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Objective: In this study, we investigated the effects of Rubus coreanus-derived lactic acid bacteria (LAB) fermented feed (RC-LAB fermented feed) and three types of LAB (Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium animalis) on the expression of transcription factors and cytokines in Th1, Th2, Th17, and Treg cells in the intestinal lymph nodes and spleens of rats. In addition, the effect on intestinal microbiota composition and body weight was investigated.Methods: Five-week-old male rats were assigned to five treatments and eight replicates. The expression of transcription factors and cytokines of Th1, Th2, Th17, and Treg cells in the intestinal lymph nodes and spleens was analyzed using real-time reverse transcriptase polymerase chain reaction assays. Intestinal tract microbiota compositions were analyzed by next-generation sequencing and quantitative polymerase chain reaction assays.Results: RC-LAB fermented feed and three types of LAB increased the expression of transcription factors and cytokines in Th1, Treg cells and Galectin-9, but decreased in Th2 and Th17 cells. In addition, the intestinal microbiota composition changed, the body weight and Firmicutes to Bacteroidetes (F/B) ratio decreased, and the relative abundance of LAB increased.Conclusion: LAB fermented feed and three types of LAB showed an immune modulation effect by inducing T cell polarization and increased LAB in the intestinal microbiota.

Atopic dermatitis (AD) is a chronic inflammatory skin disease of mainly infants and children. Currently, the development of safe and effective treatments for AD is urgently required. The present study was conducted to investigate the immunomodulatory effects of yeast-extracted β-1,3/1,6-glucan and/or Lactobacillus plantarum (L. plantarum) LM1004 against AD-like symptoms. To purpose, β-1,3/1,6-glucan and/or L. plantarum LM1004 were orally administered to AD-induced animal models of rat (histamine-induced vasodilation) and mouse (pruritus and contact dermatitis) exhibiting different symptoms of AD. We then investigated the treatment effects on AD-like symptoms, gene expression of immune-related factors, and gut microbiomes. Oral administration of β-1,3/1,6-glucan (0.01 g/kg initial body weight) and/or 2 × 1012 cells/g L. plantarum LM1004 (0.01 g/kg initial body weight) to ADinduced animal models showed significantly reduced vasodilation in the rat model, and pruritus, edema, and serum histamine in the mouse models (p < 0.05). Interestingly, β-1,3/1,6- glucan and/or L. plantarum LM1004 significantly decreased the mRNA levels of Th2 and Th17 cell transcription factors, while the transcription factors of Th1 and Treg cells, galactin-9, filaggrin increased, which are indicative of enhanced immunomodulation (p < 0.05). Moreover, in rats with no AD induction, the same treatments significantly increased the relative abundance of phylum Bacteroidetes and the genus Bacteroides. Furthermore, bacterial taxa associated with butyrate production such as, Lachnospiraceae and Ruminococcaceae at family, and Roseburia at genus level were increased in the treated groups. These findings suggest that the dietary supplementation of β-1,3/1,6-glucan and/or L. plantarum LM1004 has a great potential for treatment of AD as well as obesity in humans through mechanisms that might involve modulation of host immune systems and gut microbiota.

Atopic dermatitis (AD) is a skin disorder that causes chronic itch. We investigated the inhibitory effects of a mixture of prebiotic short-chain galacto-oligosaccharides and long-chain fructooligosaccharides (scGOS/lcFOS), inulin, or β-glucan on AD development in 1-chloro-2,4- dinitrobenzene (DNCB)-treated NC/Nga mice. Mice were randomly assigned to six groups: untreated mice, AD control, positive control (DNCB-treated NC/Nga mice fed a dietary supplement of Zyrtec), and DNCB-treated NC/Nga mice fed a dietary supplement of prebiotics such as scGOS/lcFOS (T1), inulin (T2), or β-glucan (T3). The prebiotic treatment groups (T1, T2, and T3) showed suppression of AD symptoms, Th2 cell differentiation, and AD-like skin lesions induced by DNCB. In addition, prebiotic treatment also reduced the number of microorganisms such as Firmicutes, which is associated with AD symptoms, and increased the levels of Bacteroidetes and Ruminococcaceae, which are associated with alleviation of AD symptoms. Our findings demonstrate the inhibitory effects of prebiotics on AD development by improving the Th1/Th2 cytokine balance and beneficial symbiotic microorganisms in in vitro and in vivo models.

