Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.     

Overt diabetes induced by overeating in neonatally STZ-treated impaired glucose tolerant mice: long-term follow up study

This study has investigated the effect of a long period of overeating on the glycemic control and pancreatic beta-cell function in neonatally streptozocin treated impaired glucose tolerant mice. Neonatally streptozocin (60 mg/kg) treated male ICR mice with 150-200 mg/dl of fed blood glucose levels were divided into two groups at 6 weeks of age. One group was maintained on a cafeteria diet (SZC) and the other on ordinary mouse chow (SZ) until 30 weeks of age. Normal male ICR mice were divided into a cafeteria diet group (CC) and an ordinary chow group (Cont). SZC and CC consumed 134-124% of the caloric intake in SZ and Cont throughout the study. Marked elevation of the fed blood glucose level was observed and the glucose tolerance was progressively impaired in SZC. On pancreas perfusion at 30 weeks of age, insulin secretion to 30 mM glucose in SZC was significantly decreased compared with that in SZ. That in CC was slightly decreased compared with that in Cont. The pancreatic insulin concentration in SZC was significantly less than that in SZ. We conclude that chronic hyperglycemia, induced by the long period of overeating, accelerated the selective loss of beta-cell sensitivity to glucose. Even in normal mice that did not have marked hyperglycemia, insulin secretion to glucose was suppressed, probably by chronic stimulation of the beta-cell due to the long period of dietary excess.     

H-ras gene is expressed at the G1 phase in primary cultures of hepatocytes

The expression of c-H-ras and proliferating cell nuclear antigen (PCNA) in primary cultures of rat hepatocytes was determined in order to elucidate the relationship between the c-H-ras gene and the S phase of the cell cycle. In cells treated with EGF, elevation of c-H-ras expression was detected at the 22nd, 34th, 44th, and 54th h after plating, PCNA expression and DNA synthesis were detected at the 44th and 54th h. In cells without EGF treatment, only c-H-ras expression was detected at the 44th and 54th h. In our previous report, we showed that c-myc expression increased within several hours after plating, suggesting that isolated hepatocytes traverse from G0 to G1 under culture conditions, regardless of EGF treatment. These results clearly showed that the c-H-ras gene of adult rat hepatocytes was expressed in the mid-to-late G1 phase of the cell cycle as well as in the early S phase in primary culture.     

Increased renal TXA2 synthesis in diabetes mellitus: simultaneous determination of urinary TXB2 and 2,3-dinor-TXB2

The present study was designed to determine urinary excretion of kallikrein(KAL)-kinin as well as prostaglandin (PG) E2, TXB2 and 2,3-dinor-TXB2, a major urinary metabolite of TXA2 synthesized in platelets, by specific RIAs in patients with diabetes mellitus (DM). KAL or kinin excretion in 26 type II DM did not differ from control values obtained in 18 age-matched healthy subjects (C), although DM with HbA1 greater than 11% excreted less KAL. Urinary PGE2 excretion (7.6 +/- 2.8 ng/mg creatinine, mean +/- SE) was significantly lower in DM compared to C (17.5 +/- 3.9, p less than 0.05), while DM excreted more TXB2 (0.57 +/- 0.09, p less than 0.01) and 2,3-dinor-TXB2 (0.56 +/- 0.12, N.S.) than C (0.19 +/- 0.02 or 0.33 +/- 0.01). DM with or without mild proteinuria demonstrated lower PGE2, but higher TXB2 and 2,3-dinor-TXB2 excretion. A positive correlation of TXB2/2,3-dinor-TXB2 with proteinuria was observed in this group. However, in DM with massive proteinuria over 500 micrograms/mg creatinine, TXB2 and 2,3-dinor-TXB2 excretion decreased to levels almost identical to C. As a whole, a ratio of TXB2 to PGE2 or 2,3-dinor-TXB2 in DM was significantly higher than in C. The results suggest that a relative preponderance of TXB2 to 2,3-dinor-TXB2 may indicate an augmented renal, in addition to platelet, TXA2 synthesis. An excessive vasoconstrictive and proaggregatory TXA2 renal synthesis, concomitant with a decrease in vasodilatory and antiaggregatory PGE2, may have profound effects on renal functions such as protein excretion in DM.     

Kinetic mechanism of pulmonary carbonyl reductase.

