Yukikazu Saeki

Impaired Activation of Glucose Oxidation and NADPH Supply in Human Endothelial Cells Exposed to H2O2 in High-Glucose Medium

The effects of glucose concentration on D-glucose oxidation and reduced nicotinamide adenine dinucleotide phosphate (NADPH) supply were studied during exposure of cultured human umbilical vein endothelial cells to hydrogen peroxide (H2O2). The activation of glucose oxidation via the pentose phosphate pathway (PPP), induced by exposure of cells to 200 μmol/l H2O2 for 1 h, was reduced by 50% (P < 0.01) in cells cultured for 5–7 days in 33 mmol/l D-glucose (HG) versus those cultured in 5.5 mmol/l D-glucose without (NG) or with (HR) 27.5 mmol/l D-raffinose. The intracellular NADPH content in HG cells, but not in NG or HR cells, was decreased by 42% (P < 0.01) by exposing cells to 200 μmol/l H2O2. The decrease in NADPH was dependent on D-glucose concentration in the medium and was prevented in glutathione (GSH)-depleted cells. The latter observation suggests that the decrease in NADPH is associated with activation of the GSH redox cycle. In the presence of 200 μmol/l H2O2, lactate release into the medium, NADH/NAD ratio, and phosphofructokinase activity in HG cells were 56, 53, and 68% greater, respectively, than in the NG group, which indicates that inhibition of glycolysis by H2O2 is less marked in the HG group compared with NG group. These results indicate that activation of the PPP was impaired in endothelial cells cultured under conditions of high-glucose and oxidative stress, resulting in a decreased supply of NADPH to various NADPH-dependent pathways, including the GSH redox cycle.

Deficiency of Cardiac β-Adrenergic Receptor in Streptozocin-Induced Diabetic Rats

The number of β-adrenergic receptors in cardiac myocytes isolated from rats made diabetic with streptozocin (STZ) for 10 wk was measured by use of a hydrophilic nonselective antagonist [3H]CGP 12177 and was found to decrease to 59% of the number in control rats (P < .05), without any change in affinity. Similarly, using [125I]iodocyanopindolol as a ligand, we found a decrease in the β-adrenergic-receptor number on cardiac plasma membrane isolated from the diabetic rats [29% decrease (P < .05) at 1 wk, 50% (P < .01) at 3 wk, and 49% (P < .01) at 10 wk compared with control rats]. However, the serum triiodothyronine level that had been known to modulate the β-adrenergic-receptor-adenylate cyclase system was decreased in the 1-wk-diabetic rats but not in the 10-wk-diabetic rats compared with each control group. Furthermore, there was no difference in urinary catecholamine excretion between diabetic and control groups. In the 10-wk-diabetic rats, the response of adenylate cyclase to isoproterenol was significantly defective (56% decrease compared with control rats; P < .05), although both the basal and the forskolin-stimulated maximum adenylate cyclase activities and a half-maximum concentration of isoproterenol for the stimulation of adenylate cyclase were similar in control and diabetic rats. On the other hand, both cholera toxin-dependent and islet-activating protein-dependent [32P]NAD incorporations into cardiac plasma membrane were markedly increased in the diabetic rats. The 2-wk insulin treatment improved not only the number of β-adrenergic receptors but also the response of adenylate cyclase to 10 μM isoproterenol. These results indicate that cardiac unresponsiveness to a β-adrenergic agonist in STZ-induced diabetic rats is specifically associated with a deficiency in the β-adrenergic-receptor concentration in cardiac plasma membrane.

Identification of RB1CC1, a novel human gene that can induce RB1 in various human cells.

