Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.

Uptake and oxidation of acetaldehyde by intact human erythrocytes.

Human erythrocytes were examined for acetaldehyde-oxidizing capacity of intact cells, in comparison with its uptake under various conditions. The oxidation was found to be predominant over other possible acetaldehyde-metabolizing pathways, particularly with low concentrations of acetaldehyde. Further investigation using three media which differed in the initial pyruvate level showed an independence of acetaldehyde oxidation rate up to the high cellular lactate/pyruvate concentration ratio. The apparent non-oxidative acetaldehyde uptake obtained by subtraction of oxidation from the total uptake, was reduced when pretreating the erythrocytes with pyridoxal but not with pyridoxal 5'-phosphate. Though N-ethylmaleimide also inhibited the non-oxidative uptake, another sulfhydryl blocking reagent, p-chloromercuribenzoate was without effect. Thus, in addition to the oxidation, acetaldehyde may be involved in a reaction such as binding to the red cell components, as the minor metabolizing pathway in the erythrocytes.

Apoptosis and expression of Bcl-2 and Bax proteins in invasive ductal carcinoma of the pancreas.

The Bcl-2 family of genes plays important roles in the regulation of apoptosis. The present study was designed to assess the clinicopathologic significance of apoptosis and the expression of the apoptosis-inhibitory Bcl-2 protein (pBcl-2) and the apoptosis-promoting Bax protein (pBax) in human invasive ductal carcinomas (IDCs) of the pancreas. The present study included 66 IDCs that were resected between 1982 and 1998. Apoptosis was assessed by the in situ nick end labeling method and pBcl-2 and pBax were stained immunohistochemically. Apoptosis was quantified as the apoptotic index (AI, the percentage of apoptotic cells of the total tumor cells), and a high AI (>10%) was observed in 26 of the 66 (39%) IDCs. The AI correlated significantly with the extent of nodal involvement. pBax immunoreactivity was detected in 42 of 66 IDCs (64%), and pBax expression was significantly correlated with female gender and showed a significant negative correlation with the extent of nodal involvement. pBcl-2 was expressed in 16 IDCs (24%) but did not show any correlation with the clinicopathologic factors. The AI did not correlate with the expression of pBcl-2 or pBax, but there was a significant correlation between the expression of pBcl-2 and that of pBax; 15 of the 16 pBcl-2(+)IDCs were also pBax(+), and only one pBcl-2(+)IDC was pBax(-). Univariate analysis demonstrated that the degree of apoptosis had no significant influence on the patients' prognosis, pBax or pBcl-2 expression was significantly associated with a better prognosis, and in particular, the pBax(+)pBcl-2(+) group had a significantly higher survival than the other groups. On the other hand, the survival curve of the adjuvant chemotherapy (ACT) group was also higher than that of the surgery alone (SA) group, with borderline statistical signfiicance. The ACT group showed a significantly better survival rate than the SA group for the pBax(+)IDC patients, but the AI and pBcl-2 expression were not correlated with an improved survival rate in the ACT group. Multivariate analysis showed that the AI. pBcl-2 expression, and pBax expression by themselves did not represent significant variables for death owing to IDC, but pBax expression was significantly associated with the efficacy of ACT. In conclusion, pBax expression may be essential for pBcl-2 expression. pBcl-2 and pBax expressions are not significant prognostic factors for patients with IDC, but pBax expression may be beneficial in predicting the effects of ACT on patients with IDC.

Effect of disulfiram and its reduced metabolite, diethyl-dithiocarbamate on aldehyde dehydrogenase of human erythrocytes.

Inhibition of human erythrocyte aldehyde dehydrogenase (ALDH) activity was studied using disulfiram and its reduced metabolite, diethyldithiocarbamate (DDC). The enzyme was rapidly inactivated by disulfiram and the inhibition was protected by reduced glutathione (GSH), in a concentration dependent manner when the enzyme premixed with GSH was reacted with disulfiram. Higher reactivity of the thiol group of the enzyme than that of GSH to disulfiram was suggested from the observation that half of the enzyme activity was inhibited when the ratio of disulfiram to GSH was 1:10. Although DDC alone showed no inhibitory effect on the enzyme, inactivation was mediated by a low concentration of heme-containing peroxidases, but not by methemoglobin. Under this condition, the inhibition potential was not protected, even with a high concentration of GSH. The constant reoxidation system of DDC is probably directly related to the enzyme inactivation.

