Lipase-catalyzed interesterification of Sunite sheep tail fat and flaxseed oil provides a fat having a unique fatty acid content and favorable physicochemical and nutritional properties.

The aim of this study is to combine flaxseed oil (FO), rich in α-linolenic acid (ALA), with Sunite sheep tail fat (STF) through a lipase-catalyzed transesterification reaction, in order to produce an edible oil with a fatty acid ratio suitable for human needs. Initially, the optimal conditions for esterification were determined using the Box-Behnken design, with the measurement criterion being the content of ALA at the sn-2 position. The results indicated that the highest content of sn-2 ALA was obtained under the conditions of using 6.8 wt% Lipozyme®RMIM as the catalyst, a reaction temperature of 57°C, a reaction time of 3.3 h, and a substrate mass ratio of 5.6:4.4 for STF and FO. This led to the rapid breaking and recombining of molecular bonds, resulting in the interesterified fat (IF) with the highest content of ALA at the sn-2 position. Comparing STF and FO, IF exhibited excellent fatty acid composition and content. Furthermore, IF had a lower melting point and crystallization temperature compared to STF, and its solid fat content decreased with increasing temperature, completely melting at temperatures above 30°C. Thus, IF is a synthesized fat with excellent properties from both animal and vegetable sources.

Rapeseed (canola) oil aggravates metabolic syndrome-like conditions in male but not in female stroke-prone spontaneously hypertensive rats (SHRSP).

This study was conducted to investigate whether or not there are sex differences in canola oil (CAN)-induced adverse events in the rat and to understand the involvement and the role of testosterone in those events, including life-shortening. Stroke-prone spontaneously hypertensive rats (SHRSP) of both sexes were fed a diet containing 10 wt/wt% soybean oil (SOY, control) or CAN as the sole dietary fat. The survival of the males fed the CAN diet was significantly shorter than that of those fed the SOY diet. In contrast, the survival of the females was not affected by CAN. The males fed the CAN diet showed elevated blood pressure, thrombopenia and insulin-tolerance, which are major symptoms of metabolic syndrome, whereas such changes by the CAN diet were not found in the females. Plasma testosterone was significantly lower in animals of both sexes fed the CAN diet than in those fed the SOY diet, but interestingly, the lowered testosterone was accompanied by a marked increase in plasma aldosterone only in the males. These results demonstrate significant sex differences in CAN-toxicity and suggest that those sex differences may be attributable to the increased aldosterone level, which triggers aggravation of the genetic diseases specific to SHRSP, that is, metabolic syndrome-like conditions, but only in the males. The present results also suggest that testosterone may negatively regulate aldosterone production in the physiology of the males, and the inhibition of that negative regulation caused by the CAN diet is one of the possible causes of the adverse events.

Serum Mac-2 Binding Protein Levels Associate with Metabolic Parameters and Predict Liver Fibrosis Progression in Subjects with Fatty Liver Disease: A 7-Year Longitudinal Study.

Background: Mac-2 binding protein (M2BP) is a highly glycosylated secreted glycoprotein that is involved in immune defense and regulation. Our cross-sectional studies indicated that serum M2BP was a useful liver fibrosis biomarker for nonalcoholic fatty liver disease (NAFLD). In this study, we conducted a 7-year longitudinal study to investigate the significance of serum M2BP levels (baseline and at 7-year follow-up) and their relationships with other metabolic parameters of fatty liver disease. Methods: We enrolled 715 study subjects (521 male and 194 female) during health examinations. Study subjects received blood sampling tests and abdominal ultrasound tests at baseline and follow-up. Results: Univariate analyses demonstrated that serum M2BP levels were significantly correlated with various parameters related to metabolic risk (body mass index (BMI), systolic blood pressure, triglyceride, high density lipoprotein (HDL)-cholesterol) and metabolic syndrome diseases (obesity, hypertension, dyslipidemia, diabetes mellitus, fatty liver (FL)). Multiple logistic regression analyses demonstrated that BMI and FL were independent determinants for serum M2BP levels. Baseline serum M2BP levels were significant independent determinants for changes in platelet count, Fibrosis-4 (FIB4) index, and NAFLD fibrosis score. Higher serum M2BP levels (>1.80 µg/mL) strongly correlated with changes in the FIB4-index. Conclusions: The results of this study suggest that changes in serum M2BP levels reflect changes in specific metabolic disease-related parameters, and baseline serum M2BP levels could predict changes in liver fibrosis.

