Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0011849 (diabetes)
 277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA-containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta-galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.
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PMID:A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region. 803 38

Isolation of endocrine cell precursors from the human fetal pancreas will be important to the study of islet cytodifferentiation and eventually for islet transplantation in insulin-dependent diabetes. These precursor cells, from which all four islet endocrine cell types arise, are present within fetal pancreatic ductal epithelium. After enzymatic digestion and culture of the fetal pancreas, we obtained cell clusters resembling islets, but with a high content of undifferentiated cells. Histochemical staining revealed very high acid beta-galactosidase activity in over 70% of cells within the clusters. After transplantation into athymic nude mice, the islet-like cell clusters gave rise to tissue rich in differentiated endocrine cells, but low in beta-galactosidase activity. The histochemical finding of high acid beta-galactosidase activity in endocrine precursor cells was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid beta-galactosidase was developmentally regulated, peaking at 18-24 weeks gestation and declining to low levels in adult islets. Using a fluorogenic beta-galactosidase substrate, we were able to isolate a subpopulation of cells high in acid beta-galactosidase activity using fluorescence-activated flow cytometry. Evidence identifying these cells as potential islet cell precursors includes, besides the transplantation experiments, the colocalization in vitro of tyrosine hydroxylase, a marker of embryonic islet cells. Thus, our results indicate that high acid beta-galactosidase activity serves as a marker for a population of fetal pancreatic cells with the potential to differentiate and grow into mature pancreatic endocrine cells.
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PMID:Acid beta-galactosidase: a developmentally regulated marker of endocrine cell precursors in the human fetal pancreas. 817 83

The authors investigate the in vitro component of an ex situ strategy for gene transfer into the thyroid gland using DNA complex and retroviral vectors. Canine follicular cells harvested by unilateral lobectomy and grown in low-serum media proliferated in culture and retained their differentiated state as evidenced by morphology and thyroglobulin expression. Transient and "stable" gene transfer in thyroid cells were evaluated by comparing DNA and retroviral transduction techniques. Effective gene transfer and expression was demonstrated by histochemical staining for the marker gene product beta-galactosidase. The efficiency of transduction was assessed using an amphotropic retroviral vector carrying the neomycin resistance gene and semiquantitative polymerase chain reaction (PCR) identification of integrated proviral sequences. This analysis demonstrated a proviral frequency in transduced cultures of 10% to 30%. Transduced cells showed no change in morphology or growth patterns and maintained differentiated function as assessed by antibody staining for thyroglobulin. The thyroid gland is an attractive target for somatic gene therapy because of its large protein-synthetic capacity, sensitivity to hormonal regulation, and proportionately high blood flow. Follicular cell gene therapy may be useful not only for treating congenital or acquired diseases of the thyroid, but also disorders of circulating proteins such as hypopituitarism, hemophilia, and diabetes.
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PMID:DNA- and viral-mediated gene transfer in follicular cells: progress toward gene therapy of the thyroid. 841 42

Activity of lysosomal enzymes was studied in 68 patients with Types 1 and 2 diabetes mellitus concurrent with diffuse thyroid enlargement. A decrease in activities of beta-galactosidase and DNAase and activation of cathepsins were detected in the leukocytes fraction from patients with Types 1 or 2 diabetes mellitus, thus demonstrating that metabolic impairments occurred in diabetes mellitus. These patterns were improved after intensive insulin therapy. Hyperfunction of the thyroid gland in patients with moderate Type 1 diabetes mellitus normalizes the activity of lysosomal enzymes.
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PMID:[Activity of lysosomal enzymes in diabetes mellitus patients along with diffuse enlargement of the thyroid gland]. 851 85

This study was undertaken to determine whether there are age-related changes in the specific activities of several glycosidases in fresh retinal pigment epithelial cells (RPE) isolated from the posterior pole of human donor eyes. One hundred and twenty-one pairs of eyes from human donors, between the ages of 43 and 95 years, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and the Cleveland Ohio Eye Bank within 18 to 24 h of death. None had histories of diabetes, hepatitis, HIV infection, intraocular surgery, or documented age-related macular degeneration, although several older donors with evidence of drusen were included in the study. RPE cells were isolated from the posterior third of the retina using the conventional rush method and homogenized with a glass, Broeck tissue grinder. All post-nuclear supernatants were analyzed for glycosidase activity; a smaller number of nuclear pellets were assayed to verify that the majority of the enzyme activity was associated with the post-nuclear sypernatants. Glycosidase activity was quantitated fluorometrically by measuring the enzymatic release of umbelliferone from synthetic substrate preparations, specific for each enzyme. Total protein was determined by a micro BCA protein assay. Regression analysis revealed statistically significant age-related decreases for the specific activities of alpha-mannosidase (p = 0.0001), beta-galactosidase (p = 0.0001), N-acetyl-beta-glucosaminidase (p = 0.0001), and N-acetyl beta galactosaminidase (p = 0.0001) in fresh human donor RPE cells taken from the region of the posterior third of the retina that included the macula. Mannose and N-acetyl-glucosamine are major carbohydrate monomers of the oligosaccaride chains of human rhodopsin, and a relatively high percentage of the oligosaccharide chains are galactosylated. Defects in their degradation may lead to the accumulation of undigested residual material in the RPE.
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PMID:Age-related changes of glycosidases in human retinal pigment epithelium. 867 Jul 43

