CAT mRNA Expression Research Articles

MCD diet-induced steatohepatitis is associated with alterations in asymmetric dimethylarginine (ADMA) and its transporters.

Using an experimental model of NASH induced by a methionine-choline-deficient (MCD) diet, we investigated whether changes occur in serum and tissue levels of asymmetric dimethylarginine (ADMA). Male Wistar rats underwent NASH induced by 8-week feeding with an MCD diet. Serum and hepatic biopsies at 2, 4 and 8weeks were taken, and serum enzymes, ADMA and nitrate/nitrite (NOx), were evaluated. Hepatic biopsies were used for mRNA and protein expression analysis of dimethylarginine dimethylaminohydrolase-1 (DDAH-1) and protein methyltransferases (PRMT-1), enzymes involved in ADMA metabolism and synthesis, respectively, and ADMA transporters (CAT-1, CAT-2A and CAT-2B). Lipid peroxides (TBARS), glutathione, ATP/ADP and DDAH activity were quantified. An increase in serum AST and ALT was detected in MCD animals. A time-dependent decrease in serum and tissue ADMA and increase in mRNA expression of DDAH-1 and PRMT-1 as well as higher rates of mRNA expression of CAT-1 and lower rates of CAT-2A and CAT-2B were found after 8-week MCD diet. An increase in serum NOx and no changes in protein expression in DDAH-1 and CAT-1 and higher content in CAT-2 and PRMT-1 were found at 8weeks. Hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high TBARS levels and low glutathione content. In conclusion, a decrease in serum and tissue ADMA levels in the MCD rats was found associated with a reduction in DDAH activity due to the marked oxidative stress observed. Changes in ADMA levels and its transporters are innovative factors in the onset and progression of hepatic alterations correlated with MCD diet-induced NASH.

The effects of crocin (active contstituent of saffron) treatment on brain antioxidant enzyme mRNA levels in diabetic rats / Diyabetik ratlarda safranın aktif içeriği olan krosin tedavisinin beyindeki antioksidan enzimlerin mRNA seviyeleri üzerine etkisi

Abstract Objective: The aim of the present study is to evaluate the effect of crocin on mRNA expression of antioxidant enzymes, SOD, CAT and GPX in the brain of the STZ induced diabetic rats. Methods: Thirty animals randomized in three groups containing ten animals in each group as follows; control (non-diabetic rats), DM (STZ-induced untreated diabetic rats), DM+crocin (STZ-induced diabetic rats treated with crocin,). Crocin was given at a dose of 20 mg/kg bw/day by gavage for 21 days. Results: STZ injection caused a significant increase in mRNA expression of antioxidant enzymes, SOD, CAT and GPX when compared to control group. Crocin given to diabetic rats significantly decreased mRNA expression of antioxidant enzymes, SOD, CAT and GPX when compared to DM group. Conclusion: The present study demonstrates that crocin can modulate mRNA expression of antioxidant enzymes, SOD, CAT and GPX and oxidative stress in the brain of the STZ induced diabetic rats.

Chongcao-Shencha Attenuates Liver and Kidney Injury through Attenuating Oxidative Stress and Inflammatory Response in D-Galactose-Treated Mice.

The Chongcao-Shencha (CCSC), a Chinese herbal compound formula, has been widely used as food material and medicine for enhancing physical strength. The present study investigated the possible effect of CCSC in alleviating the liver and kidney injury in D-galactose- (D-gal-) treated mice and the underlying mechanism. Mice were given a subcutaneous injection of D-gal (200 mg/kg) and orally administered CCSC (200, 400, and 800 mg/kg) daily for 8 weeks. Results indicated that CCSC increased the depressed body weight and organ index induced by D-gal, ameliorated the histological deterioration, and decreased the levels of ALT, AST, BUN, and CRE as compared with D-gal group. Furthermore, CCSC not only elevated the activities of antioxidant enzymes SOD, CAT, and GPx but also upregulated the mRNA expression of SOD1, CAT, and GPx1, while decreasing the MDA level in D-gal-treated mice. Results of western blotting analysis showed that CCSC significantly inhibited the upregulation of expression of nuclear factor kappa B (NF-κB) p65, p-p65, p-IκBα, COX2, and iNOS and inhibited the downregulation of IκBα protein expression caused by D-gal. This study demonstrated that CCSC could attenuate the liver and kidney injury in D-gal-treated mice, and the mechanism might be associated with attenuating oxidative stress and inflammatory response.

