Search

Here we characterized gene expressions in subcutaneous adipose tissue and blood metabolites of pigs with genetically low backfat (Landrace) and high backfat (Meishan). As pigs aged from 1 wk-to 3-mo old, mRNA levels of adipose-specific genes increased, although their gene expressions coding for major enzymes involved in lipid metabolism (lipoprotein lipase, fatty acid synthase, and hormone-sensitive lipase) did not differ between lean and fat pigs. Instead, there were significant effects for adiponectin and its receptor AdipoR1 mRNA levels between the two breeds of which respective expressions were lower and higher in Meishan by 3 mo of age. Contrary to changes in gene expressions, the concentrations of blood glucose, triglyceride (TG), and NEFA in both breeds decreased during growth, and 3-mo-old Meishan evidenced lower glucose with higher TG than the Landrace. The homeostasis model assessment insulin resistance (HOMA-IR) index was also calculated from the measurements of fasting glucose and insulin concentration, and Meishan showed a higher value than the Landrace. We next examined these differences in Landrace and Meishan crossbreds, which were phenotypically distinguishable by the backfat thickness as the former lean type and the latter fat type. As with the purebreds, high backfat Meishan crosses showed the characteristics of lower glucose and higher TG in circulating levels and also lower adiponectin transcripts in subcutaneous adipose tissue. Collectively, our results demonstrate that levels of adiponectin and its receptor gene expressions, blood glucose, blood lipids, and HOMA-IR in pigs vary between lean and fat. These observations strongly suggest the possibility that overall metabolic differences rather than adipocyte ability itself contribute to the fatness of genetically high backfat pigs.

One of the most characterized neurotrophic factors is brain-derived neurotrophic factor (BDNF), which regulates neuronal survival and differentiation, and functions in activity-dependent plasticity processes such as long-term potentiation, long-term depression (LTD), and learning memory. Similar to other growth factors, BDNF protein is produced by transcriptional and translational mechanisms. Nevertheless, a posttranslational mechanism, the proteolytic conversion of precursor BDNF into at least two fragments, bioactive BDNF and the prodomain, has not been well elucidated. Recently, we demonstrated that the BDNF prodomain, which is named the BDNF propeptide, was endogenously secreted from neuronal cells and facilitated a cellular mechanism of LTD, suggesting the manner through which this posttranslational mechanism multiplies the biological actions of BDNF. In this chapter, we focus on the BDNF propeptide, especially in synaptic plasticity, and discuss the role of this molecule in the brain. The findings regarding the BDNF propeptide would provide new insights for understanding the mechanisms of action of the propeptides of growth factors as well as the biological roles of neurotrophins.

Growth hormone secretion from the anterior pituitary gland is controlled by interactions between three hormone receptors, between GHRH and GHRH receptor (GHRH-R), between ghrelin and growth hormone secretagogue receptor (GHS-R1a), and between somatostatin and somatostatin receptors in the hypothalamus and anterior pituitary gland. Ghrelin-GHS-R1a is involved in many important functions, including GH secretion and appetite. We investigated age-related changes in the expressions of GHS-R1a, GHS-R1b (the truncated-type receptor), and GHRH-R mRNAs by real-time reverse transcription-PCR using 16 tissues, leukocytes, oocytes, and cumulus cells in Holstein-Friesian cattle. The tissue samples were divided into three age classes: 1) 19 to 26 d of age (preweaning calves), 2) 2 mo to 6.5 mo of age (postweaning calves), and 3) 3.2 to 8.1 yr of age (cows). The GHS-R1a mRNA was highly (P0.05) expressed in the arcuate nucleus, pituitary gland, and liver compared with that of the other tissues in all age classes. Expression of GHS-R 1a mRNA in the arcuate nucleus of postweaning calves was10-fold greater (P0.01) than those of preweaning calves and cows, and its expression level was the greatest (P0.01) in all tissues examined in age group 2. GHS-R1a and GHRH-R mRNA expressions in the pituitary gland of preweaning calves tended to be greater (P0.20 and P0.17, respectively) than those of postweaning calves and cows. GHS-R1b mRNA expression was detected in all tissues examined, and abundance was greater (P0.05) in the pancreas, pituitary gland, spleen, arcuate nucleus, adipose tissue, and leukocyte compared with that of the other tissues examined in age group 3. Interestingly, a relatively large animal-to-animal variation was observed in pancreas GHS-R 1b mRNA expression. The GHRH-R mRNA was markedly increased (P0.01) in the pituitary gland in all age groups compared with that of the other tissues. GHRH-R mRNA abundance in the arcuate nucleus, pituitary gland, liver, spleen, adipose tissue, and heart of preweaning calves tended to be greater than those of postweaning calves and cows. The GHRH-R mRNA was not detected in the mammary gland and adipose tissue of nonlactating cows.

Feline obesity generally results in aberrations to plasma metabolite levels, such as lipid concentrations and lipoprotein composition. This study sought to investigate the resultant effect of obesity on cholesterol lipoprotein composition and circulating adiponectin concentrations in cats. Plasma glucose, lipids (triglyceride, cholesterol and free fatty acid), insulin and adiponectin concentrations, and cholesterol lipoprotein composition were measured and compared between body condition score (BCS) determined normal healthy control and obese cats. Although the obese group demonstrated higher levels of plasma cholesterol, glucose, and triglycerides, as compared to healthy controls, the difference was insignificant thus indicating that the BCS determined obese cats may have been overweight and not morbidly obese. Plasma insulin levels were significantly higher (25-30%) versus healthy control animals thereby possibly hinting at the ensuing emergence of obesity induced insulin resistance. However, the BCS determined obese cat demonstrated a significant reduction (p

A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50 kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.

The hyperinsulinemic-euglycemic clamp (EGC) technique was used to investigate the effects of calcium salts of long-chain fatty acids (LCFA-Ca) and rumen-protected Met (RPM) on insulin sensitivity in the peripheral tissues of lactating cows. Six multiparous Holstein cows were used in a 3 × 3 Latin square experiment in each 14-d period. Dietary treatments were 0 (RPM0), 20 (RPM20), and 60 (RPM60) g/d of RPM, supplemented with a diet containing 1.5% of LCFA-Ca equal to 110% of the cows' ME requirement. And as a control for the 3 LCFA-Ca-containing diets, a dietary treatment without LCFA-Ca (Con) was also included. After a 10-d adaptation period, milk samples were collected for 4 d, and EGC experiments were performed on d 14 of each treatment period. Insulin solution was infused through a jugular vein catheter at a rate of 0.1, 0.2, 0.3, and 0.4 milliunits·kg BW-1·min-1 for 30 min and then at a rate of 0.5 milliunits·kg BW-1·min-1 for 60 min. Glucose solution was variably infused to maintain plasma glucose at steady state through the same catheter. Blood samples for measurements were taken using the contralateral catheter. Plasma total cholesterol, cholesterol ester, free cholesterol, and phospholipid concentrations in RPM0 and RPM20 were higher than those in Con, whereas the concentrations in RPM60 were low at the same degree of those in RPM0 (P0.05). Plasma Met concentration was greatest in RPM60 (P0.05). In the EGC experiment, the glucose infusion rate was greater in RPM60 than in RPM0 and RPM20 and an effective concentration of insulin resulting in 50% maximal glucose infusion rate was lower in RPM60 compared with RPM0 (P0.05), indicating that insulin sensitivity was intensified in RPM60. Although the insulin sensitivity evaluated from the EGC data in RPM0, RPM20, and RPM60 was not different from Con, a slight decline was observed in RPM0 and insulin sensitivity in RPM60 was higher than Con. Our results from the EGC experiment demonstrated that the feeding RPM lead to increased insulin sensitivity, which suggests that dietary Met affects lipid metabolism via insulin action in lactating dairy cows fed a LCFA-Ca-containing diet.

