Malignant melanoma cells express a melanoma-associated antigen with interspecies cross-reactivity that is recognized by monoclonal antibody M2590. This unique antigen has been shown to be a simple ganglioside, GM3 (NeuAcα2-3Galβ1-4Glcβ1-Cer), in B16 melanoma cells (Hirabayashi, Y., Hamaoka, A., Matsumoto, M., Matsubara, T., Tagawa, M., Wakabayashi, S., and Taniguchi, M. (1955): J. Biol. Chem., 260, 13328-13333). In the present study we examined the enzyme activity that catalyzes the formation of the melanoma antigen from lactosylceramide (Galβ1-4Glcβ1-Cer) and CMP-N-acetylneuraminic acid (CMP-NeuAc) using melanoma cells derived from mice (C57BL/6) injected with B16 melanoma cells. The enzyme activity was expressed strongly in the melanoma tumor cells as compared with the activity found in normal mouse tissues. The enzyme (GM3 synthase) was successfully solubilized in the presence of 1% Triton X-100. Identification of the product was performed by TLC/enzyme-immunostaining with specific monoclonal antibodies. The optimum pH of the GM3 synthase was 6.3. For maximum enzyme activity, Triton X-100 was required as a detergent. Apparent Km values for CMP-NeuAc and lactosylceramide were about 0.42 and 0.057mM, respectively. The sialyltransferase activities acting on paragloboside (Galβ1-4GlcNAcβ1-4Galβ1-4Glcβ1-Cer) and asialo-GM1 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-Cer) to form α2-3 and α2-6-sialylparagloboside (NeuAcα2-3Galβ1-4GlcNAcβ1-4Galβ1-4Glcβ1-Cer and NeuAcα2-6Galβ1-4GlcNAcβ1-4Galβ1-4Glcβ1-Cer, respectively) and GM1b (NeuAcα2-3Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-Cer) were also found in the cells. A substrate competition study suggested that the GM3 synthase is different from α2-3sialylparagloboside and GM1b synthase.
Dietary hypertriglyceridemic rats were treated with refined eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and changes in the n-6 and n-3 polyunsaturated fatty acid (PUFA) profiles in phospholipid (PL) classes in their tissues were analyzed in various aspects. The effects of EPA on the PUFA profiles in tissue PL classes were different from those of DHA. The effects were manifested differently depending on tissues and on the PL classes even in the same tissue. A decrease in the proportion of n-6 PUFA and an increase in the proportion of n-3 PUFA were both marked, particularly in the liver and heart, due to treatment with EPA and DHA; whereas these changes were somewhat slighter in the testes and were hardly observed in the brain. The variation pattern of tissue difference in the individual PUFA of each PL class differed, depending on the kind of PUFA, among the control, EPA, and DHA groups. The variation pattern of PL class difference in the individual PUFA of each tissue showed a similar tendency. Suppression of metabolic conversion from linoleic acid to arachidonic acid was not uniform depending on the tissue and also on the PL class, but the effect of DHA was more intense compared with that of EPA. On the other hand, the ratio of n-3 PUFA/total PUFA (n-3/PUFA) was largest in phosphatidylethanolamine, following by phosphatidylcholine and cardiolipin, in all the tissues, and was larger in the DHA group than in the EPA group. These findings suggest that a large uptake of n-3 PUFA by the liver PC and PE classes may decrease the secretion of VLDL-TG from the liver and may be related to the decrease of serum triglyceride.
We compared the plasma arginine-vasopressin (AVP) concentrations and hematocrit values before and after 24-h water-deprivation in normal Donryu rats and blind hereditary microphthalmic rats having a morphologically abnormal suprachiasmatic nucleus (SCN) in the hypothalamus, but a persistent circadian rhythm. We found that in the microphthalmic rats hypovolemia-induced elevation of the plasma AVP concentration was suppressed. The fact that these microphthalmic rats had a free-running circadian rhythm in their locomotor activity indicates that the cells comprising the circadian oscillator are functionally intact. Thus, we propose that SCN cells other than circadian pacemaker cells are involved in regulation of the plasma vasopressin concentration.
