Distribution of gangliosides in parenchymal and non-parenchymal cells of rat liver

Parenchymal and non-parenchymal cells were isolated from rat liver with purities of more than 90%. Total and ganglioside sialic acid contents were higher in non-parenchymal cells than in parenchymal cells. Thin-layer chromatography of gangliosides showed that the main component in rat liver was ganglioside GM3 and that this was abundant in non-parenchymal cells. Parenchymal cells had ganglioside GD1b as the main component and less GM3 than non-parenchymal cells. These results suggested that the main ganglioside of rat liver, GM3, arises mainly from non-parenchymal cells.     

Complete nucleotide sequence of human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) cDNA and a comparison with human and rat PRPS gene families.

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) were isolated from a glioblastoma cell line MGC 1 cDNA library. The longest clone contained 2,075 base pairs (bp) almost covering the 2.3-kb mRNA and the base sequence of the coding region (954 bp) had a 92.0% sequence homology with that of rat PRS I cDNA. The deduced amino acid sequences were identical between human and rat PRS I. This perfect conservation has heretofore not been reported for other enzymes involved in nucleotide metabolism and glycolysis. A comparison with other isoforms of this enzyme, PRS II and PRS III, showed that the human PRS I was 79.9 and 92.2% homologous in the coding sequence and 95.3 and 94.0% in the deduced amino acid sequence to human PRS II and PRS III, respectively. The high value of the synonymous difference between PRS I and PRS II cDNAs places their time of divergence long before that of the radiation of mammals. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago.     

The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene

In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.     

The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

Establishment and characterization of new cell lines from human renal cell carcinoma

We have characterized seven human renal cell carcinoma cell lines established from primary sites of five patients between 1987 and 1989. Two lines, OUR-20P and OUR-20S, were derived from the OUR-20 cells by cloning with a dilution method 3 months after the primary culture. These three cell lines were tumorigenic in athymic nude mice when inoculated subcutaneously. Examined by a dye uptake method, OUR-20 was highly sensitive to interferon-alpha (IFN-alpha); OUR-20P, OUR-20S and OUR-30 showed slight sensitivities, while the other three cell lines were insensitive. All seven cell lines have been maintained for more than 2 years and over 50 passages in vitro. Cytogenetic analyses performed 1.5 to 3 years after the starts of primary cultures indicated that all seven cell lines, which exhibited different morphologies in phase-contrast micrographs, were aneuploid with modal chromosome numbers 41 to 89.     

An improved method for determination of N-acetyl-L-glutamate by its function as an activator of carbamoyl phosphate synthetase I

N-Acetyl-L-glutamate has been examined with regard to its ability to activate carbamoyl phosphate synthetase I (EC 6.3.4.16). Substance(s) inhibitory to carbamoyl phosphate synthetase, present even in the partially purified preparation of rat liver extracts, interfered with the measurement of acetylglutamate. In the experiments using chelating agents, metals were apparently involved in this inhibition. When the partially purified preparation of liver extract was placed on a Chelex 100 column, the inhibitor was eliminated and accurate measurements of acetylglutamate content could be made. Evidence supporting the validity of this improved method is given. A significant difference was observed between acetylglutamate levels determined by the present method and by the one using aminoacylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) to hydrolyze acetylglutamate followed by assay of the glutamate generated. We searched for the presence of glutamate derivatives other than acetylglutamate. When impure tissue preparations containing acetylglutamate were treated with a commercial preparation of aminoacylase, there was an excess amount of glutamate apparently derived from compounds other than acetylglutamate. This can lead to an overestimation of the tissue levels of acetylglutamate.     

Selective Inhibition of Acetylcholinesterase in the Cerebellum and Hippocampus of Mice Following an Acute Treatment with Malathion

Adult male ICR mice were treated by intraperitoneal injection with 250?mg/kg of bodyweight of commercial malathion (a dose corresponding to 1/12 the LD50). After 6?h, acetylcholinesterase (AChE) activity in blood, liver, and six brain regions was determined. A statistically significant inhibition was observed in whole blood (23%), liver (21%), and, in particular, the central nervous system; the greatest degree of AChE inhibition was observed in the cerebellum (45%), followed by the hippocampus (29%). There was no significant change in AChE activity in the caudate putamen, frontal cortex, midbrain, or pons medulla. These results demonstrate that the magnitude of AChE inhibition in peripheral tissues does not accurately reflect the central-inhibitory effects of malathion on AChE activity in specific brain regions.     

Estimation of carbon biomass and community structure of planktonic bacteria in Lake Biwa using respiratory quinone analysis

The relationship between bacterial respiratory quinone (RQ) concentration and biomass was assessed for Lake Biwa bacterial assemblages to evaluate the utility of bacterial RQ concentration as an indicator of bacterial carbon. The biomass estimated from the RQ concentration correlated well with that from cell volume, indicating that RQ concentration is an appropriate indicator of bacterial biomass. The estimated carbon content per unit of RQ (carbon conversion factor) of bacteria was 0.67 mg C nmol RQ^?1. Bacterial carbon biomass, which was estimated from the RQ concentration using the conversion factor, ranged between 0.008 and 0.054 mg C L^?1 (average 0.025 mg C L^?1) at 5 m depth and between 0.010 and 0.024 mg C L^?1 (average 0.015 mg C L^?1) at 70 m depth. Ubiquinone-8-containing bacteria dominated the epilimnion and hypolimnion. Compared to conventional image analysis, bacterial RQ analysis is a less laborious method of simultaneously determining bacterial biomass and community.     

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.