High intensity interval training favourably affects antioxidant and inflammation mRNA expression in early-stage chronic kidney disease

Increased levels of oxidative stress and inflammation have been linked to the progression of chronic kidney disease. To reduce oxidative stress and inflammation related to chronic kidney disease, chronic aerobic exercise is often recommended. Data suggests high intensity interval training may be more beneficial than traditional aerobic exercise. However, appraisals of differing modes of exercise, along with explanations of mechanisms responsible for observed effects, are lacking. This study assessed effects of eight weeks of high intensity interval training (85% VO2max), versus low intensity exercise (45–50% VO2max) and sedentary behaviour, in an animal model of early-stage chronic kidney disease. We examined kidney-specific mRNA expression of genes related to endogenous antioxidant enzyme activity (glutathione peroxidase 1; Gpx1, superoxide dismutase 1; Sod1, and catalase; Cat) and inflammation (kidney injury molecule 1; Kim1 and tumour necrosis factor receptor super family 1b; Tnfrsf1b), as well as plasma F2-isoprostanes, a marker of lipid peroxidation. Compared to sedentary behaviour, high intensity interval training resulted in increased mRNA expression of Sod1 (p=0.01) and Cat (p<0.001). Compared to low intensity exercise, high intensity interval training resulted in increased mRNA expression of Cat (p<0.001) and Tnfrsf1b (p=0.047). In this study, high intensity interval training was superior to sedentary behaviour and low intensity exercise as high intensity interval training beneficially influenced expression of genes related to endogenous antioxidant enzyme activity and inflammation.

Innovative targets involved in the progression of NASH: Asymmetric-dymethylarginine (ADMA) and its cationic transporters

Background: The methionine choline-deficient (MCD) diet results in liver injury similar to humannonalcoholic steatohepatitis (NASH). Recent data demonstrated that after methionine administration an increase in plasma asymmetric-dimethylarginine (ADMA), an inhibitor of nitric oxide synthase, and a decrease in hepatic dimethylarginine-dimethylaminohydrolase (DDAH-1), enzyme involved in ADMA metabolism, occur. Aim: Using an experimental model of NASH induced by MCD diet, this study investigated whether changes in serum levels of ADMA and tissue content of DDAH-1 occur [Kusku-Kiraz Z., Genc S., Bekpinar S., et al. Circulating levels of apelin, glucagon-like peptide and visfatin in hypercholesterolemic–hyperhomocysteinemic guinea-pigs: their relation with NO metabolism. Molecular and Cellular Biochemistry 2015;400:69–75;Magne J., Huneau J.-F., Borderie D., Mathe V., Bos C., Mariotti F. Plasma asymmetric and symmetric dimethylarginine in a rat model of endothelial dysfunction induced by acute hyperhomocysteinemia. Amino Acids 2015, http://dx.doi.org/10.1007/s00726-015-1959-4]. Materials and methods: Male Wistar rats underwent to NASH induced by 8 weeks of feeding with MCD diet. Serum and hepatic biopsies at 2, 4 and 8 weeks were used: serum enzymes (AST, ALT) and ADMA were evaluated. Hepatic biopsies were used for the analysis of mRNA expression of DDAH-1 and ADMA transporters (CAT-1, CAT-2A and CAT-2B). Tissue total lipids, lipid peroxides (TBARS), glutathione and DDAH activity were also quantified. Results: As expected, an increase in serum AST and ALT and in hepatic total lipids were detected in MCD animals. A time-dependent decrease in serum ADMA and increase in mRNA expression of DDAH-1 were found in MCD group. Higher mRNA expression of CAT-1, lower mRNA expression of CAT-2A and no changes in CAT-2B were found after 8 weeks using MCD diet. The hepatic DDAH activity decreased with a concomitant increase in oxidative stress, as demonstrated by high TBARS levels and low glutathione content. Conclusions: A decrease in serum ADMA levels and an increase in DDAH-1mRNA found inMCD rats were associated with a reduction in DDAH activity due to the marked oxidative stress observed. Changes in ADMA levels and its transporters represent innovative factors involved in the onset and the progression of hepatic alteration correlated with MCD-diet-induced NASH. Funding: Supported by Fondazione Cariplo, grant n◦ 2011-0439.