Atopic dermatitis (AD) is a chronic inflammatory skin disease of mainly infants and children. Currently, the development of safe and effective treatments for AD is urgently required. The present study was conducted to investigate the immunomodulatory effects of yeast-extracted β-1,3/1,6-glucan and/or

Department of Animal Science and Biotechnology, Gyeongnam National University of Science and Technology, Jinju 52725, Korea ABSTRACT The aim of this study was to investigate the expression patterns of key genes involved in lipid metabolism in response to dietary Coenzyme Q10 (CoQ10) in hens. A total of 36 forty week-old Lohmann Brown were randomly allocated into 3 groups consisting of 4 replicates of 3 birds. Laying hens were subjected to one of following treatments: Control (BD, basal diet), T1 (BD+ CoQ10 100 mg/kg diet) and T2 (BD+ micellar of CoQ10 100 mg/kg diet). Birds were fed ad libitum a basal diet or the basal diet supplemented with CoQ10 for 5 weeks. Total RNA was extracted from the liver for quantitative RT-PCR. The mRNA levels of HMG-CoA reductase(HMGCR) and sterol regulatory element-binding proteins(SREBP)2 were decreased more than 3050% in the liver of birds fed a basal diet supplemented with CoQ10 (p

Dept. of Animal Science and Biotechnology, the Regional Animal Research Center (RAIC),Gyeongnam National University of Science and Technology, Jinju 52725, Korea,ABSTRACT This study was performed to examine the effects of dietary Acanthopanax senticosus (AS) on growth performance, immune organ weights, blood biochemical parameters and the expression of pro-inflammatory cytokines in broiler chicks. A total of 120 4-day-old birds were given a basal diet (CON) or a basal diet supplemented with 0.5% (AS1) or 1.0% (AS2) AS powder until the birds were 35 days of age. There was no difference in body weight, total gain, feed intake or immune organ weights among the treatment groups. However, the feed conversion ratio in the AS2 group was lower (p

Dept. of Animal Science and Biotechnology, Gyeongnam National University of Science and Technology, Jinju 52725, KoreaABSTRACT The present study aimed to investigate the effects of lycopene on hepatic metabolic- and immune-related gene expression in laying hens. A total of 48 25-week-old White Leghorn hens were randomly allocated into four groups consisting of four replicates of three birds: control (basal diet), T1 (basal diet + 10 mg/kg of tomato powder-containing lycopene), T2 (basal diet + 10 mg/kg of micelles of tomato powder-containing lycopene), and T3 (basal diet + 10 mg/kg of purified lycopene). Chickens were fed ad libitum for 5 weeks, and then total RNA was extracted from the livers for quantitative RT-PCR analysis. Peroxisome proliferator-activated receptor ɣ (PPARɣ) expression was decreased in the liver of chickens after lycopene supplementation (P

Preadipocyte factor-1 (Pref-1) is made as a transmembrane protein containing EGF-repeats at the extracellular domain that can be cleaved to generate a biologically active soluble form. Pref-1 is found in islet β-cells and its level has been reported to increase in neonatal rat islets upon growth hormone treatment. We found here that Pref-1 can promote growth of pancreatic tumor derived AR42J cells. To examine Pref-1 function in pancreatic islets invivo, we generated transgenic mouse lines overexpressing the Pref-1/hFc in islet β-cells using rat insulin II promoter (RIP). These transgenic mice exhibit an increase in islet mass with higher proportion of larger islets in pancreas compared to wild-type littermates. This is in contrast to pancreas from Pref-1 null mice that show higher proportion of smaller islets. Insulin expression and insulin secretion from pancreatic islets from RIP-Pref-1/hFc transgenic mice are increased also. Thus, RIP-Pref-1/hFc transgenic mice show normal glucose levels but with higher plasma insulin levels in both fasting and fed conditions. These mice show improved glucose tolerance. Taken together, we conclude Pref-1 as a positive regulator of islet β-cells and insulin production.

Chickens are exposed to the external and internal stressors such as low and high temperature, high stocking density, feed restriction and disease. There have been a few studies on gene expressions through the investigation of chickens under direct exposure to the stress of high stocking density. The objective of the present study was to determine the expressions of genes associated with stress, endoplasmic reticulum (ER)-stress, lipid and glucose metabolism in two strains of chickens, Korean Native Chicken (KNC) and White Leghorn (WL), raised in high stocking density. A total of 164 chickens aged 40 weeks were randomly allotted to a 540 cm 2 /bird stocking density (control), whereas the chickens in a high density group were assigned in a 311 cm 2 /bird stocking density with feeding ad libitum for 10 weeks. Total RNA was extracted from the live for qRT-PCR. The expression levels of hsp70 and hsp90α were higher in WL subjected to stress with high stocking density compared with those genes in control (P