Metabolite, insulin and adiponectin concentrations and LDH, AST and ALT activities were measured in plasma of 142 client-owned cats (1-13years old, 16 breeds) to set up a new criterion of hypertriglyceridemia (hyper-TG) with increased plasma insulin concentrations for early diagnosis of lipid metabolism abnormality including obesity. 25 cats with over 165mg/dl of plasma triglyceride (TG) concentrations were decided as hyper-TG with increased plasma insulin concentrations, and prevalence of hyper-TG was 16.7% in young (1-6years old) and 18.3% in old (>7years old) cats examined. In the hyper-TG cats, their plasma TG concentrations increased to 6.6-7.4-fold of the values of control cats with 35-50mg/dl of plasma TG and their plasma cholesterol, FFA and insulin concentrations and LDH and ALT activities increased significantly, whereas their plasma adiponectin concentrations decreased significantly compared to those in the control cats. Hyper-TG cats with significantly increased body weights and plasma insulin and decreased plasma adiponectin seemed to be in early stage of obesity accompanying increased plasma insulin concentrations. Increased TG, insulin, LDH and ALT and decreased adiponectin values in plasma seemed to be key factors for diagnosis of lipid metabolism abnormality at early stage in cats.

There has been rapid progress in determining the mechanism by which complement is activated by the complex formed between Mannose-Binding Lectin and its associated proteases (MASPs). MBL and the MASPs are of low abundance, but are similar to the more abundant C1q-C1r2s2 complex (C1), which has been extensively investigated. In this review we summarise recent findings on MBL-MASPs' structure. enzymic activity and regulation, and compare MBL-MASPs with C1.

The effect of ultraviolet B (UV-B) radiation and a vitamin-C-deficient (VCD) diet on guinea pig lenses was investigated. The initial lens changes in the VCD group were observed by slit-lamp examination 6 weeks after the start of the VCD treatment; after 12 weeks the changes in the posterior subcapsular region became more prominent, and the dissociation around the posterior suture became wider and slightly deeper toward the posterior cortex. The high concentration of lens oxidized glutathione (GSSG), and the low ratio of reduced glutathione (GSH) to oxidized glutathione (GSH/GSSG) on the lens posterior region correlated with density changes in the corresponding layers as measured by Scheimpflug images with linear microdensitometry. It is suggested that the strong oxidative stress of the VCD diet caused the damage at the posterior part of the lens. UV-B radiation appeared to accelerate cataract progression in lenses that lack vitamin C.

The effects of feeding and intravenous injections of glucose and VFA on blood ghrelin concentrations were investigated in calves before and after weaning. Eight Holstein bull calves were fed whole milk, allowed free access to solid feeds, and weaned at 7 wk. Measurements were carried out at 2, 4, 6, 9, 11, and 13 wk, at which time jugular blood samples were taken from 4 calves through a catheter from 10 min before to 120 min after their morning feed at 10 min intervals (Exp. 1). An additional 4 calves of the same age were intravenously injected with glucose (1.0 mmol·kg BW(-1)) and a solution of VFA (2.4 mmol·kg BW(-1), acetate:propionate:butyrate in a 6:3:1 ratio) using a catheter, and jugular blood samples were taken temporally relative to the injection time (Exp. 2). In Exp. 1, blood ghrelin concentrations decreased (P0.05) after feeding at all ages. However, preprandial ghrelin concentrations were less (P = 0.025) and the degree of postprandial depression of ghrelin tended to be greater during the postweaning period (P = 0.084) than during the preweaning period. Blood glucose concentrations increased after feeding during the preweaning period (P0.05), whereas blood acetate concentrations increased during the postweaning period (P0.05). In Exp. 2, injection of VFA induced a greater decrease in blood ghrelin concentrations than glucose injection throughout the entire period (P0.05). These results indicate that weaning reduces the basal concentration of blood ghrelin because the circulating VFA derived from ruminal fermentation may more strongly depress blood ghrelin concentrations than glucose.

BACKGROUND Previous studies have demonstrated that angiotensin II (Ang II) acts as a growth-promoting factor directly on cardiac myocytes and that angiotensin-converting enzyme inhibitor induces regression of hypertrophied hearts both in experimental animals and in humans. These results suggest that the renin-angiotensin system (RAS) is involved in the formation of left ventricular hypertrophy (LVH). To elucidate the role of RAS in the progression of cardiac hypertrophy, we evaluated the effect of an Ang II receptor antagonist on LVH in spontaneously hypertensive rats (SHRs) and investigated the molecular mechanisms by which antagonizing Ang II receptors reduces cell hypertrophy of myocytes using the in vitro model of mechanical stretch. METHODS AND RESULTS In the in vivo study, we treated SHRs with the nonpeptide Ang II receptor antagonist TCV-116 (0.1, 1, or 10 mg/kg per day) or hydralazine (10 mg/kg per day). Blood pressure was measured by the tail-cuff method, and wall thickness of left ventricle was serially monitored using M-mode echocardiography. Rats were killed at the age of 13, 17, 21, or 25 weeks, and left ventricular (LV) weight, transverse diameter of cardiomyocytes, relative amount of V3 myosin heavy chain (MHC), and degree of interstitial collagen accumulation were examined. Untreated SHRs progressively developed severe hypertension, but treatment with TCV-116 or hydralazine inhibited the increase in blood pressure. Treatment with TCV-116 reduced LV weight, LV wall thickness, transverse diameter of myocytes, relative amount of V3 MHC, and interstitial fibrosis, whereas treatment with hydralazine slightly prevented an increase in LV wall thickness but did not exert significant reduction in other parameters. In the in vitro study, neonatal rat cardiomyocytes were cultured on deformable silicone dishes and mechanically stretched with or without pretreatment of CV-11974 (an active metabolite of TCV-116), and [3H]phenylalanine incorporation, activity of mitogen-activated protein (MAP) kinase, and c-fos mRNA expression were analyzed. Pretreatment of cultured cardiomyocytes with 10(-7) mol/L CV-11974 inhibited an increase in [3H]phenylalanine incorporation, MAP kinase activity, and c-fos gene expression induced by stretch of cardiomyocytes. CONCLUSIONS The Ang II receptor antagonist TCV-116 induced regression of cardiac hypertrophy and had cardioprotective effects on hypertrophied myocardium in vivo, and antagonizing Ang II receptors inhibited intracellular signaling of stretch-mediated cardiomyocyte hypertrophy in vitro. These results suggest a crucial role of the cardiac RAS in the development of LVH produced by pressure overload.