Riboflavin attenuates lipopolysaccharide-induced lung injury in rats

Riboflavin (vitamin B2) is an easily absorbed micronutrient with a key role in maintaining health in humans and animals. It is the central component of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and is therefore required by all flavoproteins. Riboflavin also works as an antioxidant by scavenging free radicals. The present study was designed to evaluate the effects of riboflavin against acute lungs injury induced by the administration of a single intranasal dose (20 μg/rat) of lipopolysaccharides (LPS) in experimental rats. Administration of LPS resulted in marked increase in malondialdehyde (MDA) level (p < 0.01) and MPO activity (p < 0.001), whereas marked decrease in glutathione (GSH) content (p < 0.001), glutathione reductase (GR) (p < 0.001) and glutathione peroxidase (p < 0.01) activity. These changes were significantly (p < 0.001) improved by treatment with riboflavin in a dose-dependent manner (30 and 100 mg/kg, respectively). Riboflavin (100 mg/kg, p.o.) showed similar protective effects as dexamethasone (1 mg/kg, p.o.). Administration of LPS showed marked cellular changes including interstitial edema, hemorrhage, infiltration of PMNs, etc., which were reversed by riboflavin administration. Histopathological examinations showed normal morphological structures of lungs tissue in the control group. These biochemical and histopathological examination were appended with iNOS and CAT gene expression. The iNOS mRNA expression was increased significantly (p < 0.001) and levels of CAT mRNA expression was decreased significantly (p < 0.001) in the animals exposed to LPS, while treatment with riboflavin significantly (p < 0.01) improved expression of both gene. In conclusion, the present study clearly demonstrated that riboflavin caused a protective effect against LPS-induced ALI. These results suggest that riboflavin may be used to protect against toxic effect of LPS in lungs.

Differential molecular and cellular responses of GLP-1 secreting L-cells and pancreatic alpha cells to glucotoxicity and lipotoxicity

Knowledge of the effects of glucotoxic and lipotoxic environments on proglucagon producing intestinal L cells and pancreatic alpha cells is limited compared with pancreatic beta cells. This study compares the in vitro responses of these cell types to hyperglycaemia and hyperlipidaemia. Glucose (30mM) and palmitate (0.5mM) reduced GLUTag and MIN6 cell viability while alpha TC1 cells were sensitive only to lipotoxicity. Consistent with this, Cat mRNA expression was substantially higher in GLUTag and alpha TC1 cells compared to MIN6 cells. Glucose and palmitate reduced GLUTag cell secretory function while hypersecretion of glucagon was apparent from alpha TC1 cells. Glucose exposure increased transcription of Cat and Sod2 in MIN6 and GLUTag cells respectively while it decreased transcription of Cat and Gpx1 in alpha TC1 cells. Palmitate increased transcription of Cat and Sod2 in all three cell lines. Upregulation of antioxidant enzyme expression by palmitate was accompanied by an increase in Nfkb1 transcription, indicative of activation of defence pathways. Lipotoxicity activated ER stress response, evident from increased Hspa4 mRNA level in GLUTag and MIN6 cells. Glucose and palmitate-induced DNA damage and apoptosis, with substantially smaller effects in alpha TC1 cells. Thus alpha cells are resistant to gluco- and lipotoxicity, partly reflecting higher expression of genes involved in antioxidant defence. In contrast, intestinal L cells, like beta cells, are prone to gluco- and lipotoxicity, possibly contributing to abnormalities of GLP-1 secretion in type 2 diabetes.

Dietary Deficiency of Calcium and/or Iron, an Age-Related Risk Factor for Renal Accumulation of Cadmium in Mice.

The major route of cadmium (Cd) intake by non-smokers is through food ingestion. Cd is a non-essential metal absorbed through one or more transporters of essential metal ions. Expression of these transporters is affected by nutritional status. To investigate the risk factors for Cd toxicity, the effects of deficiency of essential metals on hepatic and renal accumulation of Cd were studied in mice of different ages. Mice were administered a control diet or one of the essential metal-deficient diets, administered Cd by gavage for 6 weeks, and killed; then, Cd accumulation was evaluated. Iron deficiency (FeDF) or calcium deficiency (CaDF) resulted in remarkable increases in hepatic and renal Cd accumulation compared with control-diet mice and other essential metal-deficient mice. Cd accumulation in hepatic and renal tissue was increased significantly at all ages tested in FeDF and CaDF mice. Renal Cd concentrations were higher in 4-week-old mice than in 8- and 25-week-old mice. Increase in intestinal mRNA expression of calcium transporter (CaT)1, divalent metal ion transporter-1, and metallothionein (MT)1 was also higher in 4-week-old mice than in other mice. Renal accumulation of Cd showed strong correlation with intestinal mRNA expression of CaT1 and MT1. These data suggest that CaDF and FeDF at younger ages can be a risk factor for Cd toxicity.

