Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Development and Application of Cloned DNA Probes for Clavibacter xyli subsp. xyli, the Causal Agent of Sugarcane Ratoon Stunting

Eco RI restriction fragments of genomic DNA from Clavibacter xyli subsp. xyli (CXX) were ligated with plasmid pUC18 and cloned in Escherichia coli JM109. The cloned DNA inserts from recombinant plasmids were Eco RI-excised and labeled with non-radioactive digoxigenin and used as probes. Ten specific DNA probes, RSD3, 15, 30, 31, 32, 35, 37, 41, 71, and 73 were selected for disease detection and pathogen differentiation. In the specificity tests, all of the 10 CXX DNA, probes differentiated Clavibacter xyli from other bacteria specifically. Seven out of the 10 CXX probes crossreacted with C. x. subsp. cynodontis (CXC) very weakly under moderate stringency wash conditions of hybridization. In the sensitivity tests, all of the 10 DNA probes detected the homologous DNA of CXX from 0.19 to 0.75 ng. To detect various cell numbers of CXX, the DNA probes detected 10⁴ to 10⁵ cells effectively. In Southern hybridizations, distinctly different band patterns were shown when the probes hybridized with DNA from CXX and CXC. Among these probes, RSD3, 15, 30, 31, 35, 37, and 71, efficiently detected CXX present in the sap collected from symptomless sugarcane.     

Transforming growth factor-beta(1) regulates differentiation of porcine granulosa cells in vitro

Transforming growth factor-beta (TGF-beta) is a potential regulator of ovarian function and follicular development. It is speculated that TGF-beta mediates the events in the follicle which culminate in ovulation of the oocyte. The complex processes which ultimately leads to this natural phenomenon must involve interactions between the 2 major follicular cell types, theca and granulosa cells, and the oocyte. Furthermore, a complex local regulatory system must exist to determine which follicles should undergo development and, eventually, which of those should ovulate or undergo atresia. To begin to understand this perplexing process, we must first understand the variables which control the function of each individual cell type. This study investigated the effect of TGF-beta(1) on FSH-induced porcine granulosa cell differentiation in vitro. Transforming growth factor-beta(1) was shown to inhibit progesterone production at high concentrations (0.1 and 10.0 ng/ml) after 12-, 24- and 48-hour treatment. However, TGF-beta(1) produced a biphasic effect on FSH-induced progesterone production during the 12-hour interval between the 36- and 48- hour treatment periods; TGF-beta(1) stimulated progesterone production at a low concentration (0.001 ng/ml) and inhibited production at high concentrations (0.1 and 10.0 ng/ml). The results obtained from the biphasic effect were not observed during any of the other incubation periods or intervals investigated. These results show that TGF-beta(1) has opposing effects on the differentiation of porcine granulosa cells as compared with those on rat granulosa cells. Moreover, TGF-beta(1) can produce opposing effects within the porcine granulosa cell itself which are specific to the concentration and treatment period used. The results of this study seem to suggest that TGF-beta(1) is species- and time-specific in its regulatory actions on FSH-induced porcine granulosa cell differentiation.     

Trace elements in hair of blackfoot disease

Blackfoot disease is a peripheral vascular disease resulting in gangrene of the lower extremities. Though extensive epidemiological study has implicated that high arsenic content in artesian well water of the endemic area, bears some important connection with the disease, the etiology of the disease is still unknown. In this study, attention is paid to multielement determination in order to find out whether the trace elements in hair of Blackfoot disease patients are different from those of the controls. Experimental results indicate that the concentrations of As and Se in hair of patients are significantly higher than those of the controls, but Ca and Zn are significantly lower than those of the controls. The possible connection of these elements with the etiology of the disease is discussed.     

The effect of pulsed microwaves on passive electrical properties and interspike intervals of snail neurons

The effects of pulsed microwaves (2.45 GHz, 10 μs, 100 pps, SAR: 81.5 kW/kg peak, 81.5 W/kg average) on membrane input resistance and action potential (AP) interval statistics were studied in spontaneously active ganglion neurons of land snails (Helix aspersa), at strictly constant temperature (20.8±.07°C worst case). Statistical comparison with sham-irradiated neurons revealed a significant increase in the mean input resistance of neurons exposed to pulsed microwaves (P ? .05 ). Pulsed microwaves had no visible effect on mean AP firing rate; this observation was confirmed by analysis of interspike intervals (ISIs). Using an integrator model for spontaneously active neurons, we found the net input current to be more variable in neurons exposed to pulsed microwaves. The mean input current was not affected. The standard deviation of ISIs and the autocorrelation of the input current were marginally affected, but these changes were not consistent across neurons. Although the observed effects were less obvious than those reported in other studies, they represent evidence of a direct interaction between neurons and pulsed microwaves, in the absence of macroscopic temperature changes. The data do not suggest a single, specific mechanism for such interaction. © 1993 Wiley-Liss, Inc.     

Generation of hydrogen peroxide,superoxide anion and the hydroxyl free radical from polyphenols and active benzene metabolites: Their possible role in mutagenesis

Benzene is strongly suspected of being an animal and human carcinogen, but the mechanisms by which it induces tumors of lymphoid and hematopoietic organs are unknown. Production of active oxygen species from benzene metabolites [hydroquinone (HQ), catechol and 1,2,4-benzenetriol (1,2,4-BT) and related polyphenols (resorcinol, pyrogallol and phloroglucinol) are investigated. Pyrogallol and 1,2,4-BT can produce H₂O₂, O ₂ ^– and^·OH simultaneously, and have powerful mutagenic potential. Resorcinol and phloroglucinol cannot produce all of the active oxygen species, and show no mutagenic effects. Catechol can produce H₂O₂, but cannot produce O ₂ ^– and^·OH, and has no mutagenic activity. These data strongly support the hypothesis that benzene metabolites can cause mutagenicity via the generation of oxygen radicals. Although HQ produces H₂O₂ only, and less than produced by pyrogallol and 1,2,4-BT, the mutagenicity of HQ is higher. The results indicate that HQ may act via another mechanism to cause mutagenicity. In the presence of trace metal ions, the reactivity of polyphenols is increased. The biological significance of these phenomena are investigated and discussed.     

Identification and characterization of glucocorticoid receptors in liver of nude mice

Glucocorticoids regulate the expression of many liver-specific genes via glucocorticoid receptors. The presence of glucocorticoid receptors in liver has been reported in many mammalian species but not in nude mice. In the present study, we demonstrate the presence of specific glucocorticoid receptors in nude mouse liver. The binding of ligands to these receptors could be completely inhibited by RU486, and partially blocked by hydrocortisone and progesterone, whereas estrogen and testosterone had no effect. Hydrocortisone down-regulated the level of glucocorticoid receptors in livers of nude mice and correspondingly enhanced the activities of tyrosine aminotransferase and -glutamyltransferase. Our results indicate that glucocorticoid receptors in nude mouse liver are specific, fully functional, and present at levels 28.5-fold higher than in the liver of normal inbred mice. We suggest that the nude mouse is a valuable model for studies of hepatic glucocorticoid action and may provide a clue to a putative hepatic-thymic interaction.