Chlorinated benz[a]anthracenes (Cl-BaA) are halogenated aromatic compounds (typified by dioxins) found in the environment at relatively high concentrations. Fischer 344 rats were intragastrically administered 0, 1, or 10 mg of Cl-BaA or its parent compound benz[a]anthracene (BaA) per kg of body weight for 14 consecutive days. Both chemicals at 10 mg/kg/day inhibited the gain in body weight, and consequent increase in relative liver weight. Hepatic gene expression of cytochrome P450 (CYP) 1A1, 1A2, and 1B1 was significantly stimulated by administration of BaA (10 mg/kg/day) compared with the control. After administration of Cl-BaA, only the CYP1A2 gene was significantly induced, even at the lower dosage; CYP1A1 and 1B1 mRNA levels remained unchanged in Cl-BaA-treated rats compared with controls. To elucidate the role of such Cl-BaA exposure and induced CYPs at toxicity onset, we investigated the mutagenicity of BaA and Cl-BaA using Salmonella typhimurium TA98 and TA100. BaA and Cl-BaA at 10 μg/plate produced positive results in both strains in the presence of rat S-9. Incubation of Cl-BaA with recombinant rat CYP1A2 produced a significantly higher number of revertant colonies in TA98 and TA100 than in controls, but no such change was observed for BaA. In conclusion, BaA changes its own physiological and toxicological actions by its chlorination; (1) daily exposure to Cl-BaA selectively induces hepatic CYP1A2 in rats and (2) Cl-BaA induces frameshift mutations in the presence of CYP1A2, although BaA does not exert mutagenicity. This indicates that CYP1A2 may metabolize Cl-BaA to active forms.

The roles of pancreatic polypeptide (PP) have not been determined in ruminant animals. The aim of the present study was to examine the role of PP in the regulation of ghrelin secretion in sheep. Two experiments were conducted using four 2-yr-old Suffolk wethers fed a maintenance diet of alfalfa hay cubes. In Exp. 1, the effects of feeding on blood ghrelin and PP concentrations were examined in scheduled-fed sheep. Blood samples were collected every 10 min from 30 min before to 360 min after feeding. Plasma PP concentrations were transiently increased from the preprandial average value to the values from 30 to 60 min after feeding and gradually decreased (P0.05) to stable values from 150 to 180 min. The values from 30 to 60 min were greater (P0.05) than those from 150 to 360 min. In contrast, plasma ghrelin concentrations were gradually decreased (P0.01) by feeding. The values from 60 to 360 min were less (P0.01) than the preprandial average value. In Exp. 2, the effects of continuous PP infusion on ghrelin secretion were examined in feed-deprived sheep. The animals were deprived of feed for 48 h before PP infusion. The PP-treated group intravenously received synthetic bovine PP at a rate of 10 pmol.kg(-1 )of BW.min(-1) for 180 min. Blood samples were collected every 10 min from 30 min before to 180 min after the commencement of PP infusion. Plasma PP concentrations reached a plateau within 30 min after the commencement of PP infusion. Plasma ghrelin concentrations were decreased (P = 0.002, 0.016, 0.007) by PP infusion at 160, 170, and 180 min, respectively. In conclusion, plasma ghrelin and PP concentrations were decreased and increased, respectively, in response to feeding in ruminant animals. Furthermore, PP could depress ghrelin secretion.

The permeability of the blood-ocular barrier was investigated in five monkeys using vitreous fluorophotometry (VFP). Inward permeability (Pin) of the blood-retinal barrier was calculated by a computer simulation method. Kinetic VFP was performed after intravitreal injection of fluorescein (F) or fluorescein monoglucuronide (FG). The estimated mean value of Pin (x10(-6) cm/min) was 4.8 (SD 1.2). The mean rates of loss (per hour) of F from the anterior chamber (Ka) and the vitreous (Kv) were 0.11 (SD 0.01) and 0.13 (SD 0.03), respectively, which were approximately three and four times greater than those of FG (0.04 (SD 0.01) and 0.03 (SD 0.01), respectively). Probenecid administered intraperitoneally decreased both the Ka and the Kv of F significantly but had no effect on the Ka or the Kv of FG, suggesting that F was excreted from the eye with the aid of the active transport mechanism. The results of comparative studies of the rates of loss of F from the anterior chamber (Ka) and from the vitreous (Kv) suggested that active transport was more predominant in the blood-retinal barrier than in the blood-aqueous barrier.

The aim of the present study was to investigate biochemical and morphological changes in rat lenses following long-term UV B irradiation. After an irradiation period of 156 days with follow-up documentation by means of Scheimpflug photography, section-related biochemical analyses of the lenses as well as histological investigations were performed. The video-based Scheimpflug photography (Zeiss SLC) again proved to be an excellent method for the documentation of the UV cataract induced in rats. The biochemical analyses provided indications to potential damaging mechanisms; the section-related technique used allows more precise analyses than the processing of whole lenses in a cataract type restricted to a certain layer, as is the case with UV B damage. The most prominent biochemical findings were a significant decrease in glyceraldehyde-3-phosphate dehydrogenase in the equatorial region in the group with the highest irradiation dosage and a decrease in lactate dehydrogenase in the nuclear region. The histological results reflect the local extent of the UV damage as well as its progression after a prolonged irradiation period.

The effect of energy balance on the growth hormone (GH) secretory responsiveness to growth hormone-releasing hormone (GHRH) has not been determined in ruminant animals. Therefore, we examined the effects of intravenous injections of 0, 3.3, and 6.6 microg ghrelin/kg body weight (BW), with and without GHRH at 0.25 microg/kg BW, on GH secretory responsiveness in both the fed and fasted sheep. The injections were carried out at 48 h (Fasting state) and 3h (Satiety state) after feeding. Blood samples were taken every 10 minutes, from 30 minutes before to 120 minutes after the injection. Low (3.3 microg/kg BW) and high (6.6 microg/kg BW) doses of ghrelin stimulated GH secretion significantly (P.05) greater in the Satiety state than in the Fasting state. Growth hormone-releasing hormone plus both doses of ghrelin stimulated GH secretion significantly (P.05) greater in the Satiety state than in the Fasting state. Ghrelin and GHRH exerted a synergistic effect in the Satiety state, but not in the Fasting state. Plasma ghrelin levels were maintained significantly (P.05) greater in the Fasting state than in the Satiety state except the temporal increases after ghrelin administration. Plasma free fatty acid (FFA) concentrations were significantly (P.01) greater in the Fasting state than in the Satiety state. In conclusion, the present study has demonstrated for the first time that ghrelin differentially modulates GH secretory response to GHRH according to feeding states in ruminant animals.

