Genetic association, post-translational modification, and protein-protein interactions in Type 2 diabetes mellitus

Type 2 diabetes mellitus is a complex disorder with a strong genetic component. Inherited complex disease susceptibility in humans is most commonly associated with single nucleotide polymorphisms. The mechanisms by which this occurs are still poorly understood. Here we focus on analyzing the effect of a set of disease-causing missense variations of the monogenetic form of Type 2 diabetes mellitus and a set of disease-associated nonsynonymous variations in comparison with that of nonsynonymous variations without any experimental evidence for association with any disease. Analysis of different properties such as evolutionary conservation status, solvent accessibility, secondary structure, etc. suggests that disease-causing variations are associated with extreme changes in the value of the parameters relating to evolutionary conservation and/or protein stability. Disease-associated variations are rather moderately conserved and have a milder effect on protein function and stability. The majority of the genes harboring these variations are clustered in or near the insulin signaling network. Most of these variations are identified as potential sites for post-translational modifications; certain predictions have already reported experimental evidence. Overall our results indicate that Type 2 diabetes mellitus may result from a large number of single nucleotide polymorphisms that impair modular domain function and post-translational modifications involved in signaling. Our emphasis is more on conserved corresponding residues than the variation alone. We believe that the approach of considering a stretch of peptide sequence involving a polymorphism would be a better method of defining the role of the polymorphism in the manifestation of this disease. Because most of the variations associated with the disease are rare, we hypothesize that this disease is a "mosaic model" of interaction between a large number of rare alleles and a small number of common alleles along with the environment, which is little contrary to the existing common disease common variant model.     

Clinical and experimental mycotic corneal ulcer caused by Aspergillus fumigatus and the effect of oral ketoconazole in the treatment

Aspergillus fumigatus was isolated from a case of keratomycosis. The patient, a 12-year-old boy presented with large corneal ulcer with hypopyon. The direct microscopic examination of scrapings revealed hyaline, septate mycelium. In vitro some antimycotics (amphotericin B,5-fluorocytosine, oxiconazole, amorolfine and ketoconazole) were tested against A. fumigatus by agar dilution method. Ketoconazole with minimum inhibitory concentration of 30 g/ml after 11 days of incubation was most effective against A. fumigatus. Experimental corneal ulcer was produced by injecting intralamellary spore suspension (2.5×10⁶ c.f.u.) into the right eyes of previously immunosuppressed albino and black wild types of rabbits. The extent of ocular infection was graded up to 32 days. Histopathologic examination showed infiltration and large destruction of corneal stroma. Oral ketoconazole therapy exhibited partial response followed by relapse. The black type of rabbit appeared more suitable as an animal model for mycotic keratitis.This paper was presented at the X^th congress of the International Society for Human and Animal Mycology at Barcelona, Spain from June 27 to July 1, 1988.     

The influence of insulin on circulating ghrelin

Ghrelin is a novel peptide that acts on the growth hormone (GH) secretagogue receptor in the pituitary and hypothalamus. It may function as a third physiological regulator of GH secretion, along with GH-releasing hormone and somatostatin. In addition to the action of ghrelin on the GH axis, it appears to have a role in the determination of energy homeostasis. Although feeding suppresses ghrelin production and fasting stimulates ghrelin release, the underlying mechanisms controlling this process remain unclear. The purpose of this study was to test the hypotheses, by use of a stepped hyperinsulinemic eu- hypo- hyperglycemic glucose clamp, that either hyperinsulinemia or hypoglycemia may influence ghrelin production. Having been stable in the period before the clamp, ghrelin levels rapidly fell in response to insulin infusion during euglycemia (baseline ghrelin 207 +/- 12 vs. 169 +/- 10 fmol/ml at t = 30 min, P < 0.001). Ghrelin remained suppressed during subsequent periods of hypoglycemia (mean glucose 53 +/- 2 mg/dl) and hyperglycemia (mean glucose 163 +/- 6 mg/dl). Despite suppression of ghrelin, GH showed a significant rise during hypoglycemia (baseline 4.1 +/- 1.3 vs. 28.2 +/- 3.9 microg/l at t = 120 min, P < 0.001). Our data suggest that insulin may suppress circulating ghrelin independently of glucose, although glucose may have an additional effect. We conclude that the GH response seen during hypoglycemia is not regulated by circulating ghrelin.     

Hydrogel-based protein microchips: manufacturing,properties, and applications

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.     

