Molecular genetics of alcohol-metabolizing enzymes.
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Biomedical subjects
Publications and source records attributed to A Yoshida.
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To clarify the in vivo events following administration of steroid hormones, the effect of molybdate on the cytoplasmic glucocorticoid receptor (GR) translocation in rat livers after administration of glucocorticoids was examined. After injection of hydrocortisone-succinate (Hydro) (200 micrograms/150 g rat) into the inferior vena cava of adrenalectomized rats, 3H-dexamethasone (3H-Dex) binding in liver cytosol rapidly decreased within 5 min, followed by a rapid replenishment. This decrease in binding was partially prevented by pre-treating the animals with 20 mg of sodium molybdate (Mo), which was considered to inhibit GR activation. Administration of 200 micrograms/150 g body weight of hydrocortisone caused a rapid replenishment of the binding sites following the depletion. Administration of molybdate 5 min after the hydrocortisone injection did not influence the replenishment. The GR content was increased significantly 20 min after the administration of molybdate in intact rats but not in adrenalectomized rat. It is concluded that activation of the hormone receptor complex observed in vitro also occurs in vivo after administration of glucocorticoid to animals.
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In 12 normal healthy males, the effect of preventing augmentation in tidal volume (VT) on the heart rate (HR) was examined during isocapnic progressive hypoxia. The amount of diminished tidal volume (delta VT) was 0.61 +/- 0.36 liters from the maximal VT value attained in the free breathing hypoxia. Hypoxic HR response in terms of arterial oxygen saturation (SaO2)/HR response slope (delta HR/delta SaO2) was significantly decreased from the free to controlled breathing (p less than 0.05). The effective VT range for eliciting such a HR change appeared to be more than 1,000 ml as has been demonstrated in the Hering-Breuer reflex on ventilation in humans. However, the magnitude of delta HR/delta SaO2 decrement was not significantly correlated with delta VT, suggesting that the command signal from the higher nervous center interfered with the expected response. Contrary to the HR response, the hypoxic ventilatory and occlusion pressure responses were not significantly influenced by the controlled breathing. We conclude that the hypoxic HR response is significantly depressed by the diminished VT breathing.
Mortality, major causes of moribundity, and spontaneous tumors in CD-1 mice were studied in 891 males and 890 females, which were used as controls in 11 different 2-year chronic and oncogenicity studies during the past 5 years. Average mortality of males and females at 83 weeks of age was 32.6% and 28.6%, respectively, and at 109 weeks of age was 66.4% and 63.3%, respectively. Mortality was significantly lowered in males and females born after 1980 in accordance with an abruptly decreased occurrence of systemic amyloidosis in these animals. The major cause of death or moribundity included systemic arteritis, systemic amyloidosis, auricular thrombosis, glomerulosclerosis, lymphoma, and pulmonary adenocarcinoma in both sexes. Dysuria and hepatocellular carcinoma in males and mammary adenocarcinoma in females were also critical lesions. The major tumors occurring at more than 3% incidence were systemic lymphoma, adenoma/adenocarcinoma of the lung, adenoma/carcinoma of the liver and adenoma/adenocarcinoma of the Harderian gland for males, and systemic lymphoma, adenoma/adenocarcinoma of the lung, adenoma/carcinoma of the liver, leiomyoma/leiomyosarcoma of the uterus, adenoma/adenocarcinoma of the pituitary (anterior), adenoma/adenocarcinoma of the mammary gland and adenoma/adenocarcinoma of the Harderian gland for females. Intralaboratory heterogeneities in the incidence were recorded as follows: systemic lymphoma in 1 of 11 control groups (1/11) and adenoma/adenocarcinoma in 1/11 for males, and systemic lymphoma in 3/11, adenoma/adenocarcinoma of the lung in 2/11, adenoma/adenocarcinoma of the liver in 1/11, and adenoma/adenocarcinoma in 1/11 for females.