The activity of all principal groups of lysosomal enzymes (acid phosphatase, lipase, beta-galactosidase, sulphatase and cathepsin B) was measured in the visual cortex of rabbits with experimental diabetes. In the first stage of diabetes (21 days), it was observed that enzyme activities in the free fraction and in the membrane-bound fraction are decreased as compared to the initial values determined in healthy animals. In the later stages of diabetes (90-180 days), all lysosomal enzyme activities increased except for sulphatase. This indicated a superiority of catabolic processes in visual cortex cells in the course of experimental diabetes.
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PMID:The activity of lysosomal enzymes in visual cortex of rabbits during experimental diabetes. 870 84

Gene transfer into the pancreas would be useful for the treatment of a variety of disorders, including cystic fibrosis, diabetes, cancer, and immunomodulation of pancreatic allografts. A hypothesis that various cell populations in the pancreas could be targeted by recombinant adenoviruses was developed and tested. Gene transfer into the rat ductal epithelium, acinar cells, and islets of Langerhans was accomplished with a recombinant adenovirus containing bacterial beta-galactosidase by retrograde delivery of adenovirus into the pancreaticobiliary duct. Maximal gene expression was observed at 3 days and correlated with DNA blot analysis. Histologic analysis of sections from pancreatic tissue in the adenovirus-treated rats demonstrated severe pancreatitis. Immunophenotyping of the inflammatory infiltrate with rat lymphocyte-specific markers showed CD45-, CD8-, and CD4-positive cells. Tissue injury resolved as gene expression was lost, with both features absent by 21 days. Pancreatic regeneration was documented by the presence of 5-bromo-2'-deoxyuridine-positive staining cells. Pancreatic gene transfer with first-generation recombinant adenoviruses can be accomplished by techniques applicable to clinical situations. The use of first-generation recombinant adenoviruses for pancreas-directed gene transfer is limited by the development of inflammation and transient expression.
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PMID:Adenovirus-mediated in vivo gene transfer and expression in normal rat pancreas. 874 Apr 9

The establishment of gene delivery systems that result in efficient transfection of the pancreatic beta-cells may generate an important tool for the study of IDDM and may also represent one critical step toward a clinical application of gene transfer for the prevention or early treatment of the disease. Using the reporter gene vectors pCAT and pCMV beta-gal, we have investigated the efficiency of transfection mediated by calcium phosphate precipitation, the monocationic liposome Lipofectin, the polycationic liposome Lipofectamine, and adenovirus-polylysine (AdpL) DNA complexes in human, mouse, rat, and fetal porcine islet cells. In all species studied, calcium phosphate-mediated transfection resulted in lower chloramphenicol acetyl transferase (CAT) activities than the other methods. Intact human, mouse, and rat islets were poorly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islect cells were efficiently transfected by Lipofectamine. Moreover, transfection of dispersed human and mouse islet cells using AdpL, also resulted in high CAT activities. The percentage of cells staining positively for beta-galactosidase after transfection with Lipofectamine was 49% for mouse, 56% for rat, and 57% for dispersed human islet cells. Transfection of human islet cells using AdpL, however, yielded 70% beta-gal-positive cells. Fluorescence-activated cell sorting-purified rat islet alpha- and beta-cells were transfected with similar efficiency using Lipofectamine. CAT expression in human islet cells transfected with either Lipofectamine or AdpL reached a peak value after 5-7 days, followed by a gradual decline. It is concluded that transfection with AdpL or Lipofectamine are both efficient means to achieve transient expression of gene constructs in human and mouse islet cells, while for rat and fetal porcine islet cells, Lipofectamine is the most efficient of the agents investigated in this study.
Diabetes 1996 Sep
PMID:Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes. 877 22

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.
Diabetes 1996 Sep
PMID:Effect of mitochondrial and/or cytosolic glycerol 3-phosphate dehydrogenase overexpression on glucose-stimulated insulin secretion from MIN6 and HIT cells. 877 29

Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 microU (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated 35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte [14C]glucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the beta-galactosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for beta-Gal. Approximately 80% of myotubes expressed beta-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.
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PMID:Synthesis and processing of genetically modified human proinsulin by rat myoblast primary cultures. 882 70


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