Biomedical subjects
H Minami
Publications and source records attributed to H Minami.
Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct.
A recombinant plasmid for expression of full-length human DNA ligase I (phLig-I) was constructed in a plasmid/phage chimeric vector, pTD-T7N, which was derived from pUC118 by oligonucleotide-directed mutagenesis. The insert contained a 2757-base pair coding sequence for a whole human DNA ligase I and an extra ACC codon adjacent to the ATG initiation codon. This ACC codon was required for achieving high levels of expression of full-length DNA ligase I in Escherichia coli strain BL21. The recombinant plasmid, which was designed to exploit the T7 late promoter and the ATG initiation codon for beta-galactosidase was transfected into E. coli BL21 cells that express T7 RNA polymerase. The recombinant clone produced relatively high levels of DNA ligase I with a molecular mass of 130 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. The DNA ligase was purified to near-homogeneity by the two-step column chromatographic procedure from BLphLig-I cells that had been induced with isopropyl beta-D-thiogalactoside. The specific activity, chromatographic behavior, kinetic properties, molecular mass, and antigenicity of the recombinant human DNA ligase I were indistinguishable from those of purified mammalian DNA ligase I. Metabolically labeling experiments with 32P(i) indicate that the recombinant DNA ligase I was present as an enzyme-AMP reaction intermediate, but not as a phosphoprotein, in the E. coli cells.
Differential responses of intestinal glucose transporter mRNA transcripts to levels of dietary sugars.
Dietary sugars are known to stimulate intestinal glucose transport activity, but the specific signals involved are unknown. The Na(+)-dependent glucose co-transporter (SGLT1), the liver-type facilitative glucose transporter (GLUT2) and the intestinal-type facilitative glucose transporter (GLUT5) are all expressed in rat jejunum [Miyamoto, Hase, Taketani, Minami, Oka, Nakabou and Hagihira (1991) Biochem. Biophys. Res. Commun. 181, 1110-1117]. In the present study we have investigated the effects of dietary sugars on these glucose transporter genes. A high-glucose diet stimulated glucose transport activity and increased the levels of SGLT1 and GLUT2 mRNAs in rat jejunum. 3-O-Methylglucose, D-galactose, D-fructose, D-mannose and D-xylose can mimic the regulatory effect of glucose on the SGLT1 mRNA level in rat jejunum. However, only D-galactose and D-fructose increased the levels of GLUT2 mRNA. The GLUT5 mRNA level was increased significantly only by D-fructose. Our results suggest that the increase in intestinal transport activity in rats caused by dietary glucose is due to an increase in the levels of SGLT1 and GLUT2 mRNAs, and that these increases in mRNA may be caused by an enhancement of the transcriptional rate. Furthermore, for expression of the SGLT1 gene, the signal need not be a metabolizable or transportable substrate whereas, for expression of the GLUT2 gene, metabolism of the substrate in the liver may be necessary for signalling. Only D-fructose is an effective signal for expression of the GLUT5 gene.
Calvasculin, as a factor affecting the microfilament assemblies in rat fibroblasts transfected by src gene.
Cell transformations accompany alterations in cell morphology and microfilament patterns. Calvasculin encodes mRNA termed pEL-98, 18A2, 42A, p9Ka, or mts1, found to be elevated in several metastatic cell lines. We report the elevation of calvasculin expression in SR-3Y1 cells, which show disappearance of ordered microfilaments, compared to that in 3Y1 cells and that the similar distribution of calvasculin to that of actin filaments. Interestingly, calvasculin co-sediments with F-actin and bundles actin filaments in a Ca(2+)-dependent manner. This activity, along with the elevation of calvasculin following transformation, suggests that the disorganization of filaments in SR-3Y1 cell is due to the cross-linking activity of calvasculin.
Na+/D-glucose cotransporter based bilayer lipid membrane sensor for D-glucose.