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Binding sites for mouse epidermal growth factor (EGF) were observed in purified nuclear fraction from rat liver. The binding data yielded a curvilinear Scatchard plot which was decomposed into two binding components with different binding affinities. The binding component with higher affinity (Kd approximately 10(-10) M) represented approximately 15% of the total binding, while the high affinity binding sites were less than 5% of the total in the microsomes. The ligand-dependent autophosphorylation, one of the characteristic features of EGF receptor in the plasma membrane, was not observed at the site of the receptor (170 KDa) in the nuclear fraction. The binding characteristics for EGF fluctuated during the course of liver regeneration after partial hepatectomy; the binding capacity in the nuclear fraction increased in contrast to the decrease in the microsomes. However, the binding sites in the nuclear fraction obtained in the early period after partial hepatectomy consisted only of low affinity ones.
A heritable open-eyelid mutant which arose spontaneously in the NC strain of mouse in 1982 has been maintained as the NC-eob line through brother-sister matings. When the line exceeded the 12th generation, its reproductive capabilities were initially compared with those of the NC strain. Then eye abnormalities were investigated morphologically and genetically. Although most of the reproductive parameters in the NC-eob line were comparable to those in the NC strain, a significant decrease was observed in the mean number of pups delivered, and the viability of pups at 7 days of age was reduced to 55%. The eyelids of pups were widely open at birth. The exposed cornea dried within a few hours and a bloody exudate appeared around the margin of the eyelids. At 1 day of age, the eye opening was smaller in size and covered with a scab. Histological examination demonstrated apparent inflammatory changes in the cornea and conjunctiva. At 21 days of age, the eyes revealed opacity, a rough surface, and microphthalmia. Histological findings in the cornea, such as keratosis and vascularization, coincided with the eye opacity. The weight and size of the eyeballs were almost the same as those in normal NC pups. However, a significant decrease in the length of the palpebral rima was evident and this was considered to be the cause of the microphthalmia. The results of genetic analysis indicated that these eye abnormalities were caused by an autosomal recessive gene, eob. These results suggested that the eob gene might be a new mutation manifesting semi-lethality as a pleiotropic effect.
The purpose of the present study is to determine polymorphonucleocytal elastase-alpha 1-protease inhibitor complex in bronchoalveolar lavage fluid (BALF) in order to investigate the clinical significance of the increased complex in BALF. BALF was collected by the usual method using a bronchial fiberscope and concentrated 5-10 folds by Amicon UM-2 ultrafiltration. Determination of the complex in BALF and plasma was made in accordance with the method of Neumann et al. The level of complex (microgram/mg albumin, mean value) in BALF was 0.38 in healthy non-smokers (n = 5), 3.14 in healthy smokers (n = 6), 5.61 in the patients with chronic obstructive pulmonary disease (COPD) (n = 9) and 1.78 in the patients with idiopathic pulmonary fibrosis (IPF) (n = 15), that is, higher in healthy smokers and COPD and IPF patients. The ratio of the complex in BALF vs plasma was higher in healthy smokers and in COPD patients than in healthy non-smokers. Furthermore, there was a positive correlation between the level of this complex in BALF and the number of PMN in BLAF (r = 0.68, p less than 0.01). From the above it was assumed that the release of elastase from PMN is progressed in the lower airway of smokers and COPD patients.
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The indication of tonsillectomy for recurrent tonsillitis was discussed from the standpoint of clinical immunology. Elevated serum IgG was observed in 29.8% of the patients under 8 years of age preoperatively. In contrast, in 25% of patients between 9-15 years of age, serum IgG was low preoperatively. In the patients over 16 years of age no abnormal serum IgG was found, and the elevated serum IgG returned to normal after tonsillectomy. The patients with normal serum IgG preoperatively retained normal values postoperatively. The low preoperative IgG level group still remained low postoperatively. As to postoperative improving symptoms, high and low grade fever improved most (100%), followed by cervical lymphadenopathy (93%), frequent absence from school (93%), sore throat (88%) and recurrence of bronchitis (85%). The patients with recurrent tonsillitis for 8 years showing high serum IgG had indications for tonsillectomy.
