomeric proteins indicate that, to some extent, the effects of amino acid substitutions can be predicted. However, conformational and other changes may complicate the prediction. Site-directed mutagenesis is leading both to a better understanding of protein stability and to the production of more stable proteins.     

Mechanism of photoisomerization of optically pure trans-2,3-diphenylcyclopropane-1-carboxylic acid derivatives.

The photochemistry of optically pure isomers of alpha-methylbenzylamide of trans-2,3-diphenylcyclopropane-1-carboxylic acid has been examined in isotropic solution and within zeolites. The results suggest that these isomerize through cleavage of C2-C3 bond. The direct excitation in solution leads to non-equilibrating 1,3-singlet diradical intermediates whereas triplet sensitization results in equilibrating 1,3-triplet diradical intermediates. The direct excitation within NaY zeolite seems to result in equilibrating zwitterionic intermediates. Studies on the optically pure trans isomers allow one to understand the mechanism of chiral induction during the photoisomerization of mesocis-2,3-diphenylcyclopropane-1-carboxylic acid. The current study has clarified the nature of the excited states involved during the classic (R)-N-acetyl-1-naphthylethylamine sensitized isomerization of 1,2-diphenylcyclopropane.     

Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.