A new type of amperometric blosensor for glucose was fabricated using a Na+/D-glucose cotransporter as the signal-transducing sensory element that exploits the D-glucose-triggered Na+ ion current through bilayer lipid membranes (BLMs). The planar BLM was formed by the folding method across a small aperture of a thin Teflon film. The Na+/D-glucose cotransporter, isolated and purified from small intestinal brush border membrane of guinea pigs, was embedded into BLMs through proteoliposomes. The number of the protein molecules thus incorporated in the present sensing membrane was estimated to be ca. 10(7). The sensor response was measured as an ionic current through the BLM arising from cotransported Na+ ion flux under a constant applied potential and was only induced by D-glucose above 10(-9) M, but not by the other monosaccharides except for D-galactose. The effect of applied potentials, Na+ and K+ ion concentrations, and the addition of a competitive inhibitor, phlorizin, were scrutinized to characterize the sensor output. The results were briefly discussed in terms of the potential use of the Na+/D-glucose cotransporter as a sensory element for D-glucose.
Inhibition of glucose absorption by phlorizin affects intestinal functions in rats.
BACKGROUND: To investigate the mechanism of regulation of intestinal disaccharidase activity and glucose absorption, the effect of dietary intake of phlorizin, a potent and specific inhibitor of intestinal glucose transport, on intestinal disaccharidase activity and Na(+)-dependent glucose transporter was examined in rats. METHODS: Jejunal disaccharidase activity and the number of Na(+)-dependent glucose transporters were determined in rats maintained on a low-starch diet, a high-starch diet, or low-starch diets containing various amounts of phlorizin (0.1%-0.9% wt/wt). RESULTS: Jejunal disaccharidase activity increased in a dose- and time-dependent manner. Stimulation of jejunal disaccharidase activity only occurred when phlorizin was added to starch-containing diets, not when it was added to a carbohydrate-free diet. Addition of the same amount of phloretin and glucose (constituents of phlorizin), to the diet failed to increase disaccharidase activity. The maximum binding of phlorizin to brush border membrane vesicles was increased in the rats fed phlorizin, whereas the dissociation constant remained unchanged, suggesting an increase of glucose transporter expression. CONCLUSIONS: Dietary phlorizin increased the jejunal disaccharidase activity and Na(+)-dependent glucose transporter expression. The trigger for these changes may have been due to an increased luminal glucose content.
Phase I clinical and pharmacokinetic study of a 14-day infusion of etoposide in patients with lung cancer.
PURPOSE: A phase I study was conducted to determine the maximum-tolerated dose (MTD) of a 14-day continuous infusion of etoposide, and to evaluate the pharmacokinetics in patients with lung cancer. PATIENTS AND METHODS: Etoposide was administered continuously through a central venous catheter using a pump. The starting dose level was 300 mg/m2 over 14 days, with dose escalations of 100 mg/m2 over 14 days until unacceptable toxicities occurred. Pharmacokinetic studies were performed in all patients. RESULTS: Twenty-one patients, 20 with non-small-cell lung cancer and one with refractory small-cell lung cancer, received 37 courses. No World Health Organization (WHO) grade III or greater toxicity occurred at doses up to 400 mg/m2 over 14 days. At 700 mg/m2 over 14 days, all four patients experienced grade III or IV leukocytopenia, and two developed grade III stomatitis. No cumulative toxicity was observed. A steady concentration of etoposide was achieved 24 hours after the start of chemotherapy, and it was significantly correlated with surviving fractions of leukocytes (r = -.64, P = .001) and platelets (r = -.68, P < .001). The leukocyte count at the termination of chemotherapy predicted the nadir count (r = .93, P < .001). CONCLUSION: Steady blood levels of etoposide were maintained for prolonged periods, during 14-day continuous infusions. Leukocytopenia and stomatitis were dose-limiting. Nadir counts and surviving fractions of leukocytes were predicted by the leukocyte count at the end of chemotherapy and the concentration of etoposide, respectively. The recommended dose for phase II trials is 600 mg/m2 over 14 days.
[Analysis by two-dimensional electrophoresis of the cause of myocardial dysfunction in aging rat hearts].