Department of Animal Science & Biotechnology, Gyeongnam Nationa l University of Science and Technology, Jinju 660-758, KoreaReceived September 11, 2014/Revised October 8, 2014/Accepted October 14, 2014The purpose of this study was to determine the effect of selenate on adipocyte differentiation and to identify genes involved in the modulation of adipogenesis in 3T3-L1 cells. To test the effect of selenate on adipocyte differentiation, adipogenesis was induced in cells using various concentrations (0–00 μM) of selenate. Various phases of adipogenesis were induced: postconfluent (PC), early phase (EP, d0-d2), postmitotic growth arrest (PM, d2-d4), and all period (AP). The PC cells exposed to selenate for 24 h displayed dose-dependent inhibition of intracellular lipid droplet accumulation on day 6 of adipogenesis. Two days of selenate treatment at EP or AP inhibited adipogenesis, with an approx-imately 20–80% reduction in lipid accumulation compared to that of a control (p

The aim of this study was to determine the effects of stocking density and strain on the performance and physiological adaptive responses including the plasma corticosterone content and the level of mRNA expression of pro- inflammatory cytokines and antioxidant enzymes in broiler chicks. A total of 300 birds of two strains (150 Ross strain vs. 150 Cobb strain) aged 3-d old were allotted into two stocking densities (standard stocking density, 0.046 m 2 /bird vs. high stocking density, 0.023 m 2 /bird) in battery cages by 2×2 factorial designs with ten replicates until 35 d of age. There was no significant strain effect on body weight, feed intakes and feed to gain ratio and the relative organ weights. However body weight, feed intakes and relative organ weight were found to be significantly (P

Department of Animal Science and Biotechnology, Gyeongnam National University of Science and Technology, Jinju 660-758, KoreaABSTRACT With Single Comb White Leghorn (WL) and Korean Native Chicken (KNC) breeds, we compared the stress response with chicken breeds that were subjected to a high stocking density. Stress response was analyzed by the quantity of telomeric DNA, the rate of DNA damage and the expression levels of heat shock proteins (HSPs) and hydroxyl-3- methyl-glutaryl coenzyme A reductase (HMGCR) genes on tissues and blood. The telomere length and telomere shortening rates were analyzed by quantitative fluorescence in situ hybridization on the nuclei of lymphocytes and tissues. The DNA damage rate of lymphocytes was quantified by the comet assay. The expression levels of HSP70, HSP90-α, HSP90-β and HMGCR genes were measured by quantitative real-time polymerase chain reaction in lymphocytes. There was no significant difference between KNC and WL in body weight, weight gain, telomere shortening rate and DNA damage rate. However, the growth rate significantly decreased in chickens raised under high stocking density conditions, as compared to the control group. The telomere-shortening rate, DNA damage and HSPs expression of the lymphocytes were significantly higher in the high stocking density group than the control. The stress condition and breeds had a significant effect on the expressions of HSP70, HSP90-α and HSP90-β in lymphocytes, except HMGCR. The stress response of WL was higher than that of KNC, as analyzed to the expression of HSP70 and HSP90-α. Therefore, we concluded that the chickens which were exposed to a high stocking density had increased the individual physiological stress response regardless of breeds, and White Leghorns are more susceptible to stress condition than Korean Native Chickens.(Key words : stress response, telomere, DNA damage, HSP, Korean Native Chicken, Leghorn)

The present study was carried out to investigate the effects of dietary antioxidants on pro-inflammatory cytokines, heat shock protein (HSP) and antioxidant status in broiler chicks under summer conditions. A total of 162, 3-d-old broiler chicks were randomly assigned to a basal diet (CON) and the basal diet supplemented with vitamin C (200 mg/kg diet, VCD) or vitamin E (100 mg/kg, VED) until 35 day of age. All birds were exposed to summer diurnal heat stress at average daily fluctuations of temperature between 32 degrees C to 34 degrees C at day to 27 degrees C to 29 degrees C at night for the entire feeding periods. There was no significant difference in body weight, feed to gain ratio and the relative organ weight except the thymus in response to dietary vitamin C or E supplementation. However, the mRNA expression of interleukin (IL)-1 beta, IL-6, interferon (IFN)-gamma, Toll like receptor (TLR)-4 and HSP70 in the liver of birds fed diet containing vitamin C significantly (p

