Biomedical subjects

T Mizuno

Publications and source records attributed to T Mizuno.

At least 361 records · Page 20Linked to original sources

PubMed1993

A hydrogen-bonding network modulating enzyme function: asparagine-194 and tyrosine-225 of Escherichia coli aspartate aminotransferase.

The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT). Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991). In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-->Phe enzymes. The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194. The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme. The pH-pKd relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate. The analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate.(ABSTRACT TRUNCATED AT 250 WORDS)

Asparagine↗

PubMed1993

Cytokeratins in normal thymus and thymic epithelial tumors.

BACKGROUND: Thymus consists of some distinct epithelial cells that contain different sets of cytokeratins (CK). Epithelium-derived tumors maintain the expression of some of the CK of the specific nontransformed cells. Therefore, it seems reasonable to hypothesize that thymic epithelial tumors may differentiate toward distinct subsets of intrathymic epithelial cells in terms of CK expression. METHODS: Eighty-one thymomas and 14 thymic carcinomas were studied immunohistologically using monoclonal antibodies specific for a single CK or a CK pair. RESULTS: Thymic epithelial neoplasms could not be distinguished from each other on the basis of the profile of CK expression because the degree of overlap was extensive. However, polygonal cell thymomas differentiate toward a CK13-positive cortical subset that is rare in normal thymus. Spindle cell thymomas differentiate toward a CK13-positive medullary subset. Mixed cell thymomas are comprised of a CK13-positive medullary subset and a CK13-negative medullary subset, both of which are typical in normal thymus. CK18 was expressed to a greater extent on the epithelium of thymic carcinomas than on that of thymomas. Polygonal cell thymomas more frequently were invasive than spindle and mixed cell thymomas. CONCLUSIONS: There is a possibility that the epithelium of polygonal cell thymomas is immature because it is a phenotypically unusual subset in normal thymus. A thymic carcinoma arising in a thymoma has been reported, although the relationship between the thymoma and the thymic carcinoma was not clear. Nevertheless, given the similar cellular differentiation of thymoma and thymic carcinoma, CK18-positive epithelium in thymomas may be transformed into thymic carcinoma cells in certain conditions.

Adenocarcinoma↗

PubMed1993

The Escherichia coli nucleoid protein H-NS functions directly as a transcriptional repressor.

The H-NS protein is a major constituent of the Escherichia coli nucleoid structure and is implicated in the compact organization of the chromosome. Based on recent genetic evidence, this protein appears to influence the transcription of a variety of apparently unlinked genes on the chromosome, although the underlying molecular mechanism is not fully understood. In this study, we carried out a series of in vitro transcription assays including purified H-NS with special reference to the osmotically inducible proV promoter of the proVWX operon (or proU), whose expression is known to be derepressed by lesions of the hns (osmZ) gene. Here, H-NS was revealed to selectively inhibit an early step(s) of proV transcription initiation through its direct binding to the promoter region. It was thus demonstrated that H-NS functions directly as a transcriptional repressor. Under the in vitro conditions used, this in vitro inhibitory effect of H-NS was affected by changes in the superhelical density of template DNAs and more significantly by the concentration of potassium (K+) ions. These results are also discussed with regard to the mechanism underlying regulation of the proV promoter in response to the medium osmolarity.

Bacterial Outer Membrane Proteins↗

PubMed1993

Increase of interleukin 2 receptor and CD45RO antigen on lymphocytes cultured with human cytomegalovirus.

To determine the expression of interleukin 2 receptor (IL-2R) and the change of CD45RA and CD45RO antigen on lymphocytes in response to human cytomegalovirus (HCMV), peripheral blood mononuclear cells (PBMC) were cultured with HCMV antigen, and the expression of IL-2R and of CD45RA and CD45RO antigens on lymphocytes was analyzed by flow cytometry. The expression of IL-2R alpha (p55) and -beta (p75) was significantly increased on T cells obtained from CMV-seropositive donors following culture with the CMV antigen. An increased expression of IL-2R alpha and -beta was observed on both CD4+ and CD8+ cells, but the percentage of cells that expressed IL-2R was higher in CD4+ cells. The percentage of CD45RA+ cells was decreased and the percentage of CD45RO+ cells, especially of CD4+CD45RO+ cells, was increased when cultured with the CMV antigen. These observations suggest that switching of CD45RA+ cells to CD45RO+ cells occurred following stimulation with the CMV antigen. The expression of IL-2R and the activation of memory T cells are important to the recovery from and the protection against CMV infection.

Adult↗

PubMed1993

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes of the fetal mouse in vitro.

Differentiation of functional hepatocytes and biliary epithelial cells from immature hepatocytes was analysed in vitro. When fetal mouse liver fragments containing immature hepatocytes but no bile ducts were cultured organotypically, the immature hepatocytes differentiated into large hepatocytes. Some of these expressed bile duct markers such as cytokeratin and Dolichos biflorus agglutinin-binding sites, though only to a small extent, and typical intrahepatic bile duct cells failed to differentiate. Dexamethasone stimulated immature hepatocytes to differentiate into both mature hepatocyte and biliary epithelial cell lineages. Especially in the liver fragments cultured on Matrigel, dexamethasone stimulated the expression of bile duct markers (such as cytokeratin and binding sites for two types of lectin) in the immature hepatocytes. These results support the idea that immature hepatocytes can differentiate into both mature hepatocytes and biliary epithelial cells during normal development of the mouse liver, and suggest that glucocorticoids stimulate both these differentiation pathways. It also seems that basal laminar components may play a role in bile duct differentiation.

Animals↗

PubMed1993

Anatomical variation of pancreatobiliary ducts in biliary stone diseases.

Endoscopic retrograde cholangiopancreatography examinations were prospectively analyzed to determine whether anatomical variations of ductal systems have a role in the pathogenesis of cholecystolithiasis and choledocholithiasis. Included were 140 normal examinations (control group), 102 patients with cholecystolithiasis, and 68 patients with choledocholithiasis (primary stones in the common bile duct). Low entry of the cystic duct was observed frequently in patients with cholecystolithiasis (15.7% vs. 2.1% in control, p < 0.01). No preferential type of course of the cystic duct was observed in patients with cholecystolithiasis and choledocholithiasis. Separate openings of the bile and pancreatic ducts were significantly prevalent in patients with choledocholithiasis (53.5% vs. 30.6% in control, p < 0.01). Common channel was significantly short in patients with cholecystolithiasis. Incidence of juxtapapillary duodenal diverticula was significant in patients with choledocholithiasis. These observations suggest that some of the pancreatobiliary ductal anatomy may be closely implicated in the development of gallstone diseases.

Aged↗

PubMed1993

Biliary endoprosthesis in bile duct obstruction secondary to hepatocellular carcinoma.

The effect of biliary endoprosthesis was evaluated in 13 patients with major bile duct obstruction secondary to invasion by hepatocellular carcinoma. In 12 patients major portal vein branches were also invaded by the tumors. After several days' instillation of nasobiliary or percutaneous drainage tubes to flush the bile ducts, biliary endoprosthesis was performed either endoscopically (N = 9) or percutaneously (N = 4). Significant decrease (less than 50% of initial values) of alkaline phosphatase and bilirubin levels was observed in eight and two patients on day 20, respectively. Twelve patients died of hepatic failure at 27-132 days (mean 60 days). One patient without portal vein involvement is currently alive at 300 days. Biliary endoprosthesis has a limited role in the palliation of bile duct obstruction secondary to hepatocellular carcinoma, and the prognosis may be influenced mainly by the underlying hepatic function.

Adult↗

PubMed1993

Autoregulatory expression of the Escherichia coli hns gene encoding a nucleoid protein: H-NS functions as a repressor of its own transcription.

The Escherichia coli nucleoid protein, H-NS (or H1a), appears to influence the regulation of a variety of unrelated E. coli genes and operons. To gain an insight into the regulation of the hns gene itself, we constructed in this study a hns-lacZ transcriptional fusion gene and inserted a single copy at the att lambda locus on the E. coli chromosome. Expression of hns transcription appeared to be moderately regulated in a growth phase-dependent manner. It also emerged that hns transcription is under negative autoregulation, at least in the logarithmic growth phase. The results of in vitro transcription experiments confirmed that H-NS functions as a repressor for its own transcription. Thus, H-NS was shown to exhibit relatively high affinity for the DNA sequence encompassing the hns promoter region, as compared with a non-specific sequence. These results support the view that the nucleoid protein, H-NS, can function as a transcriptional regulator.

Bacterial Outer Membrane Proteins↗

PubMed1993

Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli.

The fadL gene of Escherichia coli codes for an outer membrane protein that is involved in the uptake of long-chain fatty acids. Uptake is regulated by environmental osmolarity, and decreases when the cells are grown under conditions of high osmolarity. A temperature-sensitive mutant that requires fatty acid for growth at 42 degrees C was unable to grow at the high temperature even in the presence of fatty acid if the medium contained 10% sucrose. Promoter activity of the fadL gene in vivo was repressed by high osmolarity in a FadR repressor null mutant. Furthermore, in vitro transcription of the fadL gene was strongly repressed by the addition of OmpR and EnvZ proteins. The results of gel retardation and DNase I protection experiments indicated that OmpR, after incubation with the protein kinase EnvZ, specifically binds to at least four sites around the fadL promoter, two upstream and two downstream from the transcriptional start site. These results suggest that transcription of the fadL gene is osmotically regulated by the OmpR-EnvZ two-component system.

Bacterial Outer Membrane Proteins↗

PubMed1993

Function of the Escherichia coli nucleoid protein, H-NS: molecular analysis of a subset of proteins whose expression is enhanced in a hns deletion mutant.

The expression of numerous Escherichia coli cellular proteins was previously demonstrated to be greatly enhanced in a hns deletion background, relative to the levels in wild-type cells. In this study, a subset of such proteins, expression of which is affected by H-NS, was partially purified, and the genes coding for some of the proteins were identified and characterized. Two of the proteins thus characterized, 19K and 17K, were found to be encoded by previously predicted genes that are located adjacent to, and downstream of, the trpABCDE operon (27.6 min on the E. coli genetic map). The genes coding for the other two proteins, 10K-L and 10K-S, are located at 77.5 min on the genetic map. Their nucleotide sequences were determined and revealed that they may constitute an operon. To characterize the putative promoters for these genes, a set of promoter-lacZ transcriptional fusion genes was constructed on the E. coli chromosome. The results of such promoter-probe analyses indicated that H-NS represses the expression of these genes at the transcriptional level. Furthermore, H-NS appeared to exhibit relatively strong affinity for the putative promoter sequences in vitro. These results are compatible with the hypothesis that H-NS functions as a transcriptional repressor.

Amino Acid Sequence↗

PubMed1993

Thymic epithelial tumours: evaluation of malignant grade by quantification of proliferating cell nuclear antigen and nucleolar organizer regions.

Cellular proliferation was studied by quantification of proliferating cell nuclear antigen (PCNA) and argyrophilic nucleolar organizer regions (AgNORs) of cells in 29 thymic epithelial tumours: 8 noninvasive (7 cortical and 1 mixed) thymomas, 11 invasive/metastatic (all cortical) thymomas, and 10 thymic carcinomas. Thymic carcinoma showed the highest percentage of cells positive for PCNA (14.73 +/- 5.419%) and the largest mean number of AgNORs per nucleus (4.89 +/- 0.756). The mean percentage of PCNA-positive cells and number of AgNORs in thymoma groups were as follows: in noninvasive thymoma 2.96 +/- 1.256% and 2.73 +/- 0.647, respectively, and in invasive/metastatic thymoma 4.41 +/- 1.823% and 3.68 +/- 1.148, respectively. The differences in PCNA and AgNORs were statistically significant between thymic carcinoma and each of thymoma groups. The overlap of the values between these tumours was minimal in the PCNA stains, although it was considerable in AgNOR counts as previously noted. However, there was no statistically significant difference in these markers between noninvasive and invasive/metastatic thymomas. These results indicate that thymoma in general is a slow-growing tumour compared with thymic carcinoma and that noninvasive thymoma is similar to invasive/metastatic thymoma with regard to proliferative activity; these latter two tumours may represent an essentially identical type in different stages of progression.