A new therapeutic approach to treat Alzheimer's disease (AD) is needed, and the use of growth factors is considered to be a candidate. Hepatocyte growth factor (HGF) is a unique multifunctional growth factor, which has the potential effect to exert neurotrophic action and induce angiogenesis. In this study, we examined the effects of overexpression of human HGF plasmid DNA using ultrasound-mediated gene transfer into the brain in an Abeta-infused cognitive dysfunction mouse model. We demonstrated that HGF gene transfer significantly alleviated Abeta-induced cognitive impairment in mice in behavioral tests. These beneficial effects of HGF might be due to (1) significant recovery of the vessel density in the dentate gyrus of the hippocampus, (2) upregulation of BDNF, (3) a significant decrease in oxidative stress and (4) synaptic enhancement. A pharmacological approach including gene therapy to increase the HGF level in combination with anti-Abeta therapy might be a new therapeutic option for the treatment of AD.

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope ( residues 324-332, (1)FAPGNYPAL(9)) to promote its folding in vitro of H-2D(b). We found that H-2D(b) can be stabilized by the pentapeptide (5)NYPAL(9), which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D(b) molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove.

Objective: The effect of single-dose mitiglinide on glucose and lipid metabolism was examined in OLETF rats with spontaneous type 2 diabetes in which the early insulin response following glucose challenge is known to diminish over time and become lost with aging. Methods: (1) With catheters inserted into the portal veins, 12-wk-old prediabetic OLETF rats were given an OGTT of 1g/kg after 17 h of fasting. Eight rats each were orally given mitiglinide 1 mg/kg, nateglinide 50 mg/kg, or glibenclamide 1 mg/kg, vs 0.5% carboxy-methylcellulose (CMC) as control, and were given an OGTT immediately afterward. Following oral administration of mitiglinide, nateglinide, glibenclamide, or 0.5% CMC, the 24-wk-old overt-diabetic OLETF rats were immediately given an, OGTT of 1g/kg. (2) After 17 h of fasting, 24-wk-old OLETF rats were subjected to a fat-loading test. Eight rats each were given mitiglinide 3 mg/kg, glibenclamide 1 mg/kg, or glimepiride 1 mg/kg, vs 0.5% CMC, and were given soy oil 2 g/kg immediately afterward. They were also given mitiglinide orally and examined for LPL mRNA expression in their adipose tissue. Results: (1) After OGTT, mitiglinide produced a significant increase in portal insulin levels 15 min after its administration, as well as a significant decrease in peripheral glucose levels 15–120 min after its administration in the OLETF rats. Like-wise, nateglinide produced an increase in portal insulin levels and a decrease in peripheral glucose levels shortly after its administration in these rats. Glibenclamide increased portal insulin levels for an extended time after its administration, and significantly decreased peripheral glucose levels in the rats 120–300 min after its administration in the rats. In contrast, as in the 12-wk-old rats, a precipitous rise in insulin secretion was seen in the portal vein of 24-wk-old rats given mitiglinide, which peaked 15 min after mitiglinide administration, but the insulin levels continued to increase for 120 min or longer in the 24-wk-old rats given glibenclamide. In addition, as in the 12-wk-old rats, a significant decrease in glucose levels in peripheral blood was noted 30 and 60 min after mitiglinide administration and 300 min after glibenclamide administration in the 24-wk-old rats. (2) Mitiglinide increased LPL mRNA expression 120 min after its administration, and significantly decreased peripheral TG and chylomicron-TG levels after fat challenge in the 24-wk-old OLETF rats. Conclusion: Mitiglinide exhibited fast-onset and short-acting insulin-secretagogic effects, inhibiting post-glucose challenge increases in glucose levels and post-fat challenge increases in TG levels.

Changes in leptin and ghrelin levels occur with alterations in adiposity, but signalling may be affected by levels of the relevant receptors. We measured expression of the leptin receptor (Ob-Rb) and the ghrelin/growth hormone releasing peptide receptor (GHS-R) in the arcuate nucleus of sheep held at either high or low levels of adiposity. Plasma growth hormone (GH) levels were lower in Fat animals and higher in Lean animals. Plasma insulin and leptin levels were higher in Fat animals and lower in Lean animals. Frozen hypothalamic sections of arcuate nucleus were extracted and mRNA levels measured for mRNA for Ob-Rb and GHS-R. Gene expression for both Ob-Rb and GHS-R was higher in Lean animals than in Fat animals, with no difference in expression between Fat and Normal animals. A second group of animals (n = 4 per group) was used for double-labelling immunohistochemistry to determine whether the increase in Ob-Rb gene expression was translated into Ob-Rb protein and to ascertain whether this effect is localised to the cells of the arcuate nucleus that produce either neuropeptide Y (NPY) and/or pro-opiomelanocortin-derived peptides. Lean animals displayed a 255% increase in immunoreactive NPY cells (P0.005), a 167% increase in cells with Ob-Rb (P0.037) protein and a 344% increase in cells that were staining for both NPY and Ob-Rb (P0.02). There was no difference between the Normal and Lean animals in the number of cells that were detected with an adrenocorticotrophic hormone (ACTH) antibody or the number of ACTH-immunoreactive cells that also stained for Ob-Rb. Finally, we measured plasma ghrelin levels in Normal, Fat and Lean ewes (n = 4/group); levels were higher (P0.05) in Fat animals than in Lean animals. We conclude that lowering body weight leads to increased expression of Ob-Rb, ghrelin/GHS-R expression and proportion of NPY cells that express Ob-Rb in the arcuate nucleus. This may be an adaptive mechanism to increase responsivity to both leptin and ghrelin.

We measured changes in plasma ghrelin and GH concentrations in mature Holstein cows and 3-mo-old female Holstein calves fed at scheduled times. Our objective was to determine the characteristics of ghrelin secretion in dairy cattle and its influence on GH. Animals were fed at 0800 and 1600 for 2 wk before and during experiments. Plasma was sampled for 24 h at 2-h intervals in Exp. 1. In mature cows, plasma ghrelin concentrations decreased (P0.01) just after 0800 but not at the 1600 feeding. Ghrelin concentrations were lower (P0.01) in calves than in mature cows and they did not decrease after feeding in calves. The temporal relationship between ghrelin and GH remained unclear. In Exp. 2, plasma was sampled 2 h before and after both morning and evening feedings at 20-min intervals. Plasma ghrelin concentrations decreased (P0.05) 40 min after 0800 feeding and 60 min after 1600 feeding in mature cows. These results indicate that in mature cows, plasma ghrelin concentration decreased after feeding, but this decrease was not evident in 3-mo-old calves. Further studies are required to define the relationship between plasma ghrelin and GH concentrations.

We have previously demonstrated that ischaemia-reperfusion induces apoptosis in the intestinal mucosa. To evaluate that reactive oxygen species enhanced intestinal apoptosis after ischaemia-reperfusion, we examined whether antioxidants reduced apoptosis.Rats were infused through a duodenal tube with antioxidative agents, glutathione, rebamipide and dymethylsulfoxide during 2 h before an ischaemic insult. The superior mesenteric artery was occluded for 60 min, followed by 60 min reperfusion. Apoptosis was evaluated by percentage fragmented DNA (fragmented DNA/total DNA) and immunochemical staining.Increase in apoptosis in the intestinal mucosa after ischaemia-reperfusion was attenuated by intraduodenal infusion of antioxidative agents, but was not completely abolished.Scavenging effects of the antioxidative agents attenuated increases in intestinal apoptosis, indicating that oxidative stress after ischaemia-reperfusion plays an important role in induction of apoptosis in the intestinal mucosa.

The gene for the hibernation-specific protein HP-25 is expressed in the liver in hibernating species of the squirrel family (chipmunk and ground squirrel), but not in a nonhibernating species (tree squirrel). To investigate why the HP-25 gene is not expressed in the tree squirrel, we isolated the tree squirrel HP-25 gene and compared its gene structure and promoter activity with that of the chipmunk. The tree squirrel HP-25 gene is composed of three exons, and the gene structures are conserved between the tree squirrel and chipmunk. However, the tree squirrel HP-25 gene has an insertional mutation of 13 nucleotides in exon 2 that disrupts the ORF. In the chipmunk HP-25 gene, the 80-bp 5' flanking sequence is sufficient for the liver-specific promoter activity, and HNF-4, which binds to the sequence from nucleotides -67 to -51, is involved in its transcriptional regulation. In contrast, the corresponding tree squirrel 5' flanking sequence had almost no promoter activity in HepG2 cells, and HNF-4 did not bind to the corresponding region of the tree squirrel HP-25 gene. Furthermore, a tree squirrel-type G to A mutation at -57 in the chipmunk HP-25 gene promoter context abolished its binding to and transactivation by HNF-4. Thus, the point mutation in the HNF-4-binding site is likely to be involved in the lack of HP-25 gene expression in the tree squirrel.

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.

Growth hormone secretagogues are small synthetic molecules that act through an orphan G-protein coupled receptor(GPCR) called GHS-R. We purified its endogenous ligand from rat stomach and named it 'ghrelin'. Ghrelin is a 28-amino-acid peptide with an essential n-octanoyl modification at Ser3. The discovery of ghrelin indicates that the release of GH from the pituitary may be regulated not only by hypothalamic GHRH, but also by ghrelin from the stomach and hypothalamus. Moreover, ghrelin induces appetite-stimulating effect and adiposity in rat. These facts indicate that ghrelin is a natural leptin antagonist.

Ghrelin, a GH-releasing acylated peptide, has been recently identified from the rat stomach. The purified peptide consists of 28 amino acids in which the serine 3 residue is n-octanoylated. Here we show that ghrelin messenger RNA and ghrelin peptide are present in the human as well as in rat placentae. In human placenta, ghrelin was detected by PCR at both first trimester and after delivery. While ghrelin was not detected by immunohistochemistry in human placenta at term, it was easily identified by immunohistochemistry at first trimester being mainly expressed in cytotrophoblast cells and scarcely in syncytiotrophoblast ones. Ghrelin was also identified in a human choriocarcinoma cell line, the BeWo cells. Ghrelin was found, by immunohistochemistry, in the cytoplasm of labyrinth trophoblast of rat placenta, whereas other placental cell types seems to be negative for ghrelin immunostaining. Moreover, placental ghrelin messenger RNA, in pregnant rats, showed a characteristic profile of expression being practically undetectable during early pregnancy, with a sharp peak of expression at day 16 and decreasing in the latest stages of gestation. In conclusion, ghrelin has been detected in human and rat placenta showing a pregnancy-related time course of expression. Whether placenta-derived ghrelin is involved in the modulation of GH release, or placental cell growth and differentiation remains to be established.

Ghrelin is a recently identified endogenous ligand of the GH secretagogue (GHS) receptor. It was originally isolated from the stomach, but has also been shown to be present in the rat hypothalamus. It is a 28-amino acid peptide with an unusual octanoylated serine 3 at the N-terminal end of the molecule, which is crucial for its biological activity. Synthetic GHSs stimulate GH release via both the hypothalamus and the pituitary, and the GHS receptor (GHS-R) has been shown by us and others to be present in the pituitary. We investigated whether ghrelin messenger ribonucleic acid (mRNA) and peptide are present in the normal human hypothalamus and in normal and adenomatous human pituitary. RNA was extracted from pituitary tissue removed at autopsy and transsphenoidal surgery (n = 62), and ghrelin and GHS-R type 1a and 1b mRNA levels were investigated using real-time RT-PCR. Both ghrelin and GHS-R mRNA were detected in all samples. Corticotroph tumors showed significantly less expression of ghrelin mRNA, whereas GHS-R mRNA levels were similar to those in normal pituitary tissue. Gonadotroph tumors showed a particularly low level of expression of GHS-R mRNA. Immunohistochemistry, using a polyclonal antibody against the C-terminal end of the ghrelin molecule, revealed positive staining in the homolog of the arcuate nucleus in the human hypothalamus and in both normal and abnormal human pituitary. Pituitary tumor ghrelin peptide content was demonstrated using two separate RIA reactions for the N-terminal and C-terminal ends of the molecule. Both forms were present in normal and abnormal pituitaries, with 5 +/- 2.5% octanoylated (active) ghrelin (mean +/- SD) present as a percentage of the total. We suggest that the presence of ghrelin mRNA and peptide in the pituitary implies that the locally synthesized hormone may have an autocrine/paracrine modulatory effect on pituitary hormone release.

Ghrelin, a novel GH-releasing acylated peptide, was recently isolated from rat stomach. It stimulated the release of GH from the anterior pituitary through the GH secretagogue receptor (GHS-R). Ghrelin messenger RNA and the peptide are present in rat stomach, but its cellular source has yet to be determined. Using two different antibodies against the N- and C-terminal regions of rat ghrelin, we identified ghrelin-producing cells in the gastrointestinal tracts of rats and humans by light and electron microscopic immunohistochemistry and in situ hybridization combined with immunohistochemistry. Ghrelin-immunoreactive cells, which are not enterochromaffin-like cells, D cells, or enterochromaffin cells, accounted for about 20% of the endocrine cell population in rat and human oxyntic glands. Rat ghrelin was present in round, compact, electron-dense granules compatible with those of X/A-like cells whose hormonal product and physiological functions have not previously been clarified. The localization, population, and ultrastructural features of ghrelin-producing cells (Gr cells) indicate that they are X/A-like cells. Ghrelin also was found in enteric endocrine cells of rats and humans. Using two RIAs for the N- and C-terminal regions of ghrelin, we determined its content in the rat gastrointestinal tract. Rat ghrelin was present from the stomach to the colon, with the highest content being in the gastric fundus. Messenger RNAs of ghrelin and GHS-R also were found in these organs. Ghrelin probably functions not only in the control of GH secretion, but also in the regulation of diverse processes of the digestive system. Our findings provide clues to additional, as yet undefined, physiological functions of this novel gastrointestinal hormone.

1. The purpose of this study was to investigate the involvement of thromboxane A(2) (TXA(2)) in the cough response in a guinea-pig cough model. Here, we describe results obtained using a selective TXA(2) synthetase inhibitor, ozagrel, and a selective TXA(2) agonist, U-46619. 2. Guinea-pigs were anaesthetized and exposed to an aerosol of capsaicin (100 microM) to elicit coughing. The number of coughs was 20.0+/-5.8 during capsaicin provocation (5 min), but only 2. 8+/-0.4 during a 5-min inhalation of phosphate-buffered saline (PBS) (P:0.05). 3. TXB(2) levels in BAL were 101.4+/-8.0 and 58.4+/-8.7 pg ml(-1) following capsaicin and PBS inhalation, respectively (P:0. 01), but there was no intergroup difference in the cell populations in BAL. 4. Inhalation of U-46619 did not induce a cough response by itself at concentrations of 100 ng ml(-1) to 10 microg ml(-1). However, it caused a 2 fold increase in the number of capsaicin-induced coughs. 5. To explore the source of the TXA(2), BAL cells were stimulated with capsaicin and the supernatants collected for analysis. The TXB(2) concentration in BAL was increased dose-dependently, indicating that TXA(2) is released from BAL cells in response to capsaicin. 6. Ozagrel was administered orally 1 h before a 5 min capsaicin provocation and the number of coughs was counted during the capsaicin inhalation. Ozagrel decreased the number of coughs dose-dependently (ED(50) value, 26.3 mg kg(-1)). 7. These results show that TXA(2) modulates the capsaicin-induced cough response by increasing capsaicin-sensitivity.

The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5' flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5' flanking sequence from -74 to -46 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5' flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression.

This study aimed to examine the relationship between a harmful effect of histamine and apoptosis following ischemia-reperfusion in the rat intestine. The superior mesenteric artery was occluded for 60 min followed by reperfusion for 60 min. Rats were infused with H1-receptor antagonist (chlorpheniramine maleate) or H2-receptor antagonist (cimetidine). Additional rats were pretreated with aminoguanidine (100 mg/kg). Percent apoptosis in the intestinal mucosa increased after reperfusion, but neither H1 nor H2 antagonists had any effect on apoptosis. Aminoguanidine pretreatment inhibited activity of diamine oxidase and increased the plasma histamine concentration. Aminoguanidine attenuated the increase in mucosal apoptosis following reperfusion. Apoptosis induced by an ischemic insult to the intestinal mucosa was not related to an undesirable effect of histamine. Attenuation of increased intestinal apoptosis might be due to increased plasma histamine level and/or other pharmacological action of aminoguanidine, including inhibition of inducible nitric oxide synthase.

The aim of the present study was to investigate the density, laminar distribution, size, morphology, and neurotransmitter phenotype of rat cortical neurons expressing latexin, an inhibitor of carboxypeptidase A. Immunohistochemical analyses established that latexin-immunoreactive neurons are restricted essentially to the infragranular layers of lateral cortical areas in the rat. The overall density, laminar or sublaminar localization, and cell size distribution of latexin-positive neurons differed substantially across cytoarchitectonic areas within lateral cortex. Numerous latexin-positive neurons had the morphology of modified pyramidal cells especially of layer VI. The vast majority of latexin-positive neurons were glutamate-immunoreactive in the six lateral neocortical areas examined, while neurons immunoreactive for both latexin and GABA were virtually absent. Thus the majority of latexin-positive neurons are likely to be excitatory projection neurons. The area- and lamina-specific distribution of the latexin-expressing subpopulation of glutamate-immunoreactive neurons is a distinctive feature that may contribute to the functional specialization of the lateral cortical areas.

A novel gene, jumonji was identified by a mouse gene trap strategy. The jumonji gene encodes a protein containing a putative DNA binding domain. The mice homozygous for jumonji gene with a BALB/cA genetic background show hypoplasia of the fetal liver and embryonic lethality, suggesting impaired hematopoiesis. In the peripheral blood of jumonji mutant embryos, the number of fetal liver-derived definitive erythrocytes, but not yolk sac-derived primitive erythrocytes, showed a marked reduction, suggesting that jumonji mutants die of anemia. The defects of definitive erythrocytes in jumonji mutants seemed to be caused by a decrease in the numbers of multiple hematopoietic progenitors including colony-forming unit-spleen (CFU-S) in the fetal liver. However, hematopoietic stem cells (HSCs) in the fetal liver of jumonji mutants could reconstitute the hematopoietic system of lethally irradiated recipients. In the fetal liver, the jumonji gene is expressed in fibroblastic cells and endothelial cells, but not in Lin-/c-Kit+/Sca-1(+) cells known to include HSCs. These results suggest that an environmental defect induce the impaired hematopoiesis in the fetal liver of jumonji mutant embryos.

Erythropoietin (EPO), one of the pivotal regulators of erythrocyte production, transmits signals through the EPO receptor (EPOR). We have previously reported that human bone marrow (BM) cells express two dominant forms of the EPOR, one full-length and one truncated (EPOR-F and EPOR-T). Experiments with a cell line have shown that the EPOR-T acts as a dominant-negative regulator of EPOR-F-mediated signals. Its role in erythropoiesis in vivo, however, has yet to be clarified. Here we show the presence in mouse BM of a truncated form of the EPOR that is essentially the same as EPOR-T in humans. To investigate its role in vivo, we generated transgenic mice overexpressing mouse EPOR-T (EPOR-T-Tg mice). As a result, two independent EPOR-T-Tg lines were established. One line revealed mild anemia, but another line did not. When anemia was induced experimentally in these mice, however, both lines showed apparently poor recovery resulting in higher mortality than wild-type control mice. The impaired erythropoiesis found in these mice thus strongly suggests the EPOR-T's role as a negative regulator of erythropoiesis in vivo.

To clarify the histopath ological changes of experimentally induced steroid cataract in Brown-Norway rat eyes, a scanning electron microscopic study was performed. The biomicroscopic appearance of the cataracts, which was quite similar to that of human eyes, was induced by daily application, either topically or systemically, of prednisolone acetate for 12 months. A single dose of 2 Gy X ray was given to the right eyes of all the animals 2 weeks before drug administration. Twenty-seven rats were divided into three groups: a control group (CTL), a group receiving topically administered prednisolone (TOP), and a group receiving systemically administered prednisolone (SYS). In vivo observation was performed through a slit-lamp microscope, and the lens findings were documented and objectively analyzed by an anterior eye segment analysis system over a period of 12 months. At the end of the 12month period, X-ray-irradiated right eyes in the CTL group showed some minor lens changes on biomicroscopy, and non-X-ray-irradiated left eyes were almost normal. In the TOP and SYS groups, either with or without X-ray irradiation, lenses showed anterior and posterior subcapsular opacification; however, the grade and increase of lens opacification were higher and faster in the eyes with X-ray irradiation. Scanning electron microscopic findings of the three groups at the 12th month were as follows. In the CTL group, the X-ray-irradiated right eyes showed minor changes. In the TOP group, the X-rayirradiated right eyes showed marked damage in the lens fibers of the anterior and posterior cortices, while the nonirradiated left eyes showed minor changes. In the SYS group, the X-ray-irradiated right eyes showed prominent cataractous disorganization of lens fibers in the shallow anterior and posterior cortices, and the non-X-ray-irradiated lenses were almost the same as those in the TOP group. These findings might suggest that even a minimum invasion of a low dose of X-ray irradiation plays a cocataractogenic or syncataractogenic role during the formation of steroid cataracts.

By a randomized double blind control trial we studied the effect of vacuum cleaning of room floors, mattresses and quilts for twelve months on clinical symptoms and laboratory data of atopic dermatitis patients. All patients used the identical new vacuum cleaners. Thirty patients (3-12 years of age) with relatively stable skin conditions were randomly allocated to either of the following two groups. In the monitor group we visited patient's home every three weeks and mite specialists cleaned drastically the room floors, mattresses and quilts and the patient continued to clean in the same way in-between. In the control group we visited similarly but the cleaning was made insufficiently which was also followed by the patient. But, at 2 occasions (the first and the last visits), cleaning was made drastically also in the control group. Thus the mite numbers were counted precisely at the start and the end of the experiment both in the monitor and control groups. Each patient was seen every six weeks by the same doctor and estimated of his symptoms using our unique scoring system (Fig. 1). At the start and the end of the study, total IgE and specific IgE antibodies to house dust mites in the serum were evaluated. The monitor group showed a tendency to count smaller number of mites than the control group after a year, when there was a significant difference only in quilts. However, a statistically significant decrease in the mite counts was observed only in room floors and not in mattresses and quilts both in the monitor and control groups (Fig. 2). Clinical scores after a year significantly improved only in the monitor group and not in the control group (Fig. 3). Serum IgE value and specific antibody titer to house dust mites were not changed significantly after the trial in both groups. As a conclusion, vacuum cleaning of the patient's room improved the clinical symptoms of atopic dermatitis but this could not be related to the reduction of house dust mite number.

Exposure of atopic patients to a specific allergen evokes an immediate response which is followed, in many cases, by a late phase reaction (LPR) some hours later. Here we have examined the immunological mechanisms required for the expression of cutaneous LPR in mice.BALB/c mice were immunized by i.p. injection of ovalbumin (OVA) and alum actively or by i.v. injection of anti-OVA IgE monoclonal antibody (mAb) passively. After challenge by intradermal injection of OVA into ears, the changes in ear thickness, the number of eosinophils, and the levels of IL-4 and IFN-gamma protein at the site of antigen challenge were examined.Actively immunized mice developed a biphasic response at the site of OVA injection, while mice passively immunized with IgE anti-OVA mAb displayed a strong early response but no LPR. Cell transfer experiments using BALB/c nu/nu mice revealed that both OVA-specific IgE mAb and OVA-primed CD4 T cells were required to evoke LPR. Moreover, LPR was associated with increased levels of IL-4 production concomitant with reduced IFN-gamma production and was abolished by pretreatment with anti-IL-4 neutralizing mAb.It is suggested that murine cutaneous LPR against OVA is a type 2 inflammatory response in which both IgE antibodies and CD4 T cells play an obligatory role.

The cytologic features of cat scratch disease (CSD) from eight cases in imprint smears are presented. All patients were clinicopathologically diagnosed with CSD as follows: 1) a history of animal exposure was recorded 2 to 4 weeks before lymphadenopathy; 2) the disease occurred in the autumn and winter months; 3) a characteristic histopathology in the biopsied lymph node specimens was observed; and 4) Warthin-Starry silver stain-positive bacteria were detected in four of the seven cases examined. The characteristic cytologic finding was the presence of confluent epithelioid cells with nearby and central scattering of neutrophils against a background of polymorphic inflammatory cells. Furthermore, a varying number of medium-sized to large lymphoid cells with an appearance suggestive of monocytoid B lymphocytes (MBLs) were noted to be associated with the epithelioid cells. These cytologic findings closely paralleled the histologic patterns of epithelioid cell granulomas, with and without MBLs, which we have previously reported are probably associated with the disease.

The behavioral effects of L-dopa or cabergoline alone were compared with those of the joint administration of the two drugs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned parkinsonian cynomolgus monkeys with attention to the induction of hyperactivity and dyskinesia. Cabergoline alone at 0.2 mg/kg or less improved in a dose-dependent fashion the parkinsonism without inducing hyperactivity and dyskinesia following a single subcutaneous injection. L-dopa alone improved the parkinsonism, but induced hyperactivity and dyskinesia, depending on the dose applied. Doses required for 50% amelioration by L-dopa and cabergoline were 10 and 0.038 mg/kg, s.c., respectively. With low doses (50%-amelioration doses), cabergoline or L-dopa alone improved the parkinsonism without induction of hyperactivity and dyskinesia, but the duration of action was brief. Cabergoline in combination with L-dopa was highly effective in improving motor disability without induction of hyperactivity and dyskinesia. Moreover, the duration of action was more prolonged with the coadministration than with the single administration of each drug. These findings suggest that the combined therapy with low doses of L-dopa and cabergoline is beneficial for treating patients with advanced Parkinson's disease.