The severity and frequency of atherosclerosis, diabetes, and ischemic heart disease, which affect cardiac function, increase with aging. Although there are many reports about hemodynamic and histopathological studies about aging hearts, there are very few studies on changes in structural proteins in aging hearts. We investigated the contractile proteins of the left ventricles in rats aged 6, 12 and 125 weeks using two-dimensional electrophoresis. There were no difference in structural proteins in heart between 6-week and 12-week-old rats. The contents of myosin heavy chain, myosin light chain 2, actin, troponin-I in 125-week-old rats decreased compared with those of 12-week-old rats. Myosin heavy chain, which is one component of myosin, interacts with actin and changes chemical energy to mechanical energy. Therefore its decrease leads to a decline in myocardial contractility. These results seem to indicate one of the most important changes in the aging rat heart, as well as impairment in relaxation by the increase of interstitial fibrosis and decline of Ca uptake by sarcoplasmic reticulum.
Immunohistochemical analysis of keratin distribution in eccrine poroma.
Although eccrine poroma has been thought of as a neoplasm of the intradermal eccrine duct, this interpretation has not been entirely confirmed. In this study, twenty-five cases of eccrine poroma were retrieved and analyzed by immunohistochemical techniques, using various kinds of monoclonal antikeratin antibodies. Comparative immunohistochemical observations of eccrine poroma and normal eccrine glands revealed that the poroma cells expressed immunophenotypes similar to those of the basal cells of dermal eccrine ducts. Sweat-ductlike structures showed similar staining patterns to those observed in the inner cells of dermal eccrine ducts. Some cystic spaces were similar to those observed in the secretory cells of eccrine glands. Eccrine poroma is, therefore, speculated to originate via the proliferation and expansion of the basal cells of eccrine ducts, although it is very difficult to prove the histogenesis. Some tumor cells may differentiate toward inner cells of the eccrine ducts, forming ductal lumina, whereas other tumor cells differentiate toward eccrine secretory regions, forming some cystic spaces.
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[A case of complete response in a patient with invasive bladder cancer due to intermittent intraarterial infusion chemotherapy using the alteration of blood flow].
The patient was a 70-year-old male with invasive bladder cancer. We performed intermittent arterial infusion (ITI) combined with alteration of blood flow in the bladder wall using the contralateral arterial embolization. As for anti-tumor agents, cisplatin (CDDP) 10mg and pirarubic in (THP) 10 mg were selected, and were injected every week for 11 times. Complete response (CR) was noted by cystoscopy and biopsied specimen. In our conclusion. ITI was useful for the treatment of bladder cancer.
[A study of infusional port re-insertion in patients with liver metastasis].
We evaluated the importance and usefulness of re-insertion of the intra-arterial infusion catheter in 10 patients with liver metastasis who showed problems in the catheter or system during intra-arterial infusion chemotherapy. The route of re-insertion was laparotomy-->left subclavian method in 3 patients, left subclavian-->left subclavian method in 5, and left subclavian-->femoral method in 2. The cause of re-insertion was dislocation in 5 patients, obstruction of the system in 4, and obstruction of CHA in 1. In the patients showing dislocation, re-insertion was performed after a mean of 4 months, and CEA improvement and direct effects evaluated by imaging techniques were marked, suggesting good indication. In the patients showing system obstruction or CHA obstruction, re-insertion was performed after a mean of 8.6 months, and CEA improvement and direct effects were less than those in the patients showing dislocation. Of the 10 patients who underwent re-insertion, 5 have survived to the present for 13-20 months (mean, 17 months). The prognosis of liver metastasis may be improved by active re-insertion of the intra-arterial infusion catheter.
[A case of pulmonary asbestosis with slightly increased serum IgE concentration and histopathological changes resembling DIP].