Plant polysaccharide PSK: cytostatic effects on growth and invasion; modulating effect on the expression of HLA and adhesion molecules on human gastric and colonic tumor cell surface.

PSK is a plant polysaccharide widely used for cancer immunotherapy in Japan and other Asian countries. It is considered that its antitumor effect is derived from its immunomodulating activity on the tumor-bearing host. The present study was designed to assess the direct action of PSK on in vitro proliferation and invasion of human KATO-3 gastric and Colo205 colonic cancer cell lines, and the expression of surface molecules such as HLA and adhesion molecules on these cells. The in vitro growth of KATO-3 cells was significantly inhibited by 100 micrograms/ml of PSK 48 hrs after culture initiation, and that of Colo205 was significantly inhibited by 10 and 100 micrograms/ml of PSK 24 hrs after culture initiation. The effect of PSK on the in vitro invasion of the tumor cells, assessed with a Matrigel invasion chamber, revealed that invasion of KATO-3 and Colo205 cells was inhibited by more than 10 micrograms/ml and more than 5 micrograms/ml of PSK, respectively. KATO-3 cells expressed HLA-ABC, HLA-A2/A28, HLA-DR very weakly, at almost baseline levels, but HLA-B27, B2-microglobulin and HLA-DQ were expressed at various levels. After treatment of KATO-3 cells with PSK, the expression of HLA-B27 and beta 2-microglobulin was significantly enhanced. Colo205 cells expressed all class-I antigens tested in this study at different levels, but class-II antigens at almost baseline levels. PSK also enhanced the expression of class-I antigens on Colo205 cells. ICAM-1 was expressed on KATO-3, but not on Colo205. The expression of ICAM-1 was enhanced to a greater extent by treatment with 10 micrograms/ml than with 100 micrograms/ml of PSK. Adenocarcinoma antigen AC-81 was strongly expressed on both cell lines, but PSK-treatment significantly enhanced its expression. These results suggested that enhancement of HLA class-I expression on tumor cells after PSK treatment may be one of the mechanisms responsible for the induction of anti-tumor immunity by PSK.

Induction of tumor-specific antitumor immunity after chemotherapy with cisplatin in mice bearing MOPC-104E plasmacytoma by modulation of MHC expression on tumor surface.

Chemotherapy sometimes results in induction of specific antitumor immunity. We investigated the mechanisms responsible for the induction of antitumor immunity in mice bearing MOPC-104E plasmacytoma after chemotherapy with cisplatin (CDDP), especially the effects of CDDP on the expression of MHC on the tumor surface. BALB/c mice were subcutaneously (s.c.) inoculated with MOPC-104E cells on day 0, then intravenously (i.v.) treated with CDDP at 3.6 mg/kg on day 7. The tumors disappeared completely on day 35 and the mice rejected a second challenge with MOPC-104E, but did not reject syngeneic Meth-A fibrosarcoma. The tumors did not regress, however, when MOPC-104E was s.c. transplanted in nude mice, or when anti-T-cell monoclonal antibodies were i.v. injected into BALB/c mice before CDDP treatment on day 6. To determine which of the mice or tumor were affected by CDDP, BALB/c mice were inoculated with CDDP-treated (12.5 micrograms/ml for 3 hours in vitro) MOPC-104E cells on day 0, 7 and 14. The potential to reject MOPC-104E was lower in mice immunized with ethanol-treated MOPC-104E cells than in those immunized with CDDP-treated cells. CDDP-treated or -untreated MOPC-104E cells ultrasonicated and fractionated into soluble and insoluble fractions by centrifuging, also induced antitumor immunity. Flow cytometry demonstrated that the expression of MHC-class-I antigens H-2Dd and H-2Kd was enhanced after CDDP-treatment, but that of class-II antigens I-Ad and I-Ed was not, suggesting that CDDP induced tumor-specific antitumor immunity by enhancing the expression of MHC-class-I antigens.

Reduced invasiveness and metastasis of Chinese hamster ovary cells transfected with human interleukin-17 gene.