Multidrug resistance to anti-cancer agents (MDR) is a major barrier to successful cancer treatment. Current knowledge about genes that contribute to MDR is limited, however, and its mechanisms remain unclear. To identify genes involved in MDR, we performed differential display analysis and isolated a novel human gene, RB1CC1 (RBI-inducible Coiled-Coil 1). The 6.6-kb RB1CC1 cDNA encodes a putative 1594-amino-acid protein that contains a nuclear localization signal, a leucine zipper motif and a coiled-coil structure. Western blot analysis and immunocytochemical staining with anti-RB1CC1 antibody showed that endogenously expressed RB1CC1 protein localized to the nucleus. In MDR variants of human osteosarcoma cells, RB1CC1 expression increased in response to doxorubicin-induced cytotoxic stress and remained elevated for the duration of drug treatment. RB1CC1 expression levels correlated closely with those of RB1 (retinoblastoma 1 ) in cancer cell lines as well as in various normal human tissues. Moreover, introduction of wild-type RB1CC1 significantly induced RB1 expression in human leukemic cells. These data suggest that RB1CC1 may be a key regulator of RB1 gene expression.

Luminol chemiluminescence reaction catalyzed by a microbial peroxidase

A peroxidase produced by microorganisms belonging to the genera Arthromyces and Coprinus was found to be a potent catalyst for the chemiluminescent oxidation of luminol, the luminescence produced per unit of microbial peroxidase protein being well over 100 times as strong as that produced by horseradish peroxidase. No large difference in Km value for H2O2 in the presence of luminol was found between Arthromyces ramosus peroxidase and horseradish peroxidase (7.0 and 15.5 microM, respectively), but Vmax of the Arthromyces peroxidase was 500 times greater than that of the horseradish peroxidase. It was also found that the Arthromyces peroxidase surpasses, beyond expectation, the horseradish peroxidase in the initial velocity of the chemiluminescence reaction with the stopped-flow method. The Arthromyces peroxidase was used for the glucose and cholesterol assays, which were notably more sensitive than the corresponding assays involving the horseradish peroxidase.

Elevated contents of spermidine and spermine in the erythrocytes of cancer patients

Red blood cells (RBC) from 69 patients with advanced cancer and 37 healthy controls were subjected to polyamine determination by using high‐performance liquid chromatography. The polyamine contents in normal human RBC were spermidine 15.04 ± 3.63 nmol and spermine 8.82 ± 3.12 nmol per 1010 RBC. Spermidine and spermine levels in RBC were elevated in patients with cancer (p < 0.005). Serial studies in seven patients with cancer indicated that both polyamines in RBC were reduced after successful surgery. Our data indicate that the determination of polyamine levels in RBC is clinically useful as a marker of disease activity in patients with cancer.

Pyruvate Improves Deleterious Effects of High Glucose on Activation of Pentose Phosphate Pathway and Glutathione Redox Cycle in Endothelial Cells

In our previous study (Diabetes 44:520–526, 1995), endothelial cells cultured in high glucose condition showed impairment of an oxidant-induced activation of the pentose phosphate pathway (PPP) and a reduced supply of NADPH to the glutathione redox cycle. To gain insight into the mechanisms of this impairment, the protective effect of pyruvate was studied in human umbilical vein endothelial cells cultured in either 5.5 mmol/l glucose (normal glucose [NG] condition) or 33 mmol/l glucose (high glucose [HG] condition). Through pretreatment of cells with 0.2 mmol/l pyruvate for 5–7 days in the HG condition, glucose oxidation through the PPP and total cellular NADPH content in the presence of 0.2 mmol/l H2O2 were increased by 54 (P < 0.05) and 34%, respectively, and glutathione-dependent degradation of H2O2 in HG cells was enhanced by 41% (P < 0.01), when compared with those cells to which pyruvate was not added. The addition of pyruvate significantly reduced the fructose 1,6-bisphosphate (FDP) content and free cytoplasmic NADH/NAD ratio, estimated by increased pyruvate/lactate ratio in NG and HG cells exposed to H2O2. Furthermore, the addition of pyruvate also showed a 46% reduction (P < 0.01) of endothelial cell damage induced by H2O2 in HG cells. These results indicate that abnormalities in PPP activation and glutathione redox cycle activity induced by H2O2 in HG cells are compensated, and that the accentuated reductive stress is improved by an addition of pyruvate. These pyruvate effects are associated with protection against an oxidant-induced endothelial cell injury in the high glucose condition.