Physiological and molecular level studies on the toxicity of silver nanoparticles in germinating seedlings of mung bean (Vigna radiata L.)

In the present study, the toxic effect of silver nanoparticles was studied in seedlings of economically important food crop mung bean (Vigna radiata L.). The plants were grown in 1/2 MS agar medium containing 0, 5, 10, 20, and 50 mg/L of silver nanoparticles for 21 days. The toxic effects were studied using different parameters such as growth, chlorophyll, and proline contents, reactive oxygen species (ROS) generation, lipid peroxidation, changes in mitochondrial membrane potential (ΔΨm), and oxidative stress responsive gene expression. The shoot length and weight was significantly reduced upon exposure to 50 mg/L of silver nanoparticles. Significant reduction in root elongation and weight was observed upon exposure to 20 and 50 mg/L of silver nanoparticles. The total chlorophyll content significantly reduced after exposure to 50 mg/L of silver nanoparticles. However, proline content was increased significantly upon exposure to 20 and 50 mg/L of silver nanoparticles. The hydrogen peroxide level and lipid peroxidation levels increased significantly in roots after exposure to 20 and 50 mg/L of silver nanoparticles. Histochemical staining with nitroblue tetrazolium showed a concentration-dependant increase in superoxide formation in roots. Roots treated with 2′, 7′-dichloro-dihydro-fluorescein diacetate and 3, 3′-diaminobenzidine showed a concentration-dependant increase in ROS generation. Exposure to increasing concentrations of silver nanoparticles has resulted in changes in ΔΨm in roots as revealed by increased Rhodamine 123 fluorescence. Gene expression analysis using real-time polymerase chain reaction showed changes in the relative mRNA expression of CuZn-SOD, CAT, and APX genes indicating the plants’ antioxidative defense responses under silver nanoparticle stress.

Effects of deer velvet extract from Formosan sika deer on the embryonic development and anti-oxidative enzymes mRNA expression in mouse embryos

Ethnopharmacrological relevanceThe deer velvet or its extracts has been widely used in clinic. It has been used in promoting reproductive performances and treating of oxidation and aging process. The aim of this study is to investigate the effects of velvet extract from Formosan sika deer (Formosan sika deer; Cervus nippon taiouanus, FSD) velvet on mouse embryonic development and anti-oxidant ability in vitro. Materials and methodsMouse 4-cells embryos were divided into 16 groups for 72h in vitro incubation. The embryonic development stages and morphology were evaluated every 12h in experimental period. The quantitative real time PCR was used to measure the CuZn-SOD, GPx and CAT mRNA expression of the blastocysts. ResultsThe 4-cells embryos of hydrogen peroxide (HP) groups did not continue developing after oxidant stress challenged. The blastocyst developmental rate (90.0–90.4%, P>0.05) and normal morphological rate (84.4–85.1%, P>0.05) of the 1% and 2% DV extract groups were similar to those in the control group (90.7% and 88.8%, respectively). The embryos challenged by HP (5, 10 and 25μM) and subsequently incubated in mHTF medium with 1% and 2% of deer velvet (DV) extracts were able to continue development; the blastocyst developmental rate of these groups were similar to that in the control group. The relative mRNA expression of the focused anti-oxidative enzymes in the mouse embryos did not significantly differ among the designed DV treatment groups (P>0.05). ConclusionThe FSD velvet extract in adequate concentration could promote anti-oxidative enzymes mRNA expression followed the challenge of hydrogen peroxide, relieve the mouse embryo under oxidative stress, and maintain the blastocyst developmental ability in vitro.