Human IL-17 (hIL-17) stimulates epithelial, endothelial, fibroblastic cells and macrophages to secrete various cytokines. The present study was designed to assess the effects of the transfection of the hIL-17 gene in Chinese hamster ovary (CHO) cells. A complementary DNA (cDNA)-encoding hIL-17 was obtained by polymerase chain reaction (PCR) amplification from human CD4+ T-cell cDNA and inserted into the plasmid pRc/CMV to construct an expression vector for hIL-17. CHO cells were transduced with hIL-17 DNA-carrying cytomegalovirus (CMV)-based retroviral vectors. A clone with a high mRNA expression of hIL-17 (CHO/IL-17) was selected by Northem blotting. There was no significant difference in the in vitro growth of cells among parent CHO cells, vector-only transfected cells (CHO/neo) and CHO/IL-17 cells. A Matrigel invasion chamber assay, however, demonstrated significantly lower invasiveness by CHO/IL-17 cells than by either the parent CHO or the CHO/neo cells. There was no difference in the in vivo growth among the cells, when subcutaneously transplanted into nude mice. When injected into the tail vein, however, the number of metastatic nodules in the lungs of CHO/IL-17-injected mice was significantly smaller than that of CHO- or CHO/neo-injected mice. Furthermore, NK activity of spleen cells was significantly higher in nude mice transplanted with CHO/IL-17 cells than in mice transplanted with parent CHO or CHO/neo cells. In conclusion, the hIL-17-gene-transfected CHO cells showed a significantly lower metastatic potential to the lung by directly modulating the invasiveness and metastasis of CHO cells as well as by enhancing NK activity.

Correlation between human erythrocyte aldehyde dehydrogenase activity and sensitivity to alcohol.

Young healthy Japanese men were given 0.48 g ethanol/kg body weight orally. Those responding with a marked increase in heart rate after alcohol also exhibited facial flushing and had higher acetaldehyde levels than those not responding, in spite of similar blood alcohol levels. The activity of aldehyde dehydrogenase in erythrocytes was found to correlate significantly (r=-0.73, p < 0.01) with the increase in heart rate after alcohol drinking. It is suggested that erythrocyte aldehyde dehydrogenase activity affects or reflects blood acetaldehyde levels and physiological response to alcohol, and may prove useful as a marker for alcohol sensitivity in Orientals.

Intraperitoneally implanted artificial pancrease with transkaryotic beta-cells on microcarrier beads in a diffusion chamber improves hyperglycemia after 90% pancreatectomy in rats.

BACKGROUND: Considering the difficulties in pancreas transplantation, the development of an artificial pancreas would be of great value. We have established a transkaryotic artificial beta-cell line, CHO/I, produced by transfecting the human proinsulin gene into the Chinese hamster ovary (CHO) cell line. The present study was designed to assess the value of an artificial pancreas using a diffusion chamber containing CHO/I cells. MATERIALS AND METHODS: Wistar rats rendered diabetic by 90% pancreatectomy were treated by implanting a diffusion chamber containing CHO/I cells cultured on microcarrier beads. RESULTS: The diffusion chamber containing microcarrier beads produced 100-folds more CHO/I cells than the chamber alone, as calculated from the secreted IRI in vitro. When diffusion chambers containing CHO/I cells on microcarrier beads were implanted into the peritoneal cavity of the 90% pancreatectomized rats, the fasting serum IRI level increased and the fasting blood glucose decreased to the normal level for 12 weeks. An intraperitoneal glucose tolerance test demonstrated, however, that the diffusion-chamber-implanted rats did not respond to glucose loading. CONCLUSION: An artificial pancreas using a diffusion chamber containing human proinsulin gene transfected cells might be a promising model for future clinical application.

Sensitive fluorometric detection of heat-stable alkaline phosphatase from bloodstains for the forensic diagnosis of pregnancy.

Sensitive and simple procedures are established for the forensic pregnancy test from bloodstains. This paper describes new techniques based on heat stability of placental alkaline phosphatase. In this study the total and heat-stable alkaline phosphatase activities were determined using 4-methylumbelliferyl-phosphate as substrate. The ratio of heat-stable alkaline phosphatase to total alkaline phosphatase was calculated. A more simple screening test which was visible by exposure to UV light is described. These may be helpful in the diagnosis of pregnancy from only a slight bloodstain.

[Pharmacokinetic and clinical studies of cefuzonam in obstetric and gynecologic infections].