Eicosanoids Derived From Arachidonic Acid and Their Family Prostaglandins and Cyclooxygenase in Psychiatric Disorders.

Arachidonic acid (AA)-derived lipid mediators are called eicosanoids. Eicosanoids have emerged as key regulators of a wide variety of physiological responses and pathological processes, and control important cellular processes. AA can be converted into biologically active compounds by metabolism by cyclooxygenases (COX). Beneficial effect of COX-2 inhibitor celecoxib add-on therapy has been reported in early stage of schizophrenia. Moreover, add-on treatment of celecoxib attenuated refractory depression and bipolar depression. Further, the COX/prostaglandin E pathway play an important role in synaptic plasticity and may be included in pathophysiology in autism spectrum disorders (ASD). In this regard, plasma transferrin, which is an iron mediator related to eicosanoid signaling, may be related to social impairment of ASD. COX-2 is typically induced by inflammatory stimuli in the majority of tissues, and the only isoform responsible for propagating the inflammatory response. Thus, COX-2 inhibitors considered as the best target for Alzheimer's disease.

Effects of arachidonic acid intake on inflammatory reactions in dextran sodium sulphate-induced colitis in rats.

The aim of this study was to investigate the effects of the administration of oral arachidonic acid (AA) in rats with or without dextran sulphate sodium (DSS)-induced inflammatory bowel disease. Male Wistar rats were administered AA at 0, 5, 35 or 240 mg/kg daily by gavage for 8 weeks. Inflammatory bowel disease was induced by replacing drinking water with 3 % DSS solution during the last 7 d of the AA dosing period. These animals passed loose stools, diarrhoea and red-stained faeces. Cyclo-oxygenase-2 concentration and myeloperoxidase activity in the colonic tissue were significantly increased in the animals given AA at 240 mg/kg compared with the animals given AA at 0 mg/kg. Thromboxane B2 concentration in the medium of cultured colonic mucosae isolated from these groups was found to be dose-dependently increased by AA, and the increase was significant at 35 and 240 mg/kg. Leukotriene B4 concentration was also significantly increased and saturated at 5 mg/kg. In addition, AA at 240 mg/kg promoted DSS-induced colonic mucosal oedema with macrophage infiltration. In contrast, administration of AA for 8 weeks, even at 240 mg/kg, showed no effects on the normal rats. These results suggest that in rats with bowel disease AA metabolism is affected by oral AA, even at 5 mg/kg per d, and that excessive AA may aggravate inflammation, whereas AA shows no effects in rats without inflammatory bowel disease.

miR-182 is largely dispensable for adaptive immunity: lack of correlation between expression and function.

MicroRNA (miR)-mediated regulation of protein abundance is a pervasive mechanism of directing cellular processes. The well-studied and abundant miR-182 has previously been implicated in many aspects of T cell function, DNA repair, and cancer. In this study, we show that miR-182 is the most highly induced miR in B cells undergoing class-switch recombination. To elucidate the requirement of miR-182 in lymphocyte function, we extensively characterized mice with a targeted deletion of Mir182. We show that despite its dramatic induction, loss of miR-182 has minimal impact on B cell development, the ability of B cells to undergo class-switch recombination ex vivo and to undergo Ag-driven affinity maturation in vivo. Furthermore, in striking contrast to knockdown studies that demonstrated the requirement of miR-182 in T cell function, miR-182-deficient mice display no defect in T cell development and activation. Finally, we show that T cell-dependent immune response to experimental Listeria monocytogenes infection is intact in miR-182-deficient mice. We conclude that, contrary to previous studies, miR-182 does not play a significant role in all measured aspects of mouse adaptive immunity. This striking absence of a phenotype highlights the lack of correlation between expression pattern and functional requirement, underscores the limitations of using knockdown approaches to assess miR requirements, and suggests that miR networks may compensate for the chronic loss of specific miRs.