Crystallization and properties of aromatic amine dehydrogenase from Pseudomonas sp

An amine dehydrogenase was purified to homogeneity from an extract of a bacterium of the genus Pseudomonas grown in a medium containing beta-phenylethylamine as a sole carbon source and obtained in a crystalline form with about 100-fold purification. The purified enzyme catalyzed the oxidative deamination of various aromatic amines as well as some aliphatic amines to a lesser extent. An artificial electron acceptor such as phenazine methosulfate was required for the catalysis. The molecular weight determined by sedimentation equilibrium was 103,000 and the molecule seemed to be composed of two pairs of two nonidentical subunits (Mr 46,000 and 8000). The enzyme had a dull yellow-green color with an absorption maximum at 445 nm and this chromophore appeared to be involved in the catalytic action of the enzyme.

Characterization of a newly established human chondrosarcoma cell line, CS-OKB

Abstract A clonal cell line, CS-OKB, was derived from a human chondrosarcoma and characterized by cytogenetic study, immunocytochemical staining, and reverse transcriptase polymerase chain reaction (RT-PCR). Chromosomal abnormalities characteristic of malignant cartilaginous neoplasms were identified. CS-OKB cells were intensely stained with anti-type II collagen and anti-keratan sulphate antibodies. RT-PCR indicated that CS-OKB transcribes cartilage-specific genes such as type II, X procollagen, and aggrecan. This human chondrosarcoma cell line is stable and expresses well-differentiated chondrocyte-specific genes. It synthesizes well-differentiated chondrocyte-specific molecules in uncoated plastic dishes. CS-OKB may be useful for studies of human chondrocytes and in characterizing human chondrosarcomas.

Diurnal variation of dopamine content in the rat pineal gland.

Abstract Levels of norepinephrine and dopamine in the rat pineal gland were determined by a radioenzymatic assay with modifications to separate the reaction products. Catecholamines were converted to 3-O-methylated derivatives in the presence of catechol-O-methyltransferase (EC 2.1.1.1) and S-adenosyl-L-[methyl- 3 H]-methionine. Following solvent extraction of the labelled normetanephrine and 3-methoxytyramine, the amines were separated by high-performance liquid chromatography. Contents of both catecholamines in the pineal gland varied with a 24-hr rhythm. The content of norepinephrine was maximal at about 6 A.M. (lights on from 8 A.M. to 8 P.M.) and declined gradually thereafter. In contrast to the level of norepinephrine, the dopamine level was highest at about 0 A.M. and fell rapidly to reach a trough after the lights were turned on. These observations suggest that the diurnal variation of norepinephrine is generated by changes in the contents of dopamine in sympathetic nerve terminals innervating the pineal.

Isolation, characterization and mapping of the mouse and human RB1CC1 genes

RB1CC1 (RB1-inducible Coiled-Coil 1), a putative transcription factor implicated in the regulation of RB1 (retinoblastoma 1) expression, was recently identified in a screen for genes involved in multi-drug resistance to anticancer agents. Information about the RB1CC1 gene is limited, however, and its biological function is not determined. Here we report the isolation, characterization and mapping of the mouse RB1CC1 gene (Rb1cc1), together with further characterization of the human RB1CC1 gene. Mouse Rb1cc1 encodes 1588 amino acids, sharing 89% identity and key sequence motifs with its human counterpart. Rb1cc1 is expressed abundantly in heart and testis, with lower levels detected in lung and spleen. Immunohistochemical analysis revealed the Rb1cc1 and Rb1 proteins are co-localized in the cell nuclei of NIH3T3-3 cell and various mouse tissues. The human and mouse RB1CC1 genes, both of which contain 24 exons, span 74 kb on chromosome 8q11.2 and 57 kb on chromosome 1A2-4, respectively. Conserved sequence motifs and nuclear localization suggest that the RB1CC1 proteins function as transcription factors.