Overexpression of Slc22a18 facilitates fat accumulation in mice.

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.

Molecular characterization of feline melanocortin 4 receptor and melanocortin 2 receptor accessory protein 2.

Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein-protein interactions, and functions. We found high sequence homology (>88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells; stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.

Elevation of Glucose 6-Phosphate Dehydrogenase Activity Induced by Amplified Insulin Response in Low Glutathione Levels in Rat Liver.

Weanling male Wistar rats were fed on a 10% soybean protein isolate (SPI) diet for 3 weeks with or without supplementing 0.3% sulfur-containing amino acids (SAA; methionine or cystine) to examine relationship between glutathione (GSH) levels and activities of NADPH-producing enzymes, glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME), in the liver. Of rats on the 10% SPI diet, GSH levels were lower and the enzyme activities were higher than of those fed on an SAA-supplemented diet. Despite the lower GSH level, γ-glutamylcysteine synthetase (γ-GCS) activity was higher in the 10% SPI group than other groups. Examination of mRNAs of G6PD and ME suggested that the GSH-suppressing effect on enzyme induction occurred prior to and/or at transcriptional levels. Gel electrophoresis of G6PD indicated that low GSH status caused a decrease in reduced form and an increase in oxidized form of the enzyme, suggesting an accelerated turnover rate of the enzyme. In primary cultured hepatocytes, insulin response to induce G6PD activity was augmented in low GSH levels manipulated in the presence of buthionine sulfoximine. These findings indicated that elevation of the G6PD activity in low GSH levels was caused by amplified insulin response for expression of the enzyme and accelerated turnover rate of the enzyme molecule.

Glycerol 3-phosphate dehydrogenase 1 deficiency enhances exercise capacity due to increased lipid oxidation during strenuous exercise.

A large percentage of energy produced during high-intensity exercise depends on the aerobic glycolytic pathway. Maintenance of a cytoplasmic redox balance ([NADH]/[NAD(+)] ratio) by the glycerophosphate shuttle involves sustained aerobic glycolysis. Glycerol 3-phosphate dehydrogenase 1 (GPD1) catalyzes an oxidation reaction in the glycerophosphate shuttle. In this study, we examined whether GPD1 deficiency decreases exercise capacity due to impairment of aerobic glycolysis by using the GPD1 null mouse model BALB/cHeA (HeA). Unexpectedly, we found that exercise endurance was significantly higher in HeA mice than in BALBc/By (By) mice used as controls. Furthermore, aerobic glycolysis in HeA mice was not impaired. During exercise, lipid oxidation was significantly higher in HeA mice than in By mice, concomitant with an increase in phosphorylation of AMP-activated protein kinase (AMPK). HeA mice also showed a delay in the onset of muscle glycogen usage and lactate production during exercise. These data suggest that contribution of lipid oxidation as a fuel source for exercise is increased in HeA mice, and GPD1 deficiency enhances exercise capacity by increasing lipid oxidation, probably due to activation of AMPK. We propose that GPD1 deficiency induces an adaptation that enhances lipid availability in the skeletal muscle during exercise.

Changes in fatty acid composition in tissue and serum of obese cats fed a high fat diet.

BACKGROUND: Obesity and overweight have been frequently observed in dogs and cats in recent years as in humans. The compositions of fatty acids (FAs) in the accumulated lipids in tissues of obese animals may have important roles in the process and mechanisms related to the onset of metabolic disorders. The purpose of this study was to evaluate the effects of a high fat (HF) diet, which contained a higher proportion of saturated FAs, on FA metabolism and distribution in obese cats. Cats (N = 12) were divided into control diet group (crude fat; 16.0 %) (n = 4) or a high fat (HF) diet group (crude fat; 23.9 %) (n = 8). The HF diet contained up to 60 % of calories from fat and was rich in stearic acid. Blood samples were collected at 0, 2, 4 and 6 weeks after the feeding. Adipose and liver tissues were collected at the 6(th) week after feeding. We performed analysis of histological findings and fatty acid composition in serum and tissues. RESULTS: Body weights of the cats significantly increased in the HF group. The increased activities of hepatic enzymes and the accumulation of lipid droplets were found in hepatocytes in the HF group at the 6(th) week after feeding. In this study, the stearic acid (C18:0)-rich HF diet contained less oleic acid (C18:1n-9) and more linoleic acid (C18:2n-6) than the control. However, the composition of oleic acid in the liver was higher, and those of stearic acid and linoleic acid were lower in the HF group at the 6(th) week after feeding. The higher oleic acid:stearic acid ratio suggests an increase in the conversion from saturated FA to mono-unsaturated FAs, which may reflect the hepatic storage of FAs as a relatively harmless form. CONCLUSION: The stearic acid-rich HF diet increased hepatic lipid accumulation accompanied by the increased of hepatic oleic acid, increased serum oleic acid and activation of hepatic enzymes. These findings could be an important sign of early stages of dyslipidemia and hepatic damage. Also, the higher oleic acid:stearic acid ratio might be related to the increased activity of SCD-1, which suggests that the stearic acid-rich HF diet evoked hepatic lipogenesis in the feline liver.

