Lecithinized Cu, Zn-superoxide dismutase limits the infarct size following ischemia-reperfusion injury in rat hearts in vivo

Ghrelin is a novel gut-brain peptide that binds to the growth hormone secretagogue receptor (GHS-R), thereby functioning in the regulation of growth hormone (GH) release and food intake. Ghrelin-producing cells are most abundant in the oxyntic glands of the stomach. The regulatory mechanism that governs the biosynthesis and secretion of ghrelin has not been clarified. We report that ghrelin mRNA expression in the gastric fundus was increased, but that ghrelin peptide content decreased after a 48-h fast. Both values returned to control levels after refeeding. The ghrelin plasma concentration in the gastric vein and systemic venous blood increased after 24- and 48-h fasts. Furthermore, des-octanoylated ghrelin and n-octanoylated ghrelin were found in rat stomach, with the ratio of des-octanoylated ghrelin to n-octanoylated ghrelin markedly increased after fasting. The ghrelin mRNA level in the stomach also increased after administration of insulin and leptin. Conversely, db/db mice, which are deficient in the leptin receptor, had lower ghrelin mRNA levels than control mice. These findings suggest that this novel gastrointestinal hormone plays a role in the regulation of energy balance.     

The solubilization and biological activities of Aspergillus beta-(1 --> 3)-D-glucan

We have recently demonstrated that the cell wall beta-glucan of Candida albicans could be solubilized by sodium hypochlorite, followed by dimethylsulfoxide-extraction (NaClO-DMSO method). In this study, applying this method to Aspergillus spp., we prepared mycelial cell wall beta-glucan and examined its physical properties and immunotoxicological activity. The acetone-dried mycelia of Aspergillus spp. were oxidized by the NaClO-DMSO method. An analysis of (13)C NMR spectra revealed the preparations to be composed of alpha-(1 --> 3) and beta-(1 --> 3)-D-glucan. Also, the proportion of alpha-(1 --> 3) and beta-(1 --> 3)-D-glucan varied. Furthermore, a solubilized Aspergillus beta-glucan (ASBG) was prepared from OX-Asp by urea-autoclave treatment. ASBG showed limulus activity similar to Candida solubilized beta-glucan (CSBG), and there was little difference in the activity of ASBG between various Aspergillus spp. ASBG affected the production of IL-8 by human peripheral blood mononuclear cells (PBMC). ASBG should be useful for analyzing the clinical role of beta-glucan.     

Effect of lectins on the transport of food ingredients in Caco-2 cell cultures

We investigated the effect of several lectins, such as soy bean lectin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA), on the transport of some food ingredients (isoflavones, quercetin glycosides, carnosine/anserine) across Caco-2 cell monolayers. After incubation of food ingredients (0.03 approximately 2 mmol/L) in the presence or absence of lectins (1 approximately 180 microg/ml) on the apical side, aliquots were taken from the apical and basolateral solution, and were subjected to HPLC analysis. We also examined the effect of lectins on the permeability of the tight junction by measuring the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayer. Isoflavones, which was not transported to the basolateral solution without lectins, could be transported in the presence of lectins, whereas their aglycones were detected at the same levels with or without the lectin treatment. The transport of quercetin glycosides also increased in the presence of lectins, however, that of peptides was not affected by the lectins. Con A and WGA, but SBA, decreased the TER value, indicating that Con A and WGA increased the transport via paracellular pathway, whereas SBA did via a different pathway.     

Characterization of diabetes-related traits in MSM and JF1 mice on high-fat diet

We examined the effect of a high-fat diet on the diabetes-related traits of the Japanese Fancy mouse 1 (JF1), MSM, and C57BL/6J (B6J) mice. MSM and JF1 mice were derived from Mus musculus molossinus. B6J is a commonly used laboratory strain, with the vast majority of genome segments derived from Mus musculus domesticus and Mus musculus musculus, and is susceptible to high-fat diet-induced type 2 diabetes. None of the strains showed symptoms of diabetes or obesity when fed a laboratory chow diet. Under a high-fat diet, JF1 mice developed impaired glucose tolerance, hyperglycemia, hyperinsulinemia, and obesity. B6J mice fed a high-fat diet mildly developed these diabetes-related traits compared to JF1 mice fed a high-fat diet. JF1 mice fed a high-fat diet were classified as having type 2 diabetes and were susceptible to high-fat diet-induced diabetes and obesity. On the other hand, MSM mice were resistant to high-fat diet-induced diabetes. These results indicate that the JF1 strain, with its unique genetic origin, is a useful new animal model of high-fat diet-induced diabetes and obesity. Further investigations using JF1 mice will help to clarify the role of the high-fat diet on human diabetes and obesity.     

Structure and direct electrochemistry of cytochrome P450 from the thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7

Cytochrome P450 from thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7 (P450st) has been expressed in Escherichia coli and purified at high homogeneity. P450st was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=53.6 A, b=55.1 A, and c=130.9 A, and the structure was determined at a 3.0 A resolution. The final R-factor was 0.194 (Rfree=0.235). Structural comparison with cytochrome P450 from S. solfataricus (CYP119) suggests that the region composed of the F to G helices and the Cl- binding site is responsible for the affinity for a ligand coordinating heme iron. Direct electrochemistry of P450st in a didodecyldimethylammonium bromide (DDAB) film on a plastic formed carbon (PFC) electrode has also been demonstrated. A quasi-reversible redox response has been observed even at elevated temperatures of up to 80 degrees C.     