An IgG1 monoclonal antibody, Sulph I, reacting with sulphatide (3'-sulphogalactosylceramide), was produced by immunizing Balb/c mice with that glycolipid coated on Salmonella minnesota bacterial membrane. Radioimmunodetection of the binding of the monoclonal antibody to structurally related glycolipids adsorbed to microtitre plates or chromatographed on thin-layer plates was used to determine its binding epitope. The antibody showed similar binding avidity to three sulphated glycolipids: sulphatide, sulpholactosylceramide and seminolipid. Lysosulphatide did bind the antibody, but, compared with sulphatide, 30 times more antigen was needed for half-maximal binding. Bis(sulphogangliotriosyl)ceramide and bis-sulphogangliotetraosylceramide did not bind the antibody. These results suggest that terminal galactose-3-O-sulphate and part of the hydrophobic region of the glycolipid are recognized by the Sulph I antibody.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Induction of chromosomal aberrations in active oxygen-generating systems. II. A study with hydrogen peroxide-resistant cells in culture

Cells hyper-resistant to hydrogen peroxide (H2O2) were obtained from a Chinese hamster cell line (CHL) by repeated treatments with H2O2 at stepwise increasing concentrations. A clonal line (R-8) was approximately 10 times more resistant to H2O2 than the parental cells, and retained its resistance for about 2 months in normal medium. However, with further passages after the completion of the present study, the elevated resistance gradually decreased. Although the concentration of H2O2 required to induce chromosomal aberrations in 50% of treated cells was about 10 times higher in R-8 than in the parental cells, there were no distinct differences between the cells in the induction of chromosomal aberrations by 3 alkylating agents (N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea and mitomycin C). The catalase activity of R-8 was 10-fold in comparison with the parental cells, but no obvious differences were seen in the activities of superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase. Therefore, the elevated H2O2-resistance seemed to be associated with the enhanced catalase activity. The induction of chromosomal aberrations in two O2- generating systems--xanthine oxidase plus hypoxanthine (XO + HX), and paraquat--was compared between R-8 cells and the ordinary CHL cells. XO + HX produced chromosomal aberrations in the parental cells but not in the R-8, while paraquat induced almost the same level of aberrations in both cell lines. This finding suggests that different active oxygens are responsible for the induction of aberrations in these two O2- generating systems, i.e., H2O2 in XO + HX and O2- in paraquat.     

Evidence of intracellular and trans-acting differentiation-inducing activity in human promyelocytic leukemia HL-60 cells: its possible involvement in process of cell differentiation from a commitment step to a phenotype-expression step

We previously reported that human promyelocytic leukemia HL-60 cells, when treated with various inducers in magnesium-deficient medium, became committed to differentiate but did not express the differentiation-related phenotypes (Okazaki et al., J. Cell. Physiol., 131:50-57, 1987). In the present study we demonstrated the existence of an intracellular differentiation-inducing activity (int-DIA) in differentiation-committed phenotype-nonexpressing HL-60 cells by using cybrid formation between untreated HL-60 cells and cytoplasts from HL-60 cells treated in magnesium-deficient medium with 100 nM 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell extracts from similarly treated HL-60 cells also showed int-DIA, which when added (10 mg total protein/ml) to culture of untreated HL-60 cells, could increase the percentages of nitroblue tetrazolium (NBT)- and nonspecific esterase (NSE)-positive cells from 1% to 53%, and from 0 to 32%, respectively. They also induced differentiation of human monoblastic leukemia U-937 cells and of human myeloblastic leukemia KG-1 cells but not of erythroleukemia K-562 cells. These results suggested that the int-DIA had a common effect on differentiation induction in several human myeloid cell lines and may be involved in inducing cells to proceed from a commitment to a phenotype-expression step during human myeloid cell differentiation.     

Prerequisite for the induction of lymphokine-activated killer cells from T lymphocytes

Human blood mononuclear cells were separated into Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells by means of a combination of the Percoll gradient method and C-mediated cytolysis using mAb. When purified Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells were cultured with rIL 2 (500 U/ml) for 6 days in a medium supplemented with 10% FCS, Leu-11+7-NK cells responded at the maximum level and Leu-11-7+ cells responded moderately as shown by both cell-proliferation response and cytotoxic activity generated. On the other hand, Leu-11-7-T cells did not respond at all to rIL-2. However, when Leu-11-7-T cells were cultured with rIL-2 in a medium supplemented with 10% autologous serum, they showed considerable responsiveness to rIL-2. In addition, much greater response to Leu-11-7-T cells were produced by the addition of monocytes. Monocyte cytokines, neither IL 1, IFN-gamma, TNF, nor their combination were able to substitute for monocytes in the induction culture. In contrast, the response level of Leu-11+7- NK cells remained unchanged irrespective of supplementation with autologous serum to medium or the addition of monocytes to the culture. These results indicated that culture conditions in the experiments significantly affected the results as to determination of lymphokine-activated killer cell precursors, especially the result pertaining to the conversion of T lymphocytes to lymphokine-activated killer cells. Under appropriate conditions, not only NK cells but also T cells are important precursors of lymphokine-activated killer cells.