The C-Terminal Domain of the mGluR1 Metabotropic Glutamate Receptor Affects Sensitivity to Agonists

Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (−1a, −1b, and −1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca²⁺ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate ≫ (2 S, 1' S, 2' S )-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1 S, 3 R )-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S, 3 R )-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca²⁺]_i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I ≫ (1 S, 3 R )-ACPD ∼ quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses.     

Extracellular Striatal Dopamine and Glutamate After Decortication and Kainate Receptor Stimulation, as Measured by Microdialysis

Abstract: Disruption of corticostriatal glutamate input in the striatum decreased significantly extracellular striatal glutamate and dopamine levels. Local administration of 300 µ M concentration of excitatory receptor agonist kainic acid increased significantly extracellular striatal dopamine in intact freely moving rats. These findings support the hypothesis that glutamate exerts a tonic facilitatory effect on striatal dopamine release. The effect of kainic acid on extracellular striatal glutamate concentration in intact rats was a biphasic increase. The first glutamate increase can be explained by stimulation of presynaptic kainate receptors present on corticostriatal glutamatergic nerve terminals; the second increase is probably the result of a continuous interaction of the different striatal neurotransmitters after disturbance of their balance. Release of dopamine and glutamate was modulated differently in the intact striatum and in the striatum deprived of corticostriatal input. Dopamine release in the denervated striatum after kainate receptor stimulation was significantly lower than in intact striatum, confirming the so-called cooperativity between glutamate and kainic acid. Loss of presynaptic kainate receptors on the glutamatergic nerve terminals after decortication resulted in a loss of effect of kainic acid on glutamate release in denervated striatum. Aspartate showed no significant changes in this study.     

Assessment of the Role of the Glutathione and Pentose Phosphate Pathways in the Protection of Primary Cerebrocortical Cultures from Oxidative Stress

Abstract: Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H₂O₂). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-¹³C₂,6,6-²H₂]glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H₂O₂, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H₂O₂ caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H₂O₂ exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H₂O₂-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H₂O₂ detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.     

Desulfurization of dibenzothiophene derivatives by whole cells of Rhodococcus erythropolis H-2

Abstract Transposon mutagenesis was performed to pursue the molecular basis of carbazole catabolic pathway in a carbazple-using bacterium, Pseudomonas sp. CA10. One mutant, TD2, was capable of using anthranilic acid but not carbazole as its sole source of carbon, nitrogen, and energy. Another isolated mutant, designated as TE1, was found to have the opposite ability as TD2. TD2 could not convert carbazole to any other compound under cometabolic conditions. On the other hand, TE1 accumulated catechol and cis,cis -muconate from carbazole. The clone containing Tn 5 -flanking region from TD2, showed the meta -cleavage activity for biphenyl-2,3-diol and analysis of the DNA sequence of this region suggests that the genes involved in the degradation of aromatic compounds are clustered. Our analysis of the DNA sequence of another clone from mutant TE1 showed that the Tn 5 -Mob can be inserted into the homologous catR gene, a gene that reportedly enpodes the positive regulatory protein of the catBC operon. These data suggests that carbazole catabolic pathway comprises at least two different gene clusters (upper pathway and lower pathway) in Pseudomonas sp. CA10.     

Cloning and expression of transaldolase from potato

We have isolated a cDNA encoding transaldolase, an enzyme of the pentose-phosphate pathway, from potato (Solanum tuberosum). The 1.5 kb cDNA encodes a protein of 438 amino acid residues with a molecular mass of 47.8 kDa. When the potato cDNA was expressed in Escherichia coli a 45 kDa protein with transaldolase activity was produced. The first 62 amino acids of the deduced amino acid sequence represent an apparent plastid transit sequence. While the potato transaldolase has considerable similarity to the enzyme from cyanobacteria and Mycobacterium leprae, similarity to the conserved transaldolase enzymes from humans, E. coli and Saccharomyces cerevisiae is more limited. Northern analysis indicated that the transaldolase mRNA accumulated in tubers in response to wounding. Probing the RNA from various potato tissues indicated that the transaldolase mRNA accumulation to higher levels in the stem of mature potato plants than in either leaves or tubers. These data are consistent with a role for this enzyme in lignin biosynthesis.     

力复霉素前体甲基丙二酰CoA合成途径的研究

力复霉素合成的碳前体之一(2R)—甲基丙二酰CoA至少可以有三条酶学合成途径。三条途径中的关键酶分别为甲基丙二酰CoA转羧基酶、丙二酰CoA羧化酶、甲基丙二酰CoA变位酶和甲基丙二酰CoA消旋酶。通过比较各个酶活性的时间进程和力复霉素合成时间的相关性,以及各个酶的底物亲合力,对它们在地中海拟无枝酸菌(Amycolatopsis mediterranei)甲基丙二酰CoA合成中的贡献作了排序,发现甲基丙二酰CoA变位酶途径是主要负责酶系。但是各个途径的贡献排序并不是固定不变的,能受到环境因素的调控,丙酸盐的加入将抑制甲基丙二酰CoA变位酶活力,而使得甲基丙二酰CoA转羧基酶成为主要酶系。甲基丙二酰CoA合成途径的多样性有助于细胞对环境变化的灵活反应。此外,对各个酶的调控特性也进行了研究。     

Role ofDrosophila TRP in inositide-mediated Ca2+ entry

Inositol lipid signaling relies on an InsP₃-induced Ca²⁺ release from intracellular stores and on extracellular Ca²⁺ entry, which takes place when the Ca²⁺ stores become depleted of Ca²⁺. This interplay between Ca²⁺ release and Ca²⁺ entry has been termed capacitative Ca²⁺ entry and the inward current calcium release activated current (CRAC) to indicate gating of Ca²⁺ entry by Ca²⁺-store depletion. The signaling pathway and the gating mechanism of capacitative Ca²⁺ entry, however, are largely unknown and the molecular participants in this process have not been identified. In this article we review genetic, molecular, and functional studies of wild-type and mutantDrosophila photoreceptors, suggesting that thetransient receptor potential mutant (trp) is the first putative capacitative Ca²⁺ entry mutant. Furthermore, several lines of evidence suggest that thetrp gene product TRP is a candidate subunit of the plasma membrane channel that is activated by Ca²⁺ store depletion.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.