The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice.

Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2h and was 1.7-fold greater than that observed in the control group after 6h. The up-regulation of GPD1 began 2h after administering ethanol, and significantly increased 6h later with the concomitant escalation in the glycolytic gene expression. The incorporation of (14)C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.

Comparison of plasma lipoprotein profiles and malondialdehyde between hyperlipidemia dogs with/without treatment.

BACKGROUND: The aim of this study is to compare metabolic parameters, malondialdehyde as a lipid oxidation marker, and lipid profiles between dogs with untreated hyperlipidemia and hyperlipidemia with treatment, in order to examine the usefulness of malondialdehyde and lipid profiles as diagnostic parameters at early stages of hyperlipidemia. RESULTS: Dog samples were collected from four different veterinary clinics across Japan from March to June 2013. They were separated into three groups: control, untreated hyperlipidemia based on temporally screening, and hyperlipidemia with current anti-hyperlipidemic (statins and fibrates) treatment. Triglyceride levels of untreated hyperlipidemia dogs were significantly higher than those of control dogs. ALT levels of hyperlipidemic dogs with treatment were the highest among three groups. VLDL and LDL of both cholesterol and triglyceride of untreated hyperlipidemia dogs were the highest among three groups. HDL1 levels in triglyceride of hyperlipidemia dogs with treatment were significantly higher than those of control and untreated hyperlipidemia dog. Malondialdehyde concentrations of untreated hyperlipidemia dogs were significantly higher than those of control and hyperlipidemic dogs with treatment. CONCLUSIONS: In this study, dogs with untreated hyperlipidemia clearly showed abnormal lipid status, whereas hyperlipidemic dogs under anti-hyperlipidemia treatment showed more normal lipid status suggesting the effectiveness of the therapy. Anti-hyperlipidemics (statins and fibrates) for dogs are also effective in relieving elevated levels of lipids and lipid oxidation. Plasma lipid (triglyceride and cholesterol) profiles and malondialdehyde are useful diagnostic tools for identifying early stages of untreatment hyperlipidemia in dogs.

Age effects on plasma cholesterol and triglyceride profiles and metabolite concentrations in dogs.

BACKGROUND: In dogs, occurrence of lipid metabolism disorders such as obesity and diabetes mellitus has increased markedly in recent years. Hyperlipidemia has been regarded as a common characteristic for obese animals and hyperlipidemic condition may be associated with inflammation, oxidative stress and lipid composition changes. In this study, we investigated the changes in plasma cholesterol and triglyceride (TG) profiles and metabolite concentrations in 24 dogs (young group: 0-7 years old, n = 12, aged group: 8-13 years old, n = 12). RESULTS: Plasma adiponectin (ADN) concentrations were significantly lower in aged dogs than those in young dogs (mean ± SD, 17.2 ± 10.0 µg mL-1 vs 29.3 ± 12.5 µg mL-1, respectively; P <0.05). Although there were no significant differences statistically, aged dogs showed significantly higher plasma alpha1- acid glycoprotein (alpah1-AG) levels compared to those in young dogs. Plasma cholesterol lipoprotein and TG lipoprotein were divided into four fractions by biphasic agarose gel electrophoresis technique. The levels of the third TG-lipoprotein fraction from the positive pole (TG Fraction 3) were significantly higher in aged dogs than in young dogs (mean ± SD, 143.0 ± 109.3 mg dL-1 vs 55.2 ± 31.3 mg dL-1, respectively; P <0.05). On the correlation coefficient analysis by Peason's method, moderate positive correlations were seen between the age and TG (r = 0.446, P = 0.029), TG Fraction 3 (r = 0.516, P = 0.010), malondialdehyde (r = 0.146, P = 0.043), alpha-1 AG (r = 0.448, P = 0.028) levels, respectively. Moderate negative correlations were seen the age and total cholesterol (TC) Fraction 2 (r = -0.446, P = 0.029), glucose (r = -0.637, P = 0.001), ADN (r = -0.408, P = 0.048), respectively. CONCLUSIONS: Present data suggest biochemical characteristics of lipid metabolism disorder may be affected by aging in dogs.