An inotropic action caused by muscarinic receptor subtype 3 in canine cardiac purkinje fibers.

Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers.

MicroRNA 210 as a biomarker for congestive heart failure.

MicroRNAs (miRNAs) are endogenous small RNAs that are 18-23 nucleotides long. Recently, plasma miRNAs were reported to be sensitive and specific biomarkers of various pathological conditions. In the present study, we focused on miR-210, which is known to be induced by hypoxia and might therefore be an excellent biomarker for congestive heart failure. Plasma miR-210 levels and expression levels in mononuclear cells and skeletal muscles were elevated in Dahl salt-sensitive rats with heart failure. We also assessed miR-210 expression in patients with heart failure. The miR-210 expression levels in the mononuclear cells of patients with NYHA III and IV heart failure according to the New York Heart Association (NYHA) functional classification system were significantly higher than those with NYHA II heart failure and controls. Although no significant correlation was observed between plasma brain natriuretic peptide (BNP) and plasma miR-210 levels in patients with NYHA II heart failure, patients with an improved BNP profile at the subsequent hospital visit were classified in a subgroup of patients with low plasma miR-210 levels. Plasma miR-210 levels may reflect a mismatch between the pump function of the heart and oxygen demand in the peripheral tissues, and be a new biomarker for chronic heart failure in addition to plasma BNP concentrations.

Pex11α deficiency impairs peroxisome elongation and division and contributes to nonalcoholic fatty liver in mice.

Hepatic triglyceride (TG) accumulation is considered to be a prerequisite for developing nonalcoholic fatty liver (NAFL). Peroxisomes have many important functions in lipid metabolism, including fatty acid ß-oxidization. However, the pathogenic link between NAFL and peroxisome biogenesis remains unclear. To examine the molecular and physiological functions of the Pex11α gene, we disrupted this gene in mice. Body weights and hepatic TG concentrations in Pex11α(-/-) mice were significantly higher than those in wild-type (WT) mice fed a normal or a high-fat diet. Hepatic TG concentrations in fasted Pex11α(-/-) mice were significantly higher than those in fasted WT mice. Plasma TG levels increased at lower rates in Pex11α(-/-) mice than in WT mice after treatment with the lipoprotein lipase inhibitor tyloxapol. The number of peroxisomes was lower in the livers of Pex11α(-/-) mice than in those of WT mice. Ultrastructural analysis showed that small and regular spherically shaped peroxisomes were more prevalent in Pex11α(-/-) mice fed normal chow supplemented without or with fenofibrate. We observed a significantly higher ratio of empty peroxisomes containing only PMP70, a peroxisome membrane protein, but not catalase, a peroxisome matrix protein, in Pex11α(-/-) mice. The mRNA expression levels of peroxisomal fatty acid oxidation-related genes (ATP-binding cassette, subfamily D, member 2, and acyl-CoA thioesterase 3) were significantly higher in WT mice than those in Pex11α(-/-) mice under fed conditions. Our results demonstrate that Pex11α deficiency impairs peroxisome elongation and abundance and peroxisomal fatty acid oxidation, which contributes to increased lipid accumulation in the liver.

Classification of various muscular tissues using miRNA profiling.