This study was conducted to evaluate the effects of dietary supplementation of vitamin C or E on the expressions of endoplasmic reticulum (ER) stress, lipid and glucose metabolism associated genes in broiler chickens. A total of 216 one-day-old male broilers was randomly alloted to 4 treatments with 6 replicate pens per treatment and 9 broilers per pen for 35 days. The dietary treatments were control, vitamin C (control diet + ascorbic acid 200 mg/kg diet), vitamin E (control diet + -tocopherol 100 mg/kg diet), vitamin C + E (control diet + vitamin C 200 mg/kg + vitamin E 100 mg/kg), respectively. To evaluate gene expressions by quantitative real-time polymerase chain reaction, total RNA was extracted from the liver of the chicken at 35 days of age. Dietary supplementation of vitamins was significantly down-regulated the expression of stress marker genes including HSP70, HSP90, and HMGCR, as compared to the control (p

The aim of this study was to assess the effects of housing systems on physiological and immunological responses as stress indicators in laying hens. A total of 500 White Leghorn aged 16 weeks were allotted into ten conventional cages (10 birds/cage and 810 cm 2 /bird) and four floor pens (100 birds/pen and 2,800 cm 2 /bird) for 24 weeks. The hens housed in conventional cages with higher stocking density resulted in a significantly (P

Department of Animal Science and Biotechnology, Gyeongnam National University of Science and Technology, Jinju 660-758, KoreaABSTRACT This study was performed to investigate the investigated effects of dietary supplementation of vitamin C and E on the growth performance and stress response in broiler chickens. Stress response was analyzed by the quantity of telomeric DNA, the rate of DNA damage and the expression levels of heat shock proteins (HSPs) and hydroxyl-3-methyl-glutaryl coenzyme A reductase (HMGCR) genes on tissues and blood. The telomere length and telomere shortening rates were analyzed by quantitative fluorescence in situ hybridization on the nuclei of lymphocytes and tissues. The DNA damage rate of lymphocytes was quantified by the comet assay. The expression levels of HSP70, HSP90s and HMGCR genes were measured by quantitative real-time polymerase chain reaction in lymphocytes. In results, there was no significant difference among treatments in body weight, weight gain, feed intake and mortality. The telomere shortening rate of the lymphocytes was significantly lower in the vitamin E supplemented group than the control group. The DNA damage was also decreased supplemented with vitamin C and E, as compared to the control group. The vitamin E supplemented group had a significant positive effect on the expressions of HMGCR, HSP90-α and HSP90-β in lymphocytes, but had no significance on HSP70, as compared to the control group. We concluded that the dietary supplementation of vitamin E (100 mg/kg feed) had reduced the individual physiological stress response without stunt growth in broiler chickens.(Key words : vitamin C, vitamin E, growth performance, stress, broiler)

The present study was conducted to investigate whether myoblasts can be transdifferentiated into adipocytes by ectopic expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBPα), sterol regulatory element binding protein-1c (SREBP1c), and Krueppel-like factor 5 (KLF5), in primary bovine satellite cells. Transcription factors were transiently transfected into primary bovine myoblasts, and the cells were cultured with adipogenic differentiation medium for 2 days and then cultured on growth medium for an additional 8 days. Ectopic expression of PPARγ or C/EBPα alone was insufficient to induce adipogenesis in myoblasts. However, overexpression of both PPARγ and C/EBPα in myoblasts was able to induce adipogenic transdifferentiation as indicated by the appearance of mature adipocytes, the induction of adipogenic gene expressions, and the suppression of myogenic gene expressions. In addition, KLF5 and PPARγ co-transfected bovine myoblasts were converted to adipocytes but not in cells transfected with only KLF5 expression vector. Overexpression of SREBP1c alone was sufficient to induce transdifferentiation from myoblasts into adipocytes. These results demonstrate that primary bovine satellite cells can be transdifferentiated into adipocytes either by single ectopic expression or combined expression of adipogenic transcription factors in a culture system.