Liver fatty acid binding protein (L-FABP) is abundant in the cytosol, and is known to bind not only fatty acids but also several other endogenous and exogenous ligands. In order to determine the effects of alcohol on L-FABP synthesis and fatty acid metabolism, we studied the quantitative changes in fatty acid binding capacity (FABC) and immunoreactive L-FABP, and also the histochemical localization of L-FABP and triglycerides in the liver of rats given alcohol. Female SD rats in the control and in the two treated groups that were given 1g or 2g ethanol per kg body weight were fed the test solution of equal calories through a gastric tube every other day for 6 weeks. FABC determined by the 3H-labeled oleate binding assay and immunoreactive L-FABP measured by radioimmunoassay (RIA) increased in the treated groups (p<0.05 in FABC, p<0.005 in immunoreactive L-FABP). In the treated groups, the proportion of fatty acid-saturated L-FABP to total immunoreactive L-FABP was significantly increased in the rats fed 2g ethanol per body weight (p<0.05), and the difference of concentration of free fatty acids in the liver cytosol was not significant. In histochemical studies, L-FABP and neutral lipids were localized mainly in the cytoplasm of the periportal zone. When stained for both, the intensity and number of positive cells increased in the treated rats. These findings altogether indicated that ethanol increased fatty acid-saturated L-FABP and lipid deposition in the female rat liver, which suggests that L-FABP is involved in the mechanisms maintaining stable concentration of free fatty acids in the liver cytosol.
The effect of dietary fatty acid ethyl esters on lecithin-cholesterol acyltransferase (LCAT) activity was investigated in SD rats. The tested fatty acids were ethyl palmitate, ethyl oleate, and ethyl linoleate. The plasma VLDL cholesterol was higher in the ethyl palmitate and ethyl oleate groups and LDL-cholesterol was higher in the ethyl palmitate group than in the ethyl linoleate group. The LCAT activity was significantly higher in the rats fed ethyl linoleate than in those fed ethyl palmitate or ethyl oleate. The LCAT activity correlated positively with the percentage of linoleic acid in the plasma phospholipids and negatively with that of oleic acid in the same fraction. As studied with HDL substrate, the esterification rate for the diet groups was in the following order: ethyl linoleate>ethyl oleate>ethyl palmitate. Moreover, there was a negative correlation between the LCAT activity and the plasma total cholesterol level. These results suggest that dietary linoleic acid increases LCAT activity by influencing the substrate of this enzyme.
Pteridines are normally excreted in urine by healthy normal subjects but are significantly elevated during certain disease states associated with an activated cellular immune system. A simple method for estimation of total urinary pteridines has been developed, in which a 50-fold dilution of the urine sample was made to relieve the observed quenching of the fluorescence emitted by pteridines in the undiluted sample. The reduced pteridine derivatives were oxidized with iodine to their fluorescence-emitting fully oxidized forms, followed by direct measurement of fluorescence at 450nm with excitaiton at 360nm. The estimated total urinary pteridines were expressed as μg per mg creatinine. Employing this technique, we observed highly significant differences in excretion of total urinary pteridines between cancer patients and controls. Results were found to be as informative as those obtained by the standard selective estimation of urinary neopterin after reverse-phase high-performance liquid chromatographic separation. This rapid test requires only a small quantity of sample and has the potential to be developed into a routine clinical method for preliminary diagnosis and monitoring of cancer patients and for screening potential blood donors.
The plasma glucose and amino acid levels of a patient who had undergone total gastrectomy and total pancreatectomy 15 years previously were examined under 4 conditions determined by the method of exogenous glucagon injection. Glucagon was not injected in the first period, and 1mg of glucagon was injected twice a day in the second period. Twice a day 1mg of zinc glucagon was injected in the third period, and 2mg/day of glucagon by continuous subcutaneous glucagon infusion (CSGI) was given in the fourth period. The plasma pancreatic glucagon levels were within the normal range during CSGI. The effect of glucagon on plasma amino acid levels was greatest with CSGI. The effect became less in the order of zinc glucagon injection and twice-a-day injection of 1mg of glucagon. Furthermore, afternoon hyperglycemia and nocturnal hypoglycemia were suppressed with CSGI. Urinary nitrogen excretion was increased with glucagon injection. However, excretion of 3-methylhistidine did not show any significant increase. From these results we consider that exogenous glucagon injection has significant effects on the metabolism of glucose and amino acids after total pancreatectomy. We also conclude that both continuous subcutaneous glucagon infusion and zinc glucagon are useful in the postoperative management of total pancreatectomy.
The therapeutic efficacy of biotin was evaluated in 43 patients with non-insulin dependent diabetes mellitus. The serum biotin concentration in the patients was significantly lower than that in the 64 healthy control subjects and inversely correlated with the fasting blood glucose level. The oral administration of biotin, 9mg daily, corrected the hyperglycemia in the patients with no change in their serum insulin level. The serum levels of pyruvate and lactate decreased to their normal ranges after the administration. These observations suggest that the biotin administration ameliorates abnormal glucose metabolism in diabetic patients, presumably by enhancing the activity of the biotin-dependent enzyme, pyruvate carboxylase, with a subsequent promotion of glucose utilization for the entry into the tricarboxylic acid cycle. The administration also enhanced the response to glibenclamide in patients who had been resistant to the agent, suggesting a significant increase in the potency of the endogenous insulin action. The result demonstrates that biotin administration is effective for the treatment of the patients. Neither a relapse of clinical symptoms nor an occurrence of undesirable side effects has been observed.