MicroRNAs (miRNAs) are endogenous small RNAs of 18-23 nucleotides that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various diseases. In the present study, we explored whether miRNA expression profiling of various muscle cells may be useful for the diagnosis of various diseases involving muscle necrosis. miRNA expression profiling was assessed by miRNA array and real-time reverse-transcriptase polymerase chain reaction by using a reverse primer of a stem loop structure. Profiling of various muscle cells of mouse, including cardiac muscles, skeletal muscles, and vascular and visceral smooth muscles, indicated that profiling of miR-1, miR-133a, miR-133b, miR-145, miR-206, miR-208a, miR-208b, and miR499 were adequate to discriminate muscle cells. miR-145 was remarkably highly expressed in smooth muscles. miR-208a and miR-499 were highly expressed in cardiomyocytes. miR-133a was highly expressed in fast-twitch skeletal muscles. miR-206 and miR-208b were expressed in the slow-twitch skeletal muscles, and they can likely discriminate fast- and slow-twitch types of skeletal muscle cells. We observed that brown fat adipose cells had an miRNA expression profile very similar to those of skeletal muscle cells in the mouse. Plasma concentrations of miR-133a and miR-145 were extremely useful in diagnosing skeletal muscle necrosis in a mouse model of Duchenne muscular dystrophy and colon smooth muscle necrosis in a rat ischemic colitis model, respectively. In the present study, we investigated the miRNA expression profiles of various muscular tissues. Our results suggest that expression profiling would be useful for the diagnosis of various diseases such as muscular necrosis.

Renoprotective mechanisms of pirfenidone in hypertension-induced renal injury: through anti-fibrotic and anti-oxidative stress pathways.

Pirfenidone (PFD) is a novel anti-fibrotic agent that targets TGFß. However, the mechanisms underlying its renoprotective properties in hypertension-induced renal injury are poorly understood. We investigated the renoprotective properties of PFD and clarified its renoprotective mechanisms in a rat hypertension-induced renal injury model. Dahl salt-sensitive rats were fed a high-salt diet with or without 1% PFD for 6 weeks. During the administration period, we examined the effects of PFD on blood pressure and renal function. After the administration, the protein levels of renal TGFß, Smad2/3, TNFα, MMP9, TIMP1, and catalase were examined. In addition, total serum antioxidant activity was measured. Compared to untreated rats, PFD treatment significantly attenuated blood pressure and proteinuria. Histological study showed that PFD treatment improved renal fibrosis. PFD may exert its anti-fibrotic effects via the downregulation of TGFß-Smad2/3 signaling, improvement of MMP9/TIMP1 balance, and suppression of fibroblast proliferation. PFD treatment also increased catalase expression and total serum antioxidant activity. In contrast, PFD treatment did not affect the expression of TNFα protein, macrophage or T-cell infiltration, or plasma interleukin 1ß levels. PFD prevents renal injury via its anti-fibrotic and anti-oxidative stress mechanisms. Clarifying the renoprotective mechanisms of PFD will help improve treatment for chronic renal diseases.

P2X7 deficiency attenuates hypertension and renal injury in deoxycorticosterone acetate-salt hypertension.

The P2X(7) receptor is a ligand-gated ion channel, and genetic variations in the P2X(7) gene significantly affect blood pressure. P2X(7) receptor expression is associated with renal injury and inflammatory diseases. Uninephrectomized wild-type (WT) and P2X(7)-deficient (P2X(7) KO) mice were subcutaneously implanted with deoxycorticosterone acetate (DOCA) pellets and fed an 8% salt diet for 18 days. Their blood pressure was assessed by a telemetry system. The mice were placed in metabolic cages, and urine was collected for 24 h to assess renal function. After 18 days of DOCA-salt treatment, P2X(7) mRNA and protein expression increased in WT mice. Blood pressure in P2X(7) KO mice was less than that of WT mice (mean systolic blood pressure 133 ± 3 vs. 150 ± 2 mmHg). On day 18, urinary albumin excretion was lower in P2X(7) KO mice than in WT mice (0.11 ± 0.07 vs. 0.28 ± 0.07 mg/day). Creatinine clearance was higher in P2X(7) KO mice than in WT mice (551.53 ± 65.23 vs. 390.85 ± 32.81 µl·min(-1)·g renal weight(-1)). Moreover, renal interstitial fibrosis and infiltration of immune cells (macrophages, T cells, B cells, and leukocytes) were markedly attenuated in P2X(7) KO mice compared with WT mice. The levels of IL-1ß, released by macrophages, in P2X(7) KO mice had decreased dramatically compared with that in WT mice. These results strongly suggest that the P2X(7) receptor plays a key role in the development of hypertension and renal disease via increased inflammation, indicating its potential as a novel therapeutic target.