Erratum to: The effects of acute acetaminophen toxicity on hepatic mRNA expression of SOD, CAT, GSH-Px, and levels of peroxynitrite, nitric oxide, reduced glutathione, and malondialdehyde in rabbit

Erratum to: The effects of acute acetaminophen toxicity on hepatic mRNA expression of SOD, CAT, GSH-Px, and levels of peroxynitrite, nitric oxide, reduced glutathione, and malondialdehyde in rabbit

Study of Oxidative Stress in Human Lens Epithelial Cells Exposed to 1.8 GHz Radiofrequency Fields

ObjectivesThe aims of the present study were to determine oxidative stress and to explore possible reasons of reactive oxygen species (ROS) increase in human lens epithelial (HLE) B3 cells exposed to low intensity 1.8 GHz radiofrequency fields (RF).MethodsThe HLE B3 cells were divided into RF exposure and RF sham-exposure groups. The RF exposure intensity was at specific absorption rate (SAR) of 2, 3, or 4 W/kg. The ROS levels were measured by a fluorescent probe 2′7′-dichlorofluorescin diacetate (DCFH-DA) assay in the HLE B3 cells exposed to 1.8 GHz RF for 0.5, 1, and 1.5 h. Lipid peroxidation and cellular viability were detected by an MDA test and Cell Counting Kit-8 (CCK-8) assays, respectively, in the HLE B3 cells exposed to 1.8 GHz RF for 6, 12, and 24 h, respectively. The mRNA expression of SOD1, SOD2, CAT, and GPx1 genes and the expression of SOD1, SOD2, CAT, and GPx1 proteins was measured by qRT-PCR and Western blot assays in the HLE B3 cells exposed to 1.8 GHz RF for 1 h.ResultsThe ROS and MDA levels significantly increased (P<0.05) in the RF exposure group and that the cellular viability, mRNA expression of four genes, and expression of four proteins significantly decreased (P<0.05) compared with the RF sham-exposure group.ConclusionsOxidative stress is present in HLE B3 cells exposed to 1.8 GHz low-intensity RF and that the increased production of ROS may be related to down-regulation of four antioxidant enzyme genes induced by RF exposure.

The acute aerobic exercise‐induced increase in amino acid transporter expression adapts to exercise training in older adults

Exercise and nutritional interventions may prevent or delay frailty and sarcopenia in older adults. The OBJECTIVE of this ongoing study is to determine the acute and chronic effects of aerobic exercise +/− essential amino acid (EAA) supplementation on skeletal muscle amino acid (AA) transporter expression. Preliminary data are reported.METHODSHealthy untrained older adults (65–85 y; n=10) were randomized into one of two groups: exercise and 15 g EAA supplement (Ex+EAA) or exercise and placebo (Ex+Pla). Acute treatment consisted of 45 mins of walking exercise on a treadmill at ~70% heart rate max (Ex+EAA and Ex+Pla) immediately followed by the assigned supplement or placebo. Chronic treatment involved once daily supplementation (EAA or Pla) plus aerobic exercise 3 times/wk. Real‐time qPCR was used to measure mRNA expression of select muscle AA transporters.RESULTSAt baseline, CAT1, SNAT2, PAT1, CD98, and LAT1 mRNA expression increased more after Ex+EAA than Ex+Pla. After chronic treatment, only CD98 increased with Ex+EAA. There were no basal pre to post changes.CONCLUSIONMuscle AA transporter expression increases acutely with Ex+EAA. Exercise training+EAA induces adaptations reducing the acute AA transporter response to these stimuli. Supported by NIH/NIA R01 AG030070 and NIH/NIA R01 AG030070S1Grant Funding Source: NIH

Dual effect of nickel on L-arginine/nitric oxide system in RAW 264.7 macrophages

The immunogenic mechanisms of the potent contact allergen nickel are not completely clear. Nitric oxide (NO) serves as a fundamental signalling and effector molecule in the immune system, but little is known about its possible role in immune reactions elicited by nickel. We investigated the effects of nickel on the L-arginine/inducible NO synthase (iNOS) system in a murine macrophage cell line, RAW 264.7. Both LPS-stimulated and non-stimulated RAW 264.7 cells were incubated in the presence of 0–100μM nickel sulphate for 24h. Subsequently, NO production, iNOS protein expression, L-arginine uptake and gene expression of iNOS and cationic amino acid transporter systems (CAT) were measured. We found that 100μM NiSO4 increased LPS-induced nitrite production as well as the formation of [3H]-L-citrulline from [3H]-L-arginine in the RAW 264.7 cells. Correspondingly, the expression of iNOS gene and protein was also remarkably enhanced. Nevertheless, nickel had an inhibitory effect on L-arginine transport which disappeared gradually upon LPS-stimulation in parallel with an increase in NO output. LPS was found to significantly amplify CAT-3 as well as CAT-2 mRNA expression, mirroring the increase in L-arginine transport. In the range of 1–10μM, NiSO4 did not have any additional effect on CAT mRNA expression, but at 100μM it was able to enhance CAT-1 and CAT-3 mRNA expression upon LPS stimulation.Our data indicate that nickel interferes with macrophages' L-arginine/NOS system on multiple levels. Considering the potent biological effects of NO, these influences may contribute to nickel toxicity.