Cefuzonam (CZON, L-105), a new semisynthetic cephem antibiotic, was studied in the field of obstetrics and gynecology, pharmacokinetically and clinically. Following is a summary of the results. 1. Concentrations of CZON in serum and genital organs following intravenous drip infusion of 1 g of the drug over 60 minutes were measured. Samples were taken during 40 to 190 minutes after the end of the infusion. Ratios of concentrations of the drug transferred into genital organs to the concentration in the cubital venous serum were 0.5 to 1.5 in many cases. Levels of the drug in the genital organs examined well exceeded MICs for common causative organisms in obstetric and gynecologic infections. 2. Therapeutic effects of 1 g or 2 g of CZON by drip infusion twice daily were evaluated in 8 patients. Overall clinical efficacy was 62.5% (5/8). No side effects or abnormal laboratory findings due to the drug were noted. The results suggest that CZON is a useful antibiotic for obstetric and gynecologic infections.

[A case of recurrent pancreatic cancer brought into a complete response by a multimodal treatment with intraarterial chemotherapy and radiotherapy].

We achieved a complete response of recurrent pancreatic cancer using a multimodal treatment with intravenous, oral and intraarterial chemotherapies and radiotherapy. A 55-year-old female patient had a recurrent pancreatic cancer, which had invaded the portal vein, 2 years after pancreatoduodenectomy. Angiography demonstrated prominent stenosis of the portal vein, which was enlarged by the insertion of a metalic stent to maintain blood flow to the liver. Chemotherapy included intravenous mitomycin C, 5-FU and 4'-epirubicin (EPI), oral UFT and cyclophosphamide, and intraarterial cisplatin, 5-FU and EPI through a catheter inserted into the celiac artery. Furthermore, the patient received a total of 50 Gy radiotherapy. Four months after the initiation of therapy, a computed tomography image demonstrated a complete disappearance of the recurrent tumor and a prominent decrease in the serum CA19-9 level. At present, 11 months have passed after the initiation of therapy, and she has been followed at our outpatient department without any symptoms of recurrence.

Effects of aging on alpha 1-adrenoceptor mechanisms and the inhibitory effect of diltiazem on noradrenaline maximum response in isolated rat aortic preparation.

The effects of aging on alpha 1-adrenoceptor mechanisms in aortic preparations isolated from 3-, 6-, 10-, 18-, and 40-week-old rats were studied and compared with serotonin receptor mechanisms in the same preparations. The potency (pD2 value) of noradrenaline increased with age from 3 to 10 weeks, but decreased thereafter with age from 10 to 40 weeks. The affinity (pKA value) of noradrenaline and of prazosin (pA2 value) did not alter with aging. The change in potency or the pD2 value of noradrenaline was proportional to receptor reserve (pD2-pKA value) for noradrenaline, suggesting that the change of potency of noradrenaline with age was due to a change of receptor reserve, but not to change of drug affinity to alpha 1-adrenoceptors. The potency (pD2 value) and affinity (pKA value) of serotonin, and the affinity (pA2 value) of ketanserin, did not alter with aging, suggesting that serotonin receptor mechanisms in rat aorta did not change with age. The inhibitory effect of diltiazem on noradrenaline maximum response decreased with age from 3 to 10 weeks, but increased with age from 10 to 40 weeks. An inverse relationship between changes of diltiazem inhibition and receptor reserve of noradrenaline was found. Diltiazem's inhibitory effect on serotonin maximum response did not alter with aging.

Comparative study of phenotypes on activity and plasma concentration in the genetic system of plasminogen.

Plasma antigen concentration of plasminogen was approximately 11-13 mg/100 ml in all phenotypes. Specific activities of common PLG 1-1 and second common PLG 2-1 were 16.52 +/- 1.43 U/mg (caseinolytic activity/milligram antigen concentration, mean +/- SD) and 17.22 +/- 2.14 U/mg, respectively. Caseinolytic activity, antigen concentration and specific activity of PLG 1-B were 0.80 +/- 0.23 U/ml, 11.39 +/- 2.44 mg/100 ml and 6.95 +/- 0.96 U/mg. Plasma plasminogen levels of three rare phenotypes (PLG 3-1, PLG 1-C and PLG 1-M) were at least in the normal ranges by immunological and biological assay.