P2X(7) receptor antagonism attenuates the hypertension and renal injury in Dahl salt-sensitive rats.

The P2X(7) receptor is a ligand-gated ion channel activated by extracellular ATP, and a common genetic variation in the P2X(7) gene significantly affects blood pressure. P2X(7) receptor expression is associated with renal injury and some inflammatory diseases. Brilliant blue G (BBG) is a selective rat P2X(7) receptor antagonist. In this study, to test whether BBG has protective effects on salt-sensitive hypertension and renal injury, Dahl salt-sensitive (DS) rats fed an 8% NaCl diet were i.p. injected with BBG (50 mg kg(-1) per day) for 4 weeks. We also tested another P2X(7) receptor antagonist, namely A-438079 (100 mg kg(-1) per day), for 7 days. We found that P2X(7) antagonism markedly attenuated salt-sensitive hypertension, urinary protein or albumin excretion, renal interstitial fibrosis and macrophage and T-cell infiltration in the DS rats, and significantly improved creatinine clearance. In an in vitro experiment using macrophages, we showed that lipopolysaccharide (LPS)-primed macrophages from the DS rats released more interleukin-1 beta in response to BzATP, a P2X(7) receptor agonist, than the macrophages from Lewis rats, possibly due to higher P2X(7) expression in the DS rats. In conclusion, in vivo blockade of P2X(7) receptors attenuated salt-sensitive hypertension and renal injury in the DS rats. Thus, P2X(7) appears to be responsible for a vicious cycle of salt-sensitive hypertension and renal injury in the DS rats, through higher expression in the immune cells. Furthermore, P2X(7) antagonists can prevent the development of salt-sensitive hypertension and renal injury, thus confirming that the P2X(7) receptor is an important therapeutic target.

Locally applied TCP inhibits tumor growth via possible activation of macrophages.

The aim of this study was to investigate whether beta-tricalcium phosphate (TCP) inhibits cancer growth, because TCP, a widely used bone replacement material, is known to attract immune cells. Human colon cancer (WiDr) cells were subcutaneously injected on the backs of nude mice, and tumor growth was observed. Seven days after the injection, five animals were implanted with TCP at the tumor sites, five animals were treated by a direct application of 0.12 mg cisplatin at the sites, and four animals were not treated, as a control. Tumor size on the 43rd day of implantation was 1173 mm(3) in the TCP group and was smaller than that in the control, 1621 mm(3). This inhibition was comparable to that with cisplatin. Furthermore, tumor-growing rate in the TCP group was significantly lower than that in the control group. Histopathological examination of the tumors showed migration of macrophages only in the TCP group, with TCP particles remaining at the implantation loci. There were no between-group differences in neutrophil infiltration and angiogenesis. In another series of in vitro experiments, a concentration-dependent increase in luminol chemiluminescence was observed in isolated human peripheral neutrophils incubated with TCP, and the chemiluminescence due to phagocytosis of opsonized zymosan in the presence of TCP occurred with a lower level of TCP than when the chemiluminescence was due to TCP alone. These results suggest that subcutaneously implanted TCP inhibits tumor growth of implanted WiDr cells, and that the activation by TCP of macrophages plays a role in that inhibition.

Similar changes in clinical and pathological parameters in Wistar Kyoto rats after a 13-week dietary intake of canola oil or a fatty acid composition-based interesterified canola oil mimic.

Canola oil (CO) given as a dietary fat deteriorates hypertension-related condition and shortens the life of stroke-prone spontaneously hypertensive rats (SHRSP). Although substances other than fatty acids have been presumed as causatives, CO mimics consisting of oils other than CO also shorten the life. In this study we intended to examine whether or not fatty acid composition unique to CO participates in the adverse effect. CO or an interesterified CO mimic (ICOM) consisting of safflower oil, flaxseed oil and erucic acid was fed as a dietary fat for 13 weeks to Wistar Kyoto (WKY) rats, and clinical and pathological signs were compared. WKY rats were used to avoid the difficulty in evaluating the results in SHRSP due to irregular deterioration in conditions by stroke. Compared to a standard diet, both diets containing CO or ICOM similarly elevated blood pressure, increased plasma lipids, activated hepatic glucose-6-phosphate dehydrogenase, decreased platelets, shortened blood coagulation times and induced abnormalities in the kidney. Thus, CO-specific fatty acid composition appeared to affect the pathophysiology of the rat and produce consequent aggravation of pathological status, especially in SHRSP. However, the existence of causative factors other than fatty acids was suggested by increased neutrophil count exclusively induced by CO.