Ccdc85c encoding a protein at apical junctions of radial glia is disrupted in hemorrhagic hydrocephalus (hhy) mice.

Cortical heterotopia, a malformation of the developing cortex, are a major cause of epilepsy and mental retardation in humans. Hemorrhagic hydrocephalus (hhy) mutation on mouse chromosome 12 results in subcortical heterotopia and nonobstructive hydrocephalus with frequent brain hemorrhage. Here, we show that coiled-coil domain-containing 85C (Ccdc85c), consisting of 6 exons that encode a 420 amino acid protein, is disrupted by replacement of a 3.2-kb sequence, including exon 2 in Ccdc85c by a 1.5-kb retrotransposon-like repeat sequence in the hhy mutant. Immunoreactivity to Ccdc85C was detected predominantly at the apical junctions of radial glia in the wall of lateral ventricles of the developing brain. In the hhy brain at embryonic (E) day 18 (E18), radial glial demise followed by agenesis of the ependymal layer lining the neonatal cortex and accumulation of neuronal specific nuclear protein (NeuN)-positive postmigratory neurons in the subcortical area occurred. Accumulation of E15-born, but not of E13-born, 5-bromo-2'-deoxyuridine labeled neurons expressing special AT-rich sequence binding protein 2 was detected in both heterotopia and the superficial layers of the hhy neocortex at postnatal day 7. Ccdc85c deficiency permitted radial scattering of paired box gene 6-positive neural progenitors in the ventricular zone, likely resulting in reduced self-renewal of the progenitors in the developing hhy cortex. These findings indicate an important role of Ccdc85C in cortical development and provide a mouse model to study pathogenesis of subcortical heterotopia and hydrocephalus.

Change in mRNA expression of sirtuin 1 and sirtuin 3 in cats fed on high fat diet.

BACKGROUND: Mammalian sirtuins are homologs to the yeast silent information regulator 2 (Sir2), which is an NAD-dependent deacetylase. Sirtuins are comprised of 7 proteins, and each has different target proteins. Sirtuin 1 (SIRT1) plays important roles in maintaining metabolic functions and immune responses, and SIRT3 protects cells from oxidative stress-induced cell death. Both SIRT1 and SIRT3 are regulated by metabolic status and aging. Hence, SIRT1 and SIRT3 have been researched in metabolic diseases, such as type 2 diabetes mellitus (DM), fatty liver, and heart diseases. Although these diseases have been increasing, there is little information about relation between the diseases and SIRT1 and SIRT3 in cats. Therefore we cloned SIRT1 and SIRT3 cDNA, examined mRNA expression in cat tissues, and investigated the changes in SIRT1 and SIRT3 mRNA expression in peripheral blood leukocyte of cats fed on HFD for 6 weeks. RESULTS: Cat SIRT1 and SIRT3 contained a catalytic core region and showed high sequence homology with other vertebrate SIRT1 (>61.3%) and SIRT3 (>65.9%) amino acids. Real-time polymerase chain reaction analyses revealed that high expression levels were observed in the liver and skeletal muscle for SIRT1 and in the heart for SIRT3 in cats. In addition, both cat SIRT1 and SIRT3 expression levels in the pancreas were different between individuals. Cat SIRT1 mRNA expression in peripheral blood leukocytes was significantly elevated in obese cats fed on HFD (P < 0.05). CONCLUSIONS: Cat SIRT1 and SIRT3 genes are highly conserved among vertebrates, and HFD feeding may be related to SIRT1 mRNA expression mechanisms in cat peripheral blood leukocytes.

