Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. d-Tyrosine was used for the control. α-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Potentiation of lipid peroxides by ischemia in rat brain

Post-ischemic changes in energy metabolites and natural antioxidant compounds have been measured in rat brain in vitro concurrent with two different assays for peroxidized lipids. No exogenous free radical initiators were employed. In vitro oxygenation of minced brain preparations for periods of 10 minutes to 4 hours, following 5 minutes of preparatory ischemia, yielded increased levels of lipid conjugated dienes and TBA-reactive material, in contrast to anaerobically incubated preparations. However, either aerobic or anaerobic incubation of brain minces facilitated increased ratios of lactate: pyruvate and glutathione (oxidized): glutathione (reduced), as well as increased total ubiquinone content and loss of -tocopherol. Observation of lipid radical formation in vivo was then attempted using rats given embolic stroke in one hemisphere and left in the post-ischemic condition for times up to 24 hours. Conjugated dienes were found in lipids extracted from the ipsilateral hemisphere but not from the contralateral hemisphere. These observations of conjugated dienes in vivo (formed presumably during post-ischemic reperfusion) and in vitro (facilitated by oxygenation of brain minces), indicate that lipid radical intermediates and associated chain peroxidation processes are potentiated by ischemia and occur during tissue reoxygenation.     

Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.     

Genome-wide screening of genes required for swarming motility in Escherichia coli K-12

Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.     

Porcine brain natriuretic peptide-like immunoreactivity in rat tissues

The presence of immunoreactive porcine brain natriuretic peptide in rat tissues was studied with a specific radioimmunoassay for porcine brain natriuretic peptide-26. The cross-reactivity of the antiserum used was less than 0.001% with rat atrial natriuretic peptide, rat brain natriuretic peptide-32 and rat brain natriuretic peptide-45. Immunoreactive porcine brain natriuretic peptide was detectable in various tissues of the rat, and high concentrations of immunoreactive porcine brain natriuretic peptide were found in the brain and cardiac atrium, with the highest level in the hypothalamus (159±30 fmol/gram wet tissue, mean±SEM, n=4). Reverse phase high performance liquid chromatography showed that the immunoreactive porcine brain natriuretic peptide of the whole brain and heart extracts eluted mainly at an identical position to synthetic porcine brain natriuretic peptide-26. These findings indicate that porcine brain natriuretic peptide-like substance, distinct from rat brain natriuretic peptide, is present in high concentrations in the rat brain and cardiac atrium.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan