Prolongation of survival of a xenografted bioartificial pancreas with amesh-reinforced polyvinyl alcohol hydrogel bag employing a B-cell line (MIN6).
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Animals↗
Biomedical subjects
J Miyazaki
Publications and source records attributed to J Miyazaki.
Long survival of xenografted bioartificial pancreas with a mesh-reinforced polyvinyl alcohol hydrogel bag employing a B-cell line (MIN6).
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Animals↗
Efficient regulation of gene expression by adenovirus vector-mediated delivery of the CRE recombinase.
We have constructed an E1-defective adenovirus (Ad) vector designated AdCAG-Cre containing the Cre recombinase gene derived from bacteriophage P1 under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid (CAG) promoter. We examined the Cre-loxP-based recombination by this Ad vector in C2C12 cells bearing a reporter gene construct CAG-CAT-Z, which directs expression of the E. coli lacZ gene upon Cre-mediated excision of the CAT gene located between the CAG promoter and the lacZ gene. Nearly 100% of these cells were shown to start to produce beta-galactosidase after infection with the AdCAG-Cre vector at MOI 100. On the basis of this result, we discuss the possible use of the AdCAG-Cre vector to manipulate the gene expression in mammalian cells.
Adenoviruses, Human↗
Inhibition of pancreatic beta-cell glucokinase by antisense RNA expression in transgenic mice: mouse strain-dependent alteration of glucose tolerance.
We have generated transgenic mice, in either C57BL/6 or C3H background, expressing antisense glucokinase mRNA in beta-cells. The glucose phosphorylating activity at 60 mM glucose in transgenic islets was significantly lower than that in controls, and the insulin secretory response to glucose was lower in transgenic islets than in those of controls in both strains. Following i.p. glucose challenge, higher blood glucose levels were observed in transgenic mice than in controls in the C57BL/6 but not the C3H background. These data suggest that a beta-cell secretory defect, in combination with other undefined genetic factors, causes impaired glucose homeostasis in mice.
Animals↗
An inhibitory role for phosphatidylinositol 3-kinase in insulin secretion from pancreatic B cell line MIN6.
Phosphatidylinositol 3-kinase (PI3-kinase) has been implicated in the regulation of vesicular transport. We examined the roles of PI3-kinase in the glucose-induced insulin secretion from the pancreatic beta cell line MIN6 by using wortmannin, a potent inhibitor of PI3-kinase. Low concentrations of wortmannin markedly potentiated the glucose-induced insulin secretion. This effect was probably mediated by PI3-kinase inhibition. Furthermore, wortmannin completely canceled the suppressive effect of insulin-like growth factor-I on insulin secretion from MIN6 cells. On the basis of these results, we discuss a possible role of PI3-kinase in the negative feed-back regulation of insulin secretion.
Androstadienes↗
A novel isoform of syntaxin-binding protein homologous to yeast Sec1 expressed ubiquitously in mammalian cells.
munc-18/n-Sec1/rbSec1, a brain homologue of the yeast Sec1p protein, is thought to participate in regulating the docking and fusion of synaptic vesicles. We have screened the mouse cDNA library of an MIN6 cell line, derived from pancreatic beta cells, for its novel isoform and have identified a cDNA encoding a 593-amino acid protein having 63, 53, and 30% identity with munc-18/n-Sec1/rbSec1, Caenorhabditis elegans unc18, and Saccharomyces cerevisiae Sec1p, respectively. While munc-18/n-Sec1/rbSec1 expression has been reported to be neural-specific, RNA blot analysis has revealed that the novel isoform, which we refer to as muSec1 (mammalian ubiquitous Sec1), is expressed ubiquitously. We have also identified mouse munc-18/n-Sec1/rbSec1 from the MIN6 cDNA library, indicating that different isoforms of a protein participating in vesicular transport exist in a single cell. muSec1 bound to glutathione S-transferase-syntaxin 1A and, although with lower affinity, to glutathione S-transferase-syntaxin 4 fusion protein. These findings suggest that muSec1 is, via its binding to the syntaxin family, involved in the protein trafficking from the Golgi apparatus to the plasma membrane and that the fundamental mechanisms of protein trafficking have been conserved from yeast through virtually all mammalian cells.
Amino Acid Sequence↗
Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase.
An efficient method of transgene modulation in fertilized eggs has been developed that uses the Cre/loxP recombination system. Twelve transgenic mouse lines carrying a chicken beta-actin promoter-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase gene construct were produced. After selection of the line showing the highest expression of the CAT gene in a variety of tissues, eggs of this line were injected in the male or female pronucleus with a Cre expression vector placed under the control of the chicken beta-actin promoter and kept in a circular form to avoid genomic integration. This resulted in a transient expression of Cre in the eggs, leading to recombination of the transgene as detected by galactosidase expression and DNA analysis. Recombination was completed before the morula stage with both types of pronuclear injections and occurred with a very high frequency; no mosaicism, no incomplete recombination, and no integration of the Cre sequence were observed in 18 mice born with this modified transgene. The beta-galactosidase gene was expressed in various tissues at levels comparable to those found for the CAT gene in the founder line. This Cre transient expression system should be useful for breeding transgenic lines in which transgene expression leads to sterility or lethality--in particular, for selecting transgenic lines with high expression of a potentially lethal transgene whose full activity is difficult to explore in a conventional transgenic system because of the risk of selecting for transgenic lines carrying only poorly expressed transgenes.
Actins↗
Defective IL-5-receptor-mediated signaling in B cells of X-linked immunodeficient mice.
Murine (m) IL-5 induces proliferation and differentiation of both Ly-1+ B cells and activated conventional B cells. X-linked immunodeficient (XID) mice do not respond to thymus-independent type II antigens, and have an abnormal response to a variety of activation signals through Ig receptors, CD40 and cytokine receptors. Furthermore, XID mice show a B cell specific defect, reflected in decreased numbers of IL-5R alpha+ B cells and reduced responsiveness of IL-5R alpha+ B cells to mIL-5. We generated IL-5R alpha transgenic (5R alpha-Tg) mice in which B cells expressed recombinant IL-5R alpha. We crossed male 5R alpha-Tg mice with female XID mice and used their offspring to determine the IL-5 responsiveness of these B cells. All B cells of F1 male mice carrying the xid gene together with the transgene expressed the recombinant IL-5R alpha. However, those mice lacked Ly-1 B cells and their B cells acquired responsiveness to mIL-5. Interestingly, XID-5R alpha-Tg B cells, but not XID B cells, acquired mIL-5 proliferative and Ig-secretory responsiveness only in the presence of suboptimal doses of lipopolysaccharide. Stimulation of these B cells with mIL-5 plus phorbol myristate acetate induced proliferation, but not Ig secretion. These results indicate that the impaired mIL-5 responsiveness of B cells in XID mice is due to an abnormality of IL-5R-mediated signaling which may correlate with the xid gene mutation, alteration of a single amino acid of Bruton's tyrosine kinase.
Agammaglobulinaemia Tyrosine Kinase↗
Copy-dependent and position-independent expression of rat aldolase A gene.
In order to understand the molecular mechanisms of the temporal and spatial differences of gene expression in higher organisms, rat aldolase A gene carrying two distinct promoters was introduced into fertilized eggs and the resulting transgenic mice were analyzed. The transgene expression is tissue-specific and is developmentally regulated. In addition, the expression is regulated in a copy-dependent manner irrespective of where the transgene is integrated, suggesting that a mechanism excluding the effect of the integration site exists within the transgene itself. To explore the conformational change of this gene in the genome, the DNase I hypersensitive sites of the gene were examined. Three sites (DHS-1,2, and 3) were identified upstream and downstream of the gene and these sites were retained in the transgene as well as in the gene observed endogenously.
Animals↗
Human GLUT-2 overexpression does not affect glucose-stimulated insulin secretion in MIN6 cells.
Accumulated evidence suggests that GLUT-2, in addition to its role in glucose transport, may also have other functions in glucose-stimulated insulin secretion. As a first step in addressing this possibility, we have engineered MIN6 cells overexpressing human GLUT-2 by transfection with human GLUT-2 cDNA. Stable transformants harboring human GLUT-2 cDNA exhibited an approximately twofold increase in 3-O-methyl-D-glucose uptake at 0.5 and 15 mM. Glucokinase activity or glucose utilization measured by conversion of [5-3H]glucose to [3H]H2O was not, however, altered in the MIN6 cells overexpressing human GLUT-2. Furthermore, glucose-stimulated insulin secretion was not affected by over-expression of human GLUT-2. An abundance of GLUT-2, therefore, does not correlate with the glucose responsiveness of cells in which glycolysis is regulated at the glucose phosphorylating step. These data suggest that GLUT-2 by itself does not have significant functions other than its role in glucose transport in glucose sensing by MIN6 cells.
3-O-Methylglucose↗
Ca2+/calmodulin-dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells.
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in the regulation of insulin secretion. We obtained evidence for the presence of CaM kinase II and its substrate, a 84-kilodalton (kDa) protein, in mouse insulinoma MIN6 cells. CaM kinase II from MIN6 cells has one subunit of 55 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is autophosphorylated in a Ca2+/CaM-dependent manner, and phosphorylates several substrates that serve for rat brain CaM kinase II. In the membrane fraction of MIN6 cells, we identified a 84-kDa protein that was immunoreactive with the antirat brain synapsin I antibody. One-dimensional phosphopeptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the sites of the phosphorylation by cAMP-dependent protein kinase (cAMP kinase) and that by CaM kinase II to be site 1 (10 kDa) and site 2 (30 kDa), respectively, therefore, the same as for rat brain synapsin I. In this context, we tentatively termed it synapsin I-like protein. In 32P-labeled cells, nonfuel insulin secretagogues, such as ionomycin, KCl, and tolbutamide, and a fuel secretagogue, glucose, stimulated autophosphorylation of CaM kinase II and the phosphorylation of synapsin I-like protein. These secretagogues potentiated the Ca(2+)-independent activity of CaM kinase II and secretion of insulin from MIN6 cells. The 84-kDa protein is apparently a newly identified member of the synapsin family. We suggest that CaM kinase II regulates insulin secretion via phosphorylation of synapsin I-like protein.
Animals↗
Morphologic study of the laryngeal taste buds in the cat.
The distribution of laryngeal taste buds (TBs) and their neutral components in the cat were investigated by immunohistochemistry and electron microscopy. The antisera used in this study were against cytokeratin, protein gene product 9.5 (PGP9.5), neuron-specific enolase (NSE), S-100 protein, calbindin D, calcitonin gene-related peptide (CGRP), and substance P (SP). Taste bud cells were specifically immunoreactive to the antibodies of human cytokeratin subtypes 8 and 18 (CAM5.2). On observation with CAM5.2, TBs were seen distributed on the laryngeal surface of the epiglottis and spread caudally along the aryepiglottic folds, reaching peak density at the laryngeal side of the arytenoid tubercle. The PGP9.5 and NSE immunoreactivities were recognized in TB cells and nerve fibers, both within the TBs and in the subepithelial connective tissue. S-100 protein immunoreactivities were not found in any of the cells in the TBs but were found exclusively in the subepithelial neural elements. The calbindin-D, CGRP, and SP immunoreactivities were confined to a part of the neural elements that was very thin. Taste pores, taste villi, neuronal varicosity, and synapselike structures were observed by scanning and transmission electron microscopic study. From these results it is considered that the TBs act as a chemical receptor.
Animals↗
Nitric oxide is important for mouse beta-cell line killing by peritoneal exudate cells obtained from cyclophosphamide treated non-obese diabetic mice.
Macrophages from recent onset non-obese diabetic (NOD) mice showed cytotoxicity against the NOD mouse derived beta-cell line, MIN6N-9a. In this report, we examined whether nitric oxide is associated with beta-cell destruction. Peritoneal exudate cells (PEC), obtained from cyclophosphamide treated NOD mice showed higher cytotoxicity against MIN6N-9a compared to PECs from saline injected NOD mice (P < 0.01). This effect was suppressed in cells incubated with 0.5 mmol/l NG-methyl-L-arginine, a nitric oxide synthase inhibitor (P < 0.001). In addition, the nitrite concentration of the co-culture medium, as an index of nitric oxide production, increased in MIN6N-9a cells co-cultured with peritoneal exudate cells from cyclophosphamide injected NOD mice but not in co-culture with saline injected NOD mice (P < 0.05). Thus, nitric oxide plays an important role in beta-cell line destruction of macrophages obtained from NOD mice.
Amino Acid Oxidoreductases↗
Formation of islet amyloid fibrils in beta-secretory granules of transgenic mice expressing human islet amyloid polypeptide/amylin.
To investigate the relationship between human islet amyloid polypeptide (IAPP)/amylin expression and islet amyloid deposits in the pathogenesis of human non-insulin-dependent diabetes mellitus (NIDDM), we developed transgenic mice using a human IAPP cDNA connected to an insulin promoter. Ribonucleic acid blotting and immunohistochemistry revealed the expression of the transgene in the pancreatic beta cells. Immunogold electron microscopy showed that beta-secretory granules contained the human C-terminal flanking peptide of the IAPP precursor. Reverse-phase HPLC demonstrated human and mouse IAPP amide in the pancreas. Electron microscopy showed the accumulation of fibril-like material in a considerable number of beta-secretory granules. These results suggest that in transgenic mice, the human IAPP precursor is expressed in beta cells and becomes normally sorted into beta-secretory granules in which normal conversion to mature human IAPP takes place. The human IAPP molecules, because of their amyloidogenesis, aggregate into amyloid fibrils in secretory granules. Glucose tolerance was normal at 7 months old and islet amyloid was not observed. A longer time may be required for islet amyloid deposits and hyperglycemia to develop in mice. Our working hypothesis is that in human NIDDM, IAPP aggregates into amyloid fibrils in beta-secretory granules, and that the fibrils are released into the extracellular space and islet amyloid deposits become substantial with time.
Amyloid↗
Glucose induces calcium-dependent and calcium-independent insulin secretion from the pancreatic beta cell line MIN6.
The present study was undertaken to determine whether there are Ca2+ -dependent and -independent pathways of glucose-induced insulin secretion from the pancreatic beta cell line MIN6. Glucose at a concentration of 16.7 mmol/l caused marked increases in cellular free calcium ([Ca2+]i) and insulin secretion, which depended on glucose metabolism. When cells were pretreated with 20 mmol/l mannoheptulose, an inhibitor of glucokinase, the 16.7 mmol/l glucose induced a rise in [Ca2+]i and insulin secretion disappeared. Also, L-leucine and L-arginine increased [Ca2+]i and induced insulin secretion. Under Ca2+ -free conditions, insulin release was still induced, without any change in [Ca2+]i, by these three different stimulants. The cumulative values of insulin secretion were 13.7-29.3% of the control, which were significantly less than that in the presence of Ca2+. Cellular alkalinization occurred in response to all these stimulants, irrespective of the presence of Ca2+. Forskolin, a diterpene activator of adenylate cyclase, produced insulin secretion independently of [Ca2+]i, which accompanied cellular alkalinization. Also, a high glucose level increased cellular cyclic AMP (cAMP) production in the presence and absence of Ca2+, and the effect was diminished by approximately 73% in Ca2+ -free conditions. These results indicate that a high glucose level stimulates both Ca2+ -dependent and -independent insulin secretion from pancreatic beta cells. We suggest that the cAMP production and the cellular alkalinization participate in the Ca2+ -independent mechanism. Spermatogonial proliferation is under the control of FSH, whereas the survival of germ cells is dependent on Sertoli cell function. The observed rise in the number of mitotically inactive Ad-spermatogonia can be explained by a transformation of Ap-spermatogonia into resting Ad-spermatogonia.
Alkalies↗
[The expression of the E-cadherin in human urinary bladder carcinoma].
We examined immunohistochemically the expression of E-cadherin which is Ca2+ dependent intercellular adhesion molecules in bladder carcinoma and investigated the correlation among the expression of E-cadherin, pathological examination, clinical findings and course. Fifty cases of bladder carcinoma were examined except one squamous cell carcinoma. The pattern of the immunohistochemical staining by E-cadherin antibody were classified into 4 groups as follows. The tumor, over 75% of which cells were stained like normal epithelium, was regarded as (2+). When from 50% to 75% of the carcinoma cells were stained, it was (+). When from 25% to 50% of the carcinoma cells were stained, it was (+/-). The tumor showing that under 25% of the cells were stained or lack of staining was regarded as (-). It was demonstrated that the percentage of positive staining was significantly lower in cases of high grade or high stage tumors compared with those of low grade or low stage. As the pattern of invasion, 88% of the cases showing INF alpha was observed as (2+) or (+), while all cases with INF gamma showed (+/-) or (-). The patients with superficial tumors showing (+/-) or (-) tended to have the higher local recurrence rate of the carcinoma compared with those showing (2+) or (+) staining. Immunoblotting analysis demonstrated no evidence of gross alteration of E-cadherin molecules between normal and carcinoma cells of the bladder. In conclusion, the decrease of E-cadherin expression may contribute to the tumor grade and invasiveness of bladder carcinoma.
Adult↗
Assessment of pore size of a semipermeable membrane for immunoisolation on xenoimplantation of pancreatic B cells using a diffusion chamber.
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Animals↗
Colonoscopic diagnosis of dysplasia and early cancer in longstanding colitis.
Five colitic cancers were detected among 40 patients with longstanding total colitis. The colitic cancers did not show the common polypoid or ulcerated appearance in the early stage, often being flat or plaque-like. It was not easy to detect these lesions endoscopically, and it was often impossible to do so radiologically. The flat or plaque-like early cancers were often surrounded by granular and/or red mucosa. We believed that the colonoscopic detection of this colitic cancer and dysplasia was difficult because: (1) the morphology of the lesions was difficult to determine, (2) the background mucosa was not normal. When the lesions were small, it was more difficult to detect them on the colitic mucosa than on the normal mucosa. The contrast between the lesion and the background mucosa was not clear in the latter condition. In surveillance colonoscopy (using a TV colonoscope) for longstanding ulcerative colitis, careful scrutiny throughout the large intestine is required to detect colitic cancers and dysplasia at an early state.
Adult↗