Distinct pattern of allelic loss and inactivation of cadherin 1 and 5 genes in mammary carcinomas arising in p53(+/-) mice.

p53 is one of the most frequently mutated genes in mammary carcinomas (MCs). To detect tumor suppressor genes cooperating with a hetero-deficient p53 gene in mammary carcinogenesis, we first examined allelotypes in MCs from (BALB/cHeA x MSM/Ms) F(1)- p53(+/-) and (BALB/cHeA x 129/SvEv) F(1)- p53(+/-) female mice, and then surveyed down-regulated genes in the allelic loss regions. Genome-wide screening at 42 loci identified frequent (more than 30%) loss of heterozygosity (LOH) on chromosomes 5, 8, 11, 12, 14 and 18 in the MCs from either of the F(1) mice. The MCs in the p53(+/- )mice indicated highly frequent LOH, especially on chromosomes 8, 11 and 12, distinct from other mouse tumors. More than 60% of the 38 MCs from (BALB/cHeA x MSM/Ms) F(1)- p53 (+/-) mice showed LOH in a region ranging from D8Mit85 (105.0 Mb from centromere) to D8Mit113 (111.8 Mb) on chromosome 8, a region syntenic to human chromosome 16q22.1, on which LOH has been found in breast cancers. RT-PCR analyses revealed that the LOH of chromosome 8 was associated with the reduced and/or complete loss of expression of Cdh1 and Cdh5 genes in 15 (58%) and 8 (31%) of 26 MCs derived from the F(1) mice, respectively. Thus, inactivation of Cdh1 and Cdh5 is likely to cooperate with the loss of p53, suggesting a possible tumor suppressive function of these genes in mammary carcinogenesis.

Sirtuin 1 suppresses nuclear factor κB induced transactivation and pro-inflammatory cytokine expression in cat fibroblast cells.

Nuclear factor κB (NF-κB) is a key factor in the development of chronic inflammation and is deeply involved in age-related and metabolic diseases development. These diseases have become a serious problem in cats. Sirtuin 1 (SIRT1) is associated with aging and metabolism through maintaining inflammation via NF-κB. In addition, fibroblasts are considered an important factor in the development of chronic inflammation. Therefore, we aimed to examine the effect of cat SIRT1 (cSIRT1) on NF-κB in cat fibroblast cells. The up-regulation of NF-κB transcriptional activity and pro-inflammatory cytokine mRNA expression by p65 subunit of NF-κB and lipopolysaccharide was suppressed by cSIRT1 in cat fibroblast cells. Our findings show that cSIRT1 is involved in the suppression of inflammation in cat fibroblast cells.

Glycerol-3-phosphate dehydrogenase 1 deficiency induces compensatory amino acid metabolism during fasting in mice.

BACKGROUND: Glucose is used as an energy source in many organs and obtained from dietary carbohydrates. However, when the external energy supply is interrupted, e.g., during fasting, carbohydrates preserved in the liver and glycogenic precursors derived from other organs are used to maintain blood glucose levels. Glycerol and glycogenic amino acids derived from adipocytes and skeletal muscles are utilized as glycogenic precursors. Glycerol-3-phosphate dehydrogenase 1 (GPD1), an NAD+/NADH-dependent enzyme present in the cytosol, catalyzes the reversible conversion of glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP). Since G3P is one of the substrates utilized for gluconeogenesis in the liver, the conversion of G3P to DHAP by GPD1 is essential for maintaining blood glucose levels during fasting. We focused on GPD1 and examined its roles in gluconeogenesis during fasting. METHODS: Using GPD1 null model BALB/cHeA mice (HeA mice), we measured gluconeogenesis from glycerol and the change of blood glucose levels under fasting conditions. We also measured gene expression related to gluconeogenesis in the liver and protein metabolism in skeletal muscle. BALB/cBy mice (By mice) were used as a control. RESULTS: The blood glucose levels in the HeA mice were lower than that in the By mice after glycerol administration. Although lack of GPD1 inhibited gluconeogenesis from glycerol, blood glucose levels in the HeA mice after 1-4h of fasting were significantly higher than that in the By mice. Muscle protein synthesis in HeA mice was significantly lower than that in the By mice. Moreover, blood alanine levels and usage of alanine for gluconeogenesis in the liver were significantly higher in the HeA mice than that in the By mice. CONCLUSIONS: Although these data indicate that a lack of GPD1 inhibits gluconeogenesis from glycerol, chronic GPD1 deficiency may induce an adaptation that enhances gluconeogenesis from glycogenic amino acids.

Demethylation of methylmercury and the enhanced production of formaldehyde in mouse liver.

Methylmercury (MeHg) is gradually changed to inorganic Hg after demethylation in animal tissues, and a selective quantification of inorganic Hg in the tissues is necessary to detect the reaction. We detected inorganic Hg formation in liver and kidney of mouse as early as 24 hr after MeHg injection. As an example of biological demethylation, the cytochrome P450 (P450)-mediated N-demethylation of drugs has been well documented, and formaldehyde was detected as a reaction product. Here we incubated mouse liver homogenate with added MeHg and observed a dose-dependent production of formaldehyde, as well as inorganic Hg formation. Since the amount of formaldehyde was approx. 500 times higher than that of the inorganic Hg that formed, the formaldehyde production would be stimulated by inorganic Hg formed from MeHg. We observed that inorganic Hg caused formaldehyde production, and it was enhanced by L-methionine and sarcosine. Thus, some biomolecules with S-methyl and N-methyl groups may function as methyl donors in the reaction. Using subcellular fractions of mouse liver, we observed that microsomal P450 did not participate in the demethylation of MeHg, but the greatest activity was located in the mitochondria-rich fraction. The addition of superoxide anion in the reaction mixture significantly enhanced the formaldehyde production, whereas Mn-superoxide dismutase depressed the reaction. Our present findings demonstrated that inorganic Hg formed by MeHg demethylation in mouse liver stimulated the endogenous formaldehyde production, and we observed that MeHg demethylation could be estimated by a formaldehyde analysis. Our results also suggested that superoxide anion is involved in the reaction.

Impact of feline AIM on the susceptibility of cats to renal disease.

Renal failure is one of the most important social problems for its incurability and high costs for patients' health care. Through clarification of the underlying mechanism for the high susceptibility of cats to renal disease, we here demonstrates that the effective dissociation of serum AIM protein from IgM is necessary for the recovery from acute kidney injury (AKI). In cats, the AIM-IgM binding affinity is 1000-fold higher than that in mice, which is caused by the unique positively-charged amino-acid cluster present in feline AIM. Hence, feline AIM does not dissociate from IgM during AKI, abolishing its translocation into urine. This results in inefficient clearance of lumen-obstructing necrotic cell debris at proximal tubules, thereby impairing AKI recovery. Accordingly, mice whose AIM is replaced by feline AIM exhibit higher mortality by AKI than in wild-type mice. Recombinant AIM administration into the mice improves their renal function and survival. As insufficient recovery from AKI predisposes patients to chronic, end-stage renal disease, feline AIM may be involved crucially in the high mortality of cats due to renal disease. Our findings could be the basis of the development of novel AKI therapies targeting AIM-IgM dissociation, and may support renal function in cats and prolong their lives.

Atm heterozygous deficiency enhances development of mammary carcinomas in p53 heterozygous knockout mice.

INTRODUCTION: Ataxia-telangiectasia is an autosomal-recessive disease that affects neuro-immunological functions, associated with increased susceptibility to malignancy, chromosomal instability and hypersensitivity to ionizing radiation. Although ataxia-telangiectasia mutated (ATM) heterozygous deficiency has been proposed to increase susceptibility to breast cancer, some studies have not found excess risk. In experimental animals, increased susceptibility to breast cancer is not observed in the Atm heterozygous deficient mice (Atm+/-) carrying a knockout null allele. In order to determine the effect of Atm heterozygous deficiency on mammary tumourigenesis, we generated a series of Atm+/- mice on the p53+/- background with a certain predisposition to spontaneous development of mammary carcinomas, and we examined the development of the tumours after X-irradiation. METHODS: BALB/cHeA-p53+/- mice were crossed with MSM/Ms-Atm+/- mice, and females of the F1 progeny ([BALB/cHeA x MSM/Ms]F1) with four genotypes were used in the experiments. The mice were exposed to X-rays (5 Gy; 0.5 Gy/min) at age 5 weeks. RESULTS: We tested the effect of haploinsufficiency of the Atm gene on mammary tumourigenesis after X-irradiation in the p53+/- mice of the BALB/cHeA x MSM/Ms background. The singly heterozygous p53+/- mice subjected to X-irradiation developed mammary carcinomas at around 25 weeks of age, and the final incidence of mammary carcinomas at 39 weeks was 31% (19 out of 61). The introduction of the heterozygous Atm knockout alleles into the background of the p53+/- genotype significantly increased the incidence of mammary carcinoma to 58% (32 out of 55) and increased the average number of mammary carcinomas per mouse. However, introduction of Atm alleles did not change the latency of development of mammary carcinoma. CONCLUSION: Our results indicate a strong enhancement in mammary carcinogenesis by Atm heterozygous deficiency in p53+/- mice. Thus, doubly heterozygous mice represent a useful model system with which to analyze the interaction of heterozygous genotypes for p53, Atm and other genes, and their effects on mammary carcinogenesis.