Antigens, Neoplasm↗

PubMed1993

Partial sequencing of two forms of concanavalin A-unbound collagenase inhibitor from bovine gingiva.

Three forms of collagenase inhibitor, one ConA-bound and two ConA-unbound, were extensively purified from bovine gingiva by sequential column chromatography. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that inhibitory activity resides in proteins with M(r) of 26000-28000 and 22000 for ConA-bound and two ConA-unbound inhibitors, respectively. Of these, two ConA-unbound inhibitors were partially sequenced in the first 12 amino acids and found to have an identical sequence. The NH2-terminal sequence had 100% identity with TIMP-2 or MI.

Amino Acid Sequence↗

PubMed1993

Accumulation of cardiac lipofuscin in crab-eating monkeys (Macaca fasicularis): the same rate of lipofuscin accumulation in several species of primates.

Previously, we have reported that the aging process begins at sexual maturation (Nakano, M. et al., Mech. Ageing Dev., 52 (1990) 93-106). In this paper, we reported the cardiac lipofuscin accumulation of crab-eating monkeys. The first appearance of cardiac lipofuscin was around sexual maturation, and the rate of accumulation in crab-eating monkey was 0.45. Several primates which have different life spans show the same rate of lipofuscin accumulation in the life stage. Namely, even in a different life span, the amount of lipofuscin accumulation in a given period of life such as puberty, middle age, old age was the same. From these results, it is suggested that the amount of lipofuscin accumulation is the same in the life span of primates having different life spans.

Aging↗

PubMed1993

The acupuncture point and its connecting central pathway for producing acupuncture analgesia.

Characteristics of the acupuncture point in producing acupuncture analgesia (AA) were examined by the inhibition of noxious responses in the brain stem reticular formation, potentials, and neuronal activity in the dorsal periaqueductal central gray (D-PAG), and analgesia caused by low frequency stimulation of the acupuncture point. As a result, stimulation of the muscle beneath the acupuncture point was found to be effective in producing AA. AA measured by tail flick, vocalization, and writhing tests was abolished by hypophysectomy, and by antiserum of beta-endorphin administered into the 3rd ventricle. The pathway from the D-PAG to the anterior hypothalamus (AA-AH) in the AA afferent pathway from the acupuncture point to the pituitary gland was determined. The lateral hypothalamus, lateral septum, cingulate bundle, dorsal-hippocampus, and habenulo-interpeduncular tract were found, in addition to regions previously found, to belong to the AA afferent pathway. A network of divergence and convergence in their rostral and caudal relations was observed. The AA afferent pathway diverges from the D-PAG, converges to the HP, and then projects to the AA-AH.

Acupuncture Analgesia↗

PubMed1993

Novel members of the two-component signal transduction genes in Escherichia coli.

A variety of adaptive response systems in prokaryotes often involve two families (two components) of signal transduction proteins, namely, sensory kinases and response-regulators. To extend the list of such sensor/regulator genes for Escherichia coli, we adopted a random screening method in this study. In particular, we isolated a series of recombinant plasmids that are able phenotypically to suppress mutational lesions of both the envZ and phoR/creC genes, each of which encodes a well-characterized sensory-kinase. Among the recombinant plasmids thus isolated, two clones (named pSN11 and pSN25) were subjected to characterization in detail. These analyses allowed us to identify the genetic loci specifying novel members of the sensor/regulator families. One (pSN11) is located around 45 min on the E. coli genetic map, that contains two adjacent coding-sequences (baeS and baeR). The other (pSN25) is located around 93 min of the genetic map, that also comprises two adjacent coding-sequences (basS and basR). These two pairs of gene-products, thus newly identified, were revealed to belong to typical members of the sensor/regulator families. Furthermore, they were demonstrated to exhibit the in vitro phosphotransfer reaction in the presence of ATP, that is also a characteristic of the sensory-kinase and response-regulator proteins.

Amino Acid Sequence↗

PubMed1993

Functional reconstitution of the putative Escherichia coli osmosensor, KdpD, into liposomes.

Expression of the Escherichia coli kdpABC operon, which is responsible for a high-affinity potassium-uptake system, is regulated in response to a change in the medium osmolarity. We present the first in vivo evidence that the putative osmosensor (kinase), KdpD, plays a crucial role in the osmotic regulation of kdpABC expression. We then isolated the intact form of KdpD from the cytoplasmic membrane with a mild non-ionic detergent, dodecyl maloside, as a soluble form. The solubilized KdpD itself did not exhibit the ability to phosphorylate the cognate response-regulator, KdpE. Upon reconstitution into liposomes, however, KdpD exhibited its kinase activity. Thus, using this partially purified form of KdpD, we succeeded in reconstituting KdpD-bearing liposomes that are fully functional in terms of their ability to phosphorylate the cognate positive regulator, KdpE. By adopting this in vitro method, some in vivo conditions were mimicked in order to address the molecular mechanism underlying signal transduction mediated by KdpD. Some catalytic properties of KdpD in liposomes are also presented.

Bacterial Proteins↗

PubMed1993

Hepatic parenchymal changes after the intraarterial injection of lipiodol in tumor-bearing rabbits.

RATIONALE AND OBJECTIVES: The influence of lipiodol injection on hepatic morphology and function was investigated in 119 rabbits with hepatic VX2 tumors. METHODS: The rabbits were divided into the following three groups, according to the materials injected via the proper hepatic artery: group 1 received doxorubicin (Adriamycin [ADM]); group 2 received lipiodol (LPD); and group 3 received LPD plus ADM. Groups 2 and 3 were each divided into three subgroups, according to the dose of LPD injected (0.3, 0.6, and 1.2 mL). Non-tumorous regions in the tumor-bearing lobe and the tumor-free lobe were examined histologically; low-kilovolt x-ray studies and computed tomography (CT) also were performed in 84 rabbits. Liver function tests were performed in 35 rabbits. RESULTS: Elevation of the serum aminotransferase levels was observed after the administration of LPD. Hepatocellular degeneration was marked in the tumor-free lobe, but was much more severe in the tumor-bearing lobe in groups 2 and 3. The degree of degeneration depended on the dose of LPD. Both low-kilovolt x-ray studies and CT showed the heavy accumulation of LPD in the tumor-bearing lobe. CONCLUSIONS: The current study shows that the histologic and functional adverse effects of oily chemoembolization depend on the dose of LPD administered to the liver.

Animals↗

PubMed1993

Cell proliferation kinetics in acetic acid-induced gastric ulcer evaluated by immunohistochemical staining of proliferating cell nuclear antigen.

A monoclonal antibody to proliferating cell nuclear antigen (PCNA) has been previously shown to be capable of identifying proliferating cells. We investigated proliferative activity in the healing process of acetic acid-induced gastric ulcer by immunohistochemical staining of PCNA and 5-bromo-2-deoxyuridine (BrdU), and the two methods were compared. Cell proliferative activity of regenerated mucosa around ulcers showed continuous acceleration for 42 days, and PCNA-labeled cells had stained nuclei as clearly discernible as those of BrdU-labeled cells. In addition, immunohistochemical staining of PCNA provided reproducible and quantifiable results without the requirement of pretreatment. We conclude that immunohistochemical staining of PCNA may represent a useful technique for analysis of proliferative activity during healing of gastric ulcers.

Acetates↗