A new type soluble guanylyl cyclase, designated as ksGC (kinase-like domain containing soluble guanylyl cyclase), was identified from a kidney cDNA library. By Northern blot analysis, expression of this novel guanylyl cyclase mRNA was found in lung, kidney and skeletal muscle. Although ksGC is a soluble type guanylyl cyclase, it is not a member of soluble alpha or beta guanylyl cyclases. The ksGC contains in its N-terminal region a protein kinase-like domain, which is usually a characteristic feature only of particulate type guanylyl cyclases. This unique feature of ksGC suggests a novel pathway of cGMP synthesis.

Chickens fed on semi-purified low (7%) or high (61%) protein-energy diets for 14 or 17 d were used for determinations of oxidative phosphorylation and specific amounts of mitochondrial protein in liver. The ADP:oxygen (ADP:O) values obtained when pyruvate+malate were used as substrates were significantly reduced in the high-protein-fed group after the 4th day compared with those for the group fed the low-protein diet, while the differences in ADP:O values between the two treatments when L-glutamate was used as substrate were found to be significant on the 14th day. At any feeding period no significant differences in ADP:O values were observed between the two groups when alpha-ketoglutarate, malate, or octanoate+malate were used as substrates, nor in specific amounts of mitochondrial protein in liver. The dependency of the pyruvate+malate-supported respiration rate on the temperature in the reaction medium was also determined. The results of an Arrhenius plot showed that transition temperatures, and the lower and upper energies of activation, were similar for the groups fed on low- and-high-protein diets. Furthermore, no morphological changes in mitochondria were observed among chickens fed on diets with various protein levels for 14 d. From these results we concluded that the reduction of ADP:O value with pyruvate+malate of L-glutamate substrates in chickens fed on a high-protein diet was substrate-specific, and was not due to functional damage to the respiratory chain for electron flow from NAD-linked substrates to the ubiquinone pool, nor to modulation of properties of the inner mitochondrial membrane.

In order to induce steroid cataracts in rat lenses, prednisolone acetate was administered together with a single subliminal dose of X-irradiation, which was applied unilaterally before steroid application started. The rats were divided into a control group (without prednisolone acetate administration), a group with a topically administered daily dose of 1 mg/kg prednisolone acetate suspension and a group with a systemically applied daily dose of 0.8-1.0 mg/kg prednisolone acetate suspension. Changes in the lens were objectively evaluated with in vivo Scheimpflug slit images during a 30-week period. Although body weight increase was significantly affected in both groups administered prednisolone, animals survived until the end of the observation period. The initial changes in the lens were the dissociation of the Y-suture and a slight increase in scattering light intensity at the posterior embryonic nucleus after both topical and systemic steroid administration.

A new compound containing the cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide was synthesized, i.e. tetrahydrofurantetracarboxylic acid (THFTCA)-RGDS conjugate [THFTCA- (RGDS)3, FC-243], and the inhibitory effect of FC-243 on lung metastasis of B16-BL6 melanoma in mice was examined in combination with or without the anticancer agent doxorubicin (DOX). FC-243 showed an inhibitory effect on lung metastasis of melanoma cells in a dose-dependent manner. A mixture of THFTCA and RGDS peptide or THFTCA alone did not show any inhibitory effect on experimental lung metastasis as compared with FC-243 on a molar basis. RGDS peptide, however, required a higher dose to obtain a sufficient antimetastatic effect. Intermittent IV administration of FC-243 after the inoculation of B16-BL6 cells caused significant inhibition of spontaneous lung metastasis as compared with multiple administration of RGDS or untreated control. The in vitro tumor invasion study showed that FC-243 as well as RGDS+THFTCA on a molar basis resulted in similar inhibition of the invasion of B16-BL6 cells into reconstituted basement membrane Matrigel. Combined treatment with FC-243 and DOX significantly inhibited lung metastasis of melanoma as compared with either treatment alone or the untreated control. Administrations of FC-243 and DOX in combination substantially prolonged the survival time of mice. These results demonstrate that combination therapy of the anti-cell adhesive FC-243 and the anticancer agent DOX, i.e. antiadhesion therapy and chemotherapy, is a new approach that offers enhanced inhibitory effects on tumor metastasis and invasion.

1. A piecewise exponential three-state model previously introduced by the authors in 5.4 mmol/l was intended to apply to states with different [K+]o and with a different drug. Using the conventional microelectrode technique the effects of 20 mumol/l mexiletine (MEX), 5 mumol/l aprindine (APR), 20 mumol/l quinidine (QUI), 5 mumol/l flecainide (FLE), 5 mumol/l E-0747 (dl-6-chloro-2,2'-dimethyl-1'-[3-(4-hydroxypiperizino)propyl]spiro [chroman-4,4'-imidazolidine]-2',5'-dione hydrochloride and 100 mumol/l QX-222, a quaternary derivative of lidocaine, on action potentials (APs) in guinea-pig papillary muscles were studied. Specific objects of the study were (1) steady state Vmax values at various frequencies (all drugs), (2) the recovery process of Vmax in premature responses (MEX) and (3) Vmax changes during a train of stimulation at 1 Hz after a rest period (all other drugs) in 2.7 and 10 mmol/l [K+]o. Further, those of APR alone and APR plus 1 mmol/l nicorandil (NIC), which shortened action potential durations (APDs) specifically, were investigated on the above items (1) and (3) in 5.4 mmol/l [K+]o. 2. All the drugs reduced the Vmax of APs frequency-dependently and, except QX-222, more markedly in 10 mmol/l [K+]o than in 2.7 mmol/l [K+]o at 1 Hz. 3. The rate constants estimated from the model fitting characterized MEX and APR and the other drugs as predominantly inactivated and activated channel blockers, respectively. The calculated rate of onset of block, lambda T, does not differ much between the three [K+]o levels. lambda T shows that APR belongs to class Ia rather than class Ib.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to find an appropriate model of experimentally induced streptozotocin diabetic cataract rat for evaluation of the anti-cataract agents. Experimental diabetic cataracts were induced in 3 rat strains (Sprague-Dawley SD, Brown Norway BN, Wistar WIS), age 5 and 7 weeks old, by intravenous injection of 70 mg/kg streptozotocin. The cataract progression was followed by morphological and biochemical assessment. Two types of initial cataractous change were observed in SD and WIS rats which mainly consisted of water-vacuole formation in the peripheral area and a diffuse cloudiness progressing around the anterior Y-suture area. The initial change of BN rats was a Y-suture dissociation and fine water-clefts with dot-like vacuoles. Those initial changes in SD and WIS rats differed from those in BN. No difference in aldose reductase activities among the three strains was found, but BN rats had significantly higher sorbitol dehydrogenase activity than SD or WIS rats. It seems that 7-week-old BN rats are more suitable for experimental diabetic cataract models than SD or WIS.