Preliminary Analysis of Modified Low-Density Lipoproteins in the Serum of Healthy and Obese Dogs and Cats.

Oxidized low-density lipoprotein (LDL) is thought to play an important role in the inflammatory response associated with human obesity. The purpose of this preliminary study was to determine oxidized LDL concentrations in healthy dogs and cats, and to evaluate whether obesity affects oxidized LDL concentration, using 39 cats and 19 dogs that had visited two different veterinary clinics in Japan. We hypothesized that oxidized LDL concentrations measured against body condition score (BCS) may have a potential value in evaluating the qualities of accumulated or circulating lipids in obese dogs and cats that do not show signs of metabolic diseases. The mean oxidized LDL value in BCS3 dogs (2.4 ± 0.9 µg/dl) was very similar to that of BCS5 dogs (2.2 ± 0.3 µg/dl). The mean oxidized LDL value of BCS4 dogs was 7.2 ± 10.3 µg/dl and the highest among three groups. BCS4 dogs included two dogs whose oxidized LDL values were higher than the mean oxidized LDL value of healthy humans (11.2 ± 0.3 µg/dl). On the other hand, the mean oxidized LDL value of BCS3 cats was 2.5 ± 0.9 µg/dl, and those of BCS4 and 5 cats were higher than that of BCS3, but there was no significant difference. The BCS4 cat group included one cat with a higher oxidized LDL value, and the BCS5 group also included two cats with oxidized LDL values higher than the mean oxidized LDL value of healthy humans. Interestingly, the oxidized LDL values in two obese dogs and three obese cats were indeed higher than the mean oxidized LDL value of humans with coronary artery disease (20.1 ± 1.1 µg/dl). In conclusion, this preliminary study showed reference ranges of oxidized dogs and cats against BCS. Obesity alone does not appear to have any direct effect on serum oxidized LDL values in healthy dogs and cats.

Short communication: molecular characterization of dog and cat p65 subunits of NF-kappaB.

Nuclear factor kappa B (NF-κB) plays an important role in the immune system. The p65 subunit is an important part of NF-κB unit, and studies of dog and cat p65 subunits of NF-κB (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (>87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-κB promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function.

Hemorrhagic hydrocephalus (hhy): a novel mutation on mouse chromosome 12.

A novel mouse hemorrhagic hydrocephalus mutation (hhy) inherited in an autosomal recessive manner on chromosome 12 has been found at the Osaka Prefecture University. The hhy homozygous mutant had dilated lateral ventricles and a communicating aqueduct, with no histological abnormalities either in the subarachnoid space or in the choroid plexus. Multiple hemorrhages in the meninges and throughout the brain parenchyma of the mutant were relevant to advanced stages of hydrocephalus. Subcortical heterotopia was detected unexceptionally in the mutants. Thus, the hhy mutation is characterized by three different abnormalities, i.e. hydrocephalus, intracranial hemorrhage and subcortical heterotopia.

Generation of large homozygous chromosomal segments by mitotic recombination during lymphomagenesis in F1 hybrid mice.

The loss of heterozygosity (LOH) has been reported in numerous neoplasms in both human and animals, and has often been observed in chromosomal regions, which contain tumor-suppressor genes. We previously found frequent LOH on chromosomes 4, 12 and 19 in radiation-induced lymphomas from (BALB/cHeA x STS/A)F1 hybrid mice by allelotype analysis at polymorphic microsatellite loci. In this study, to elucidate the nature of allelic losses, we refined the loss regions on chromosomes 4, 12 and 19 of the tumors from the F1 mice and then analyzed them cytogenetically. The results represent evidence of a wide range of allelic losses owing to mitotic recombination on chromosomes 4 and 19 in the tumors, possibly reflecting functional losses of putative tumor-suppressor genes. It is suggested that the generation of these large homozygous chromosomal segments probably containing the affected genes is one of the genetic alterations responsible for tumorigenesis.