Synthesis and evaluation of substituted 4-alkoxy-2-aminopyridines as novel neuropeptide Y1 receptor antagonists

A series of substituted 4-alkoxy-2-aminopyridines 2, which were formally derived from neuropeptide Y1 antagonist 1 by replacing the morpholino portion with alkoxy groups, were synthesized and evaluated as neuropeptide Y Y1 receptor antagonists. Primary structure-activity relationships and identification of potent 4-alkoxy derivatives are described.     

Evidence that exogenous substances can be phagocytized by alveolar epithelial cells and transported into blood capillaries

Since the ability of alveolar epithelial cells to ingest inhaled fine particles has not been characterized in detail, the present study seeks to evaluate this physiological activity. We used a 0.2% suspension of intact or lecithin-coated polystyrene latex beads (240 nm in diameter). A 5-ml suspension of intact or lecithin-coated latex beads was intratracheally administered to rats using a compressor nebulizer. Thereafter, the lungs were perfused intratracheally with glutaraldehyde solution and cut into small pieces. The samples were postfixed with osmium tetroxide, embedded in epoxy resin and examined under an electron microscope. Both lecithin-coated and uncoated beads were incorporated into alveolar macrophages. Some of the ingested beads in the alveolar macrophages were sequestered within lysosomes. Types I and II alveolar epithelial cells selectively incorporated only lecithin-coated beads, which were also observed within the cytoplasm of monocytes in the capillary lumen. These findings suggest that alveolar epithelial cells can incorporate exogenous particles, which are then transferred from the alveoli to intravascular spaces by transcytosis.     

An oxidized purine nucleoside triphosphatase,MTH1, suppresses cell death caused by oxidative stress

MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and thus protects cells from damage caused by their misincorporation into DNA. In the present study, we established MTH1-null mouse embryo fibroblasts that were highly susceptible to cell dysfunction and death caused by exposure to H2O2, with morphological features of pyknosis and electron-dense deposits accumulated in mitochondria. The cell death observed was independent of both poly(ADP-ribose) polymerase and caspases. A high performance liquid chromatography tandem mass spectrometry analysis and immunofluorescence microscopy revealed a continuous accumulation of 8-oxo-guanine both in nuclear and mitochondrial DNA after exposure to H2O2. All of the H2O2-induced alterations observed in MTH1-null mouse embryo fibroblasts were effectively suppressed by the expression of wild type human MTH1 (hMTH1), whereas they were only partially suppressed by the expression of mutant hMTH1 defective in either 8-oxo-dGTPase or 2-OH-dATPase activity. Human MTH1 thus protects cells from H2O2-induced cell dysfunction and death by hydrolyzing oxidized purine nucleotides including 8-oxo-dGTP and 2-OH-dATP, and these alterations may be partly attributed to a mitochondrial dysfunction.     

Genistein administration decreases serum corticosterone and testosterone levels in rats

The phytochemical flavonoid genistein has been shown to act as a potent competitive inhibitor of human adrenocortical 3beta-hydroxysteroid dehydrogenase and cytochrome P450 21-hydroxylase activities in vitro [J. Steroid Biochem. Molec. Biol. 2002; 80: 355-363]. In the present study, we evaluated the effects of large amounts of genistein continuously administered to weanling rats, particularly on steroidogenesis at the pubertal stage in vivo. Serum concentrations of free and total genistein were significantly higher in the 40 mg/kg genistein administration group when compared with the control group. In genistein administered rats, adrenal weight was significantly higher. Furthermore, a clear expansion of cells was observed in hematoxylin and eosin stained tissue at the zona fasciculata and zona reticularis of the adrenal cortex. However in the testis, no differences in weights or histologic changes were observed. Serum corticosterone concentration significantly decreased to 50% of control levels by 40 mg/kg genistein administration and testosterone also tended to decrease with this dose of genistein. On the other hand, although serum follicle stimulating hormone was unchanged, adrenocorticotropic hormone and luteinizing hormone levels increased with genistein administration. These results suggest a significant effect of genistein on steroidogenesis in the adrenal gland and testis of rats, and this effect appeared to be more evident on steroid production in adrenals than in testis in vivo.     

Gliclazide protects pancreatic beta-cells from damage by hydrogen peroxide

Oxidative stress is induced under diabetic conditions and possibly causes various forms of tissue damage in patients with diabetes. Recently, it has become aware that susceptibility of pancreatic beta-cells to oxidative stress contributes to the progressive deterioration of beta-cell function in type 2 diabetes. A hypoglycemic sulfonylurea, gliclazide, is known to be a general free radical scavenger and its beneficial effects on diabetic complications have been documented. In the present study, we investigated whether gliclazide could protect pancreatic beta-cells from oxidative damage. One hundred and fifty microM hydrogen peroxide reduced viability of mouse MIN6 beta-cells to 29.3%. Addition of 2 microM gliclazide protected MIN6 cells from the cell death induced by H(2)O(2) to 55.9%. Glibenclamide, another widely used sulfonylurea, had no significant effects even at 10 microM. Nuclear chromatin staining analysis revealed that the preserved viability by gliclazide was due to inhibition of apoptosis. Hydrogen peroxide-induced expression of an anti-oxidative gene heme oxygenase-1 and stress genes A20 and p21(CIP1/WAF1), whose induction was suppressed by gliclazide. These results suggest that gliclazide reduces oxidative stress of beta-cells by H(2)O(2) probably due to its radical scavenging activity. Gliclazide may be effective in preventing beta-cells from the toxic action of reactive oxygen species in diabetes.