CDNA cloning and mRNA expression of cat and dog Cdkal1.

The cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1) gene encodes methylthiotransferase, and the gene contains risk variants for type 2 diabetes in humans. In this study, we performed complementary DNA cloning for Cdkal1 in the cat and dog and characterized the tissue expression profiles of its messenger RNA. Cat and dog Cdkal1 complementary DNA encoded 576 and 578 amino acids, showing very high sequence homology to mammalian CDKAL1 (>88.4%). Real-time polymerase chain reaction analyses revealed that Cdkal1 messenger RNA is highly expressed in smooth muscle and that tissue distribution of Cdkal1 is similar in cats and dogs. Genotyping analysis of single-nucleotide polymorphism for cat Cdkal1 revealed that obese cats had different tendencies from normal cats. These findings suggest that the cat and dog Cdkal1 gene is highly conserved among mammals and that cat Cdkal1 may be a candidate marker for genetic diagnosis of obesity.

Effects of dietary protein and amino acid levels on the expression of selected cationic amino acid transporters and serum amino acid concentration in growing pigs

The absorption of lysine is facilitated by leucine, but there is no information regarding the effect of crude protein, lysine and leucine levels on the expression of cationic amino acid transporters in pigs. Therefore, an experiment was conducted with 20 pigs (14.9 ± 0.62 kg initial body weight) to evaluate the effect of two protein levels, and the content of lysine, threonine, methionine and leucine in low crude protein diets on the expression of b0,+ and CAT-1 mRNA in jejunum, Longissimus dorsi and Semitendinosus muscles and serum concentration of amino acids. Treatments were as follows: (i) wheat–soybean meal diet, 20% crude protein (Control); (ii) wheat diet deficient in lysine, threonine and methionine (Basal diet); (iii) Basal diet plus 0.70% l-lysine, 0.27% l-threonine, 0.10% dl-methionine (Diet LTM); (iv) Diet LTM plus 0.80% l-leucine (Diet LTM + Leu). Despite the Basal diet, all diets were formulated to meet the requirements of lysine, threonine and methionine; Diet LTM + Leu supplied 60% excess of leucine. The addition of lysine, threonine and methionine in Diet LTM increased the expression of b0,+ in jejunum and CAT-1 in the Semitendinosus and Longissiums muscles and decreased CAT-1 in jejunum; the serum concentration of lysine was also increased (p < 0.01). Further addition of l-leucine (Diet LTM + Leu) decreased the b0,+ expression in jejunum and CAT-1 in the Longissimus dorsi muscle (p < 0.05), increased the serum concentration of leucine and arginine and decreased the concentration of isoleucine (p < 0.05). Pigs fed the Control diet expressed less b0,+ in jejunum, and CAT-1 in the Semitendinosus and Longissiums muscles expressed more CAT-1 in jejunum (p < 0.05) and had lower serum concentration of isoleucine, leucine and valine (p < 0.05), but higher lysine concentrations (p < 0.01) than those fed Diet LTM. These results indicated that both, the level and the source of dietary amino acids, affect the expression of cationic amino acid transporters in pigs fed wheat-based diets.

The relationship between gene expression of cationic and neutral amino acid transporters in the small intestine of chick embryos and chick breed, development, sex, and egg amino acid concentration