Ascorbic acid blocks the growth inhibitory effect of tumor necrosis factor-alpha on endothelial cells

Impaired endothelial cell proliferation has been proposed to be an early, critical defect contributing to the development of atherosclerosis. Recent studies show that high plasma tumor necrosis factor (TNF)-alpha levels and low serum ascorbic acid (AA) levels correlate with atherosclerosis severity. Additionally, AA has been reported to have potential beneficial effects in preventing atherosclerosis. Based on these studies, we investigated the role of AA (< or =1mM) on TNF-alpha-mediated vascular endothelial cell growth inhibition in vitro. In accordance with previous reports, we found that TNF-alpha alone inhibited endothelial cell proliferation. Further studies revealed that AA alone enhanced endothelial cell proliferation and that AA blocked endothelial cell growth inhibition induced by TNF-alpha. By contrast, we observed no effect of AA on endothelial cell activation or nuclear entry of nuclear factor-kappaB in response to TNF-alpha. The protective effect of AA on endothelial cell proliferation was not simply the result of its antioxidant activity but did correlate with collagen IV expression by endothelial cells. AA pre-treatment of proliferating endothelial cells promoted retinoblastoma protein (Rb) phosphorylation and decreased p53 levels when compared to untreated cells. Furthermore, the addition of AA to TNF-alpha-treated proliferating endothelial cells blocked both the inhibition of retinoblastoma protein phosphorylation and enhanced p53 expression induced by TNF-alpha. Consistent with these results, we found that AA protects endothelial cells against TNF-alpha-induced apoptosis. These studies highlight the potential therapeutic role of AA in promoting endothelial cell proliferation during inflammatory conditions, such as atherosclerosis and cardiovascular disease.     

LDL induces intracellular signalling and cell migration via atypical LDL-binding protein T-cadherin

Cadherins are a superfamily of adhesion molecules that mediate Ca²⁺-dependent cell–cell adhesion. T-cadherin (T-cad), a unique glycosylphosphatidylinositol-anchored member of the cadherin superfamily, was initially identified by immunoblotting of vascular cell membranes as an atypical low affinity low density lipoprotein (LDL)-binding protein. It is not known whether this heterophilic interaction is physiologically relevant. Expression of T-cadherin is upregulated in vascular cells during atherosclerosis, restenosis and tumour angiogenesis, conditions characterized by enhanced cell migration and growth. Elevated levels of serum low density lipoproteins (LDL), which result in cholesterol accumulation in vascular wall, is a widely accepted risk factor in atherosclerosis development. Additionally to its metabolic effects, LDL can produce hormone-like effects in a number of cell types. This study has utilized HEK293 cells and L929 cells stably transfected with T-cadherin cDNA to investigate T-cad-dependent responses to LDL. Stable expression of T-cad in both HEK293 and L929 cells results in significantly (p < 0.05) elevated specific surface binding of [I¹²⁵]-LDL. Compared with mock-transfectants, cells expressing T-cad exhibit significantly (p < 0.01) enhanced LDL-induced mobilization of intracellular Ca²⁺-stores and a significantly (p < 0.01) increased migration toward an LDL gradient (0.1% BSA + 60 g/ml LDL) in Boyden chamber migration assay. Thus LDL-binding to T-cad is capable of activating physiologically relevant intracellular signaling and functional responses.     

Hydrogel glycan microarrays

The technology of hydrogel microchips manufacturing, which was developed previously for covalent immobilization of DNA and proteins, was applied for the preparation of glycochips and combined glyco/protein chips. Microchips consist of hydrogel drops separated with hydrophobic surface. Spacered amino-saccharides and polyacrylamide glycoconjugates were used for immobilization. Gel elements were approximately 1 nl in volume (150 microm in diameter and 25 microm in height), and the amount of covalently immobilized saccharide in the glycoarray was 0.4-1.7 pmol per gel element. Hydrogel glycan microchips were used for quantitative assay of antibodies against blood group antigens and assay of lectins with fluorescent detection. In all cases, only specific interaction with chip-immobilized saccharides was observed, whereas the background signal was very low. The detection limit of on-chip assays was comparable to that of the standard 96-well plate assays. Mixing of reaction solution allowed us to decrease the duration of the assays significantly: 2-3 h for incubation and development steps and 10 min for washing. A method for determination of association constants for binding of compounds with chip-immobilized ligands from the kinetics of their binding is proposed. Combined microchips containing different types of biomolecules can be designed and used for simultaneous detection of different compounds.