Tracheal instillation of diesel exhaust particles component causes blood and pulmonary neutrophilia and enhances myocardial oxidative stress in mice.

The influences of inhaling particulate air-pollutants on hematopoiesis and myocardial oxidative stress were investigated in mice by intratracheal instillation (IT) of diesel exhaust particles (DEP), its dichloromethane soluble-component (DMSC) or residual particle-component (RPC). After IT, time courses of cytokine levels in bronchial alveolar lavage fluid (BALF), peripheral blood cell count, myocardial myeloperoxidase (MPO) activity and myocardial chemokine levels were observed for 24 hr. RPC caused sustained blood neutrophilia while that caused by DEP and DMSC was transient. RPC also caused sustained elevations of granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-6 levels in BALF. Furthermore, IL-1beta level in BALF in the RPC group was significantly elevated at 24 hr after IT. Significant positive correlations were observed between blood neutrophil count and IL-6/G-CSF levels in BALF. MPO activity in the myocardium was increased by RPC at 12 and 24 hr after IT while the activities in the kidney and the liver were not affected. Significant correlation was also observed between myocardial MPO activity and blood neutrophil count at 12 hr after IT, for all three substances. From these results, it was concluded that particle component of DEP may enhance myocardial oxidative stress via blood neutrophilia and the elevation of cytokine levels in BALF.

Dietary intake of rapeseed oil as the sole fat nutrient in Wistar rats--lack of increase in plasma lipids and renal lesions.

Dietary rapeseed (canola) oil (CO) given as the only fat nutrient shortens life in stroke-prone spontaneously hypertensive rats (SHRSP), compared with SHRSP given soybean oil (SO) instead of CO. CO ingestion increases plasma lipids and causes renal lesions in SHRSP and in spontaneously hypertensive rats (SHR), and increases plasma lipids also in Wistar Kyoto (WKY) rats, a normotensive counterpart of SHR. This study examined whether or not such unfavorable effects of CO are restricted to these closely related strains. For this purpose Wistar rats, the strain from which these strains were derived, were fed a diet containing 10% CO or SO as the sole fat nutrient for 10 weeks, and changes in clinical signs, urinalysis, blood biochemistry and pathology were compared. CO ingestion did not induce any abnormalities in Wistar rats, except significant increases in plasma concentrations of aldosterone and Na(+), compared with the SO group. Thus, the unfavorable effects of CO ingestion appear to be restricted to SHRSP and its closely related strains. The role of increased aldosterone and Na(+ )in the unfavorable events caused by CO in SHRSP, SHR and WKY rats, and any factors which could induce such increases in aldosterone and Na(+), remain to be elucidated.

Changes in the presence of urine Helicobacter pylori antibody in Japanese children in three different age groups.

BACKGROUND: The rates of acquisition and spontaneous eradication of Helicobacter pylori infection in children has yet to be established. To determine these rates in children living in an urban region of Japan, the levels of urine H. pylori antibodies in children of three different age groups were measured. METHODS: A urine-based enzyme-linked immunosorbent assay (ELISA) was used to detect H. pylori antibodies twice within a 12 month interval over 2 years in 452 healthy children living in Tokyo. The subjects were divided into three groups: ages 4, 7, and 10 years. RESULTS: The prevalence of H. pylori infection was not different among the groups, being between 4.0% and 6.7%. The rate of turn to positivity for H. pylori infection was 1.5% per year and the rate of turn to negativity was 1.1%, but in the 10 year age group the rates were markedly lower than in the younger children. CONCLUSION: The prevalence of H. pylori infection in Tokyo was 4.0-6.7% and was not different among 4, 7, and 10 year age groups.