A 68-year-old male presented with cough and sputum. He had suffered from these symptoms for ten years prior to admission. Chest roentgenogram revealed reticulonodular shadows in the lower fields of both lungs. CT scan of the chest revealed an interstitial pattern in the lower field of both lungs. Honeycombing and bullous pattern were also present in the subpleural area. The patient had a history of dust and asbestos inhalation while working as an electrician. Eosinophilia of the peripheral blood and BALF, and a slightly increased serum IgE concentration were noted. Open lung biopsy revealed interstitial fibrosis with intra-alveolar macrophage accumulation and asbestos bodies. The histopathological features resembled UIP and DIP, although DIP is uncommon in pulmonary asbestosis. The slightly increased serum IgE concentration was considered to be an additional effect of asbestos. This is a case of pulmonary asbestosis with intriguing immunological and histopathological features.
Role of liver-type glucose transporter (GLUT2) in transport across the basolateral membrane in rat jejunum.
To obtain information on the regulation of glucose transport across the basolateral membrane (BLM) of intestinal epithelial cells, we measured the number of [3H]cytochalasin B binding sites and the level of liver-type glucose transporter (GLUT2) protein in the BLM in the jejunum of rats (i) with diabetes (ii) given a high-carbohydrate diet or (iii) with experimental hyperglycemia (12 h infusion of a high-glucose solution). A glucose uptake and the number of D-glucose inhibitable [3H]cytochalasin B binding sites in BLM vesicles were significantly increased in all three conditions. Western blot analysis showed that the amount of GLUT2 protein in BLM vesicles was increased in rats with diabetes and those given a high-carbohydrate diet, but not in those with experimental hyperglycemia. These results suggest that there is a mechanism for rapid regulation of glucose transport in the BLM that does not depend on change in the amount of GLUT2.
Specific binding of CAP-50 to calcyclin.
CAP-50, a calcyclin-associated protein with an apparent molecular mass of 50 kDa, was purified and proved to be a novel annexin [Tokumitsu, H. et al. (1992) J. Biol. Chem. 267, 8919-8924]. We examined the binding of CAP-50 to other Ca(2+)-binding proteins which have two of four EF-hand structures, by a co-precipitation assay with phospholipid (phosphatidylserine). Among nine Ca(2+)-binding proteins (calcyclin, S-100 proteins, p11, calgizzarin, calvasculin, calmodulin and troponin C) examined, only calcyclin interacted with CAP-50. These results clearly show that the interaction of CAP-50 to calcyclin is specific, i.e. other Ca(2+)-binding proteins with the EF-hand structure could not substitute for calcyclin, thereby suggesting the possible role in specific regulation of the function of CAP-50 by Ca2+/calcyclin.
CAP-50, a newly identified annexin, localizes in nuclei of cultured fibroblast 3Y1 cells.
A 50-kDa protein, which binds to the growth-regulated gene (2A9) product, calcyclin in a calcium-dependent manner, was purified from bovine lung. Partial amino acid sequencing of the protein revealed it to be the bovine equivalent of rabbit lung CAP-50 (calcyclin-associated protein, 50 kDa), which is a member of the annexin family and binds to calcyclin in a calcium-dependent manner. Specific polyclonal antibodies to bovine lung CAP-50 were prepared. Comparative studies between CAP-50 and synexin (annexin VII) on the immunoreactivity against anti-CAP-50 antibodies and the ability of binding to calcyclin revealed that CAP-50 was a distinct molecule from synexin. Using specific polyclonal antibodies to bovine lung CAP-50, tissue distribution and subcellular distribution of CAP-50 were investigated. In most rat tissues, except those in the central nervous systems and kidney, CAP-50 is expressed at a high or moderate level. Both studies by subcellular fractionation and by indirect immunofluorescence staining of the rat embryonic fibroblast cell line, 3Y1, revealed that CAP-50 mainly localized in nuclei. Moreover, between the cells at interphase and at mitotic phase, different distributions of CAP-50 were observed. That is, in the cells at interphase, CAP-50 seemed to localize throughout the nucleoplasm. On the other hand, in the cells during mitosis, CAP-50 was concentrated at the loop-like structure around the mitotic apparatus. CAP-50 was found in isolated 3Y1 nuclei lacking outer nuclear membranes, and approximately 50% of CAP-50 was extracted from the nuclei by chelating calcium. Thus, CAP-50, a unique annexin, localizes in nuclei.