Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Primary structure of rat liver alkaline phosphatase deduced from its cDNA.

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.     

A disease with features of cutis laxa and Ehlers-Danlos syndrome

Summary A mother and daughter are described with light and electron microscopic, and biochemical abnormalities of their connective tissue characteristic of both cutis laxa and the Ehlers-Danlos syndrome. The mother was clinically normal, while her 8-year-old daughter exhibited loose, wrinkled skin and other clinical features of cutis laxa, and also fragility, bruisability and hyper-extensibility of the skin and poor healing of wounds, leaving cigarette paper scars, features characteristic of the Ehlers-Danlos syndrome. Light and electron microscopic studies of skin biopsy specimens and cultured skin fibroblasts from both individuals revealed reduced and distorted elastic fibres, a finding usually seen in cutis laxa. Electrophoretic studies of collagen excreted from cultured skin fibrobasts revealed in both individuals and alpha 2(I) chain with a molecular size smaller than usual. The father and elder daughter were normal by clinical, light and electron microscopic and electrophoretic studies. It was concluded from these findings that the mother and daughter represented a hitherto undescrbed disease of the connective tissue with dominant inheritance and variable expressivity.     

Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord

Axons containing both serotonin-like (5-HT)-LI and substance P-like (SP)-LI immunoreactivity were identified in all laminae of the cat spinal cord at the level of the lumbar enlargement. Using an immunologically-specific, double immunofluorescence method, coexistent 5-HT-LI and SP-LI immunoreactivity could be visualized in the same tissue section with appropriate FITC and rhodamine fluorescent filter sets. The fewest number of coexistent axons were observed in the superficial laminae of the dorsal horn, while their number increased in the more ventral dorsal horn laminae. Numerous coexistent axons were observed in the area adjacent to the central canal. The greatest number of coexistent axons was found in the ventral horn, especially in the motoneuronal cell groups. This study demonstrates that axons containing coexistent 5-HT-LI and SP-LI immunoreactivity are found in all laminae of the cat lumbar spinal cord and are thus involved in both sensory and motor functions. Their more frequent occurrence in the ventral horn suggests a greater role for coexistent 5-HT and SP in motor function. Since axons containing coexistent 5-HT and SP, and those containing only 5-HT, likely originate from different populations of neurons, our observations provide evidence for a diverse origin of descending 5-HT afferents to the different spinal laminae.     

Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

Clinical studies on cell-mediated immunity in patients with renal cell carcinoma: interleukin-2 and interferon-γ production of lymphocytes

Summary The authors examined interleukin-2 (IL-2) production and interferon (IFN) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, 5 cm and >5 cm. The production of IL-2 and IFN was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN production was impaired. However, in the patients with tumors 5 cm, IFN production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN production.     

Effects of chronic bifemelane hydrochloride administration on receptors for N-methyl-d-aspartate in the aged-rat brain

We assayed N-methyl-d-aspartate (NMDA) receptors [³H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([³H]CPP) bindings) and evaluated their distribution in the brain by quantitative autoradiography in young adult and aged rats. In the young adult rats, NMDA receptors were present at relatively high concentrations in the cerebral cortex and hippocampus. In the aged rats, NMDA receptors were decreased in the nealy all areas of the brain, especially in the cerebral cortex and hippocampus. Chronic administration of bifemelane hydrochloride, a drug for sequela of cerebrovascular diseased, at a dose of 15 mg/kg/day for 14 days, markedly attenuated these decrease in NMDA receptors. Since NMDA receptors are considered to be involved in memory and learning processes, our results suggest that bifemelane hydrochloride may be applicable to the treatment of disturbed memory and learning.     

Generation of 19 STS markers that can be anchored at specific sites on human chromosome 21.

Sequence-tagged sites (STSs) are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR) and can be used to construct long-range physical maps of chromosomal DNA. These STSs can be detected by PCR assays developed by reference to data obtained from the sequencing of restriction fragment length polymorphism-DNA markers for chromosome 21, which were derived from recombinant lamba-phage and plasmid clones made from DNA of a human-hamster hybrid cell line. In this report, we describe the generation of 19 new STSs that are specific for human chromosome 21.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.