M-GTFI, originally screened as an inhibitor of Streptococcus mutans glucosyltransferase, strongly inhibited alpha-glucosidase, in a non-competitive manner especially when the synthetic substrate p-nitrophenyl-alpha-D-glucopyranoside was used. It also inhibited beta-glucosidase, beta-amylase and, to a lesser extent, beta-glucuronidase. The inhibitor was stable in neutral and alkaline pH ranges and dependency of the inhibition on pH and temperature was not observed. Some proteinases and polysaccharides-hydrolyzing enzymes as well as human saliva did not inactivate the inhibitor. There was a correlation between the release of sulfate anions from the inhibitor molecule on incubation with HCl (0.2 N) at 100 degrees C and loss of inhibitory properties of the molecule. It is suggested that the presence of sulfate ester linkages in the inhibitor molecule play an important role in the inhibition process.
Metabolic changes in lipids, ascorbic acid, and hepatic microsomal cytochrome P-450 by feeding polychlorinated biphenyls (PCB) were investigated in streptozotocin-induced diabetic rats. Streptozotocin (STZ, 60 mg/kg body weight) was injected in Wistar male rats intraperitoneally. Diabetic and non-diabetic rats were fed ad libitum a 20% casein-based control diet or a PCB-containing diet (200 mg/kg diet) for 9 days. Body weight decreased significantly in STZ-induced diabetic rats with or without PCB (groups PD and D, respectively). In rats of group D, urinary ascorbic acid excretion was 15 times higher than that in non-diabetic rats fed a control diet (group C). Dietary PCB caused 30-fold higher urinary ascorbic acid excretion in non-diabetic rats (group P) than that in group C. In group PD, urinary ascorbic acid was nearly 60 times higher than that in group C. Ascorbic acid in liver and kidney was significantly lower in group D than in group C, and it was significantly lower in group PD than in group P. Liver microsomal cytochrome P-450 and b5 were both increased by dietary PCB in group P. Addition increase in these enzymes was observed in diabetic rats by PCB. Serum total cholesterol was 1.8 times higher in group P than in group C. Further increase in serum total cholesterol was observed in group PD. These data suggest that metabolic changes in lipids and ascorbic acid induced by the dietary xenobiotic were magnified in STZ-induced diabetic rats.
The in vitro growth of Plasmodium falciparum malaria parasites was assayed in mutant red cells deficient in either diphosphoglycerate mutase (DPGM) or phosphoglycerate kinase (PGK). In addition, cDNA probes developed for human DNA sequences coding for these enzymes were used to examine the parasite genome by means of restriction endonuclease digestion and Southern blot analysis of parasite DNA. In both types of enzymopathic red cells, parasite growth was normal. In infected DPGM deficient red cells, no DPGM activity could be detected, and in normal red cells, DPGM activity declined slightly in a manner suggestive of parasite catabolism of host protein. However, in infected PGK deficient red cells, there was a 100-fold increase in PGK activity, and in normal red cells, a threefold increase in PGK activity was observed. Parasite PGK could be recovered from isolated parasites, and a marked increase in heat instability of parasite PGK as compared with the host cell enzyme was noted. Neither cDNA probe was found to cross-react with DNA sequences in the parasite genome. It is concluded that the parasite has no requirement for DPGM, and probably has no gene for this enzyme. On the other hand, the parasite does require PGK, (an adenosine triphosphate [ATP] generating enzyme) and synthesizes its own enzyme, which must have been encoded in the parasite genome. The parasite PGK gene most likely lacks sufficient homology to be detected by a human cDNA probe. Enzymopathic red cells are useful tools for elucidating the glycolytic enzymology of parasites and their co-evolution with their human hosts.
Two common variants of glucose-6-phosphate dehydrogenase (G6PD), i.e., A(+) and A(-), exist in blacks in high frequencies. The mutation of the A(+) gene is a single nucleotide transition, A/G in equilibrium Asp) in the G6PD protein and produces an additional FokI cleavage site of the mutation site. Thus, the FokI fragment types detected by the genomic clone that contain the mutation site differ in the normal B(+) DNA and the variant A(+) DNA. The FokI fragment type of the variant A(-) is the same as that of the A(+). Since A(+) and A(-) enzymes differ at the protein level, the A(-) gene was presumably evolved by stepwise mutations through the A(+) gene.
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