This study was conducted to investigate the gene expression of cationic and neutral amino acid (AA) transporters in the small intestine of chick embryos with different genetic backgrounds [Wenshi Yellow-Feathered chick (WYFC) and White Recessive Rock chick (WRRC)]. The study also investigated the correlation between the abundance of AA transporter mRNA and the AA content of fertilized eggs. Intestinal samples were collected on embryonic d 9, 12, 14, 17, and 19 and the day of hatch. The results showed that, before incubation, the AA content of WRRC eggs was lower (P < 0.05) than the AA content of WYFC eggs. In WYFC, the mRNA abundance of CAT-1 [solute carrier (SLC) family 7 member 1], CAT-4 (SLC family 7 member 4), rBAT (SLC family 3 member 1), y(+)LAT-1 (SLC family 7 member 7), y(+)LAT-2 (SLC family 7 member 6), LAT-4 (SLC family 43 member 2), and SNAT-2 (SLC family 38 member 2), as detected by real-time reverse transcriptase PCR, was greater (P < 0.05) than the mRNA abundance detected in the WRRC samples. The mRNA abundance of all measured AA transporters was affected (P < 0.05) by embryonic age. Sex had the largest effect (P < 0.05) on the mRNA expression of CAT-1, CAT-4, y(+)LAT-2, and LAT-4 in WYFC and on CAT-4 and B(0)AT-1 (SLC family 6 member 19) mRNA expression in WRRC. In WYFC, only CAT-1 mRNA expression was negatively correlated (r = -0.68 to -0.84, P < 0.05) with all AA content. However, few correlations were detected between AA content and the mRNA expression of multiple transporters in WRRC. These findings provide a comprehensive profile of the temporal and spatial mRNA expression of AA transporters in the small intestine of chick embryos. Few correlations were detected between the AA content of the eggs and mRNA expression of specific AA transporters in the small intestine.

Effects of LED light spectra on oxidative stress and the protective role of melatonin in relation to the daily rhythm of the yellowtail clownfish, Amphiprion clarkii

The present study aimed to test the effects of melatonin on oxidative stress in the yellowtail clownfish, Amphiprion clarkii, as produced by light emitting diodes (LEDs): red, green, and blue. We investigated the effects of the different LEDs on oxidative stress by measuring the mRNA expression of arylalkylamine N-acetyltransferase (AANAT2), the expression and activities of antioxidant enzymes (superoxide dismutase, SOD (EC 1.15.1.1); and catalase, CAT (EC 1.11.1.6)), and plasma H2O2 and plasma melatonin levels. In red light, the expression of AANAT2, SOD, and CAT mRNA was significantly higher than those under the other light spectra. SOD and CAT activities and plasma H2O2 and melatonin levels were also significantly higher for the red spectra than those for the other light spectra. These results indicate that red light induces oxidative stress. To investigate the effects of melatonin on oxidative stress, we injected melatonin into live fish (in vivo) or treated cultured pineal organ (in vitro) with melatonin. We found that AANAT2, SOD, and CAT mRNA expression levels, SOD and CAT activities, and plasma H2O2, lipid peroxidation (LPO) and melatonin levels were significantly lower than those for the controls. Therefore, our results indicate that red light induces oxidative stress and melatonin plays the role of a strong antioxidant in yellowtail clownfish.

Nitric oxide suppression of cellular proliferation depends on cationic amino acid transporter activity in cytokine-stimulated pulmonary endothelial cells

Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing L-arginine (L-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-α (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM L-leucine [L-leu; a competitive inhibitor of L-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by L-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with L-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by L-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with L-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with L-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular L-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction.

Effect of titanium particles on osteoclast activity in vitro

Titanium particles generated from orthopedic and dental implants are suspected to play an important role in peri-implant osteolysis. Elucidation of the mechanisms involved in the effects of titanium particles on osteolysis may provide methods for preventing osteolysis. Therefore, in the present study, we investigated the effect of titanium particles on osteoclast activity in vitro. We evaluated bone resorption pits on dentin slices and osteoclast F-actin rings after treatment with titanium particles. The influence of titanium particles on the expression of TRAP, Cat K and CAII mRNA was also evaluated. We found that osteoclast bone resorption activity was enhanced at a lower concentration of titanium particles, but inhibited by a higher concentration of titanium particles. Titanium particles can be phagocytosed by osteoclasts, but the F-actin ring was not destroyed after phagocytosis. In addition, a lower concentration of titanium particles enhanced the mRNA expression of TRAP and CatK, while higher concentrations of titanium particles decreased their expression. By contrast, the mRNA expression of CAII was decreased at all concentrations of titanium particles. These results suggest that direct exposure to titanium particles at a low concentration enhances osteoclast activity, while exposure to a high concentration inhibits their activity. This understanding of the direct effect of titanium particles on osteoclast activity suggests that aseptic loosening is caused not only by the generation of osteoclasts, but also by enhanced osteoclast activity.