To investigate the effect of dietary supplementation of freeze-dried plum (Prunus mume Siebold and Zucc., PMS) or omija meal (Schizandra chinensis Baill.; SCB) on growth performance, organ weights, blood biochemical profiles and antioxidant defense system, a total of 96, 3-day-old male broiler chickens were assigned to three dietary groups: (1) control diet, (2) control diet supplemented with PMS at 0.2%, (3) control diet supplemented with SCB at 0.2%. In vitro antioxidant activity, plum and omija extracts showed a significantly higher radical scavenging activity (RSA). In particular, omija extract showed much higher RSA than plum extract. Dietary addition of plum or omija did not affect body weight, feed intake, feed conversion and the relative weight of digestive organ in birds. Plasma triglyceride significantly (P

A total of 21 male SD rats were divided into three groups to investigate the effects of consecutive cyclic heat stress or vitamin C under heat stress on heat shock protein (HSP) 70, inflammatory cytokines, and antioxidant systems. The heat stress (HS) and vitamin C supplementation during heat stress (HS+VC) groups were exposed to cyclic heat stress (23 to 38 to 23°C) for 2 h on each of seven consecutive days. The HS+VC group had free access to water containing 0.5% vitamin C throughout the experiment. Hepatic HSP70 mRNA in the HS group was significantly (P

A study was conducted to investigate the effects of the combined stressor induced by high stocking density with feed restriction on immunological parameters such as leukocyte differential counts and cytokine expression in laying hens. A total of forty White Leghorn laying hens were randomly allotted into the control (12 kg of body weight/m) and the stress (44 kg of body weight/m) groups, and then birds of the stress group were given 75% of voluntary intake of the control birds for 12-d on a daily basis. There was a significant decrease in body weight without affecting the relative weights of the liver and spleen after 12-d of the combined stressor. In hematological values, no significant difference in leukocyte differential counts including heterophils (H), lymphocytes (L), monocytes and H:L ratio was observed between the two groups. In cytokines, hepatic lipopolysaccharide-induced tumor necrosis factor-α (LITNFα) and inducible nitric oxide synthase (iNOS) expression levels in the stress group were significantly (p

Serum lipid (SL) is a commercially available cholesterol-rich, proteinaceous compound extracted from bovine serum. Here we investigated the adipogenic transdifferentiation potential of SL on bovine myogenic satellite cells. Exposure of satellite cells to SL could generate lipid droplets on day 2, and further exposure to SL increased cytoplasmic lipid accumulation giving adipocyte morphology. The expression analysis of PPAR gamma and GPDH adipocyte markers along with Oil-red-O staining results confirmed the transdifferentiation potential of SL. When cells were treated at different concentrations (5, 10, 20, 40 μl/ml) of SL, the results indicated that even levels as low as 5 μl SL /ml could induce transdifferentiation, and maximum induction was obtained at 20 μl SL/ml. After treatment with SL at different concentrations the expression levels of PPAR gamma varied significantly (p

To investigate the effect of dietary supplementation of Acanthopanax senticosus (AS) and Eucommia ulmoides (EU) on antioxidant defense system in laying hens, a total of three hundreds sixty 20-wk old Hyline brown commercial laying hens were assigned to five dietary groups for 10-wk: (1) control diet, (2) control diet supplemented with AS at 0.5%, (3) control diet supple- mented with AS at 1.0%, (4) control diet supplemented with EU at 0.5% and (5) control diet supplemented with EU at 1.0%. Total antioxidant status (TAS) in blood and antioxidant enzymes including superoxide dismutase (SOD), gluthathione -S- transferase (GST) and glutathione peroxidase (GSH-Px), and lipid peroxidation in the small intestine and liver were measured. There were no changes in body weight for 10-wk dietary treatment. TAS in blood significantly (P

To be economically profitable, the poultry industry demands an increase in stocking density, which could adversely affect chicken welfare. The current study was performed to investigate the effect of stocking density on stress-related, heat shock protein genes (HSP70 and HSP90), 3-hydroxyl-3-methyl-glutaryl coenzyme A reductase (HMGCR) gene and telomere length in broiler chickens. Seven-day-old broiler chickens were housed at High (0.0578 m 2 /bird), Standard (0.077 m 2 /bird) and Low (0.116 m 2 /bird) stocking densities with 8 replicates each until 35 d of age. The growth performance, such as body weight gain and average daily feed intake, was found to be significantly (p

Department of Animal Science and Biotechnology, Jinju National University, Jinju 660-758, KoreaABSTRACT The present study was conducted to determine the effect of energy restricted (ER) diet on the expressions of lipogenic genes in liver and myogenic genes in muscle tissue of broiler chickens. Energy restriction was accomplished by providing chicks with 70% (ER70) or 85% (ER85) energy level of control diet intake. Energy restricted groups of chickens were restricted for 7 days, starting at 8 days of age. Ad libitum feeding was resumed after the restriction period, and continued through the end of the experiment. The body weight of chickens in the restricted groups gained less during the energy restriction period (P

The molecular mechanism of adipocyte differentiation has been well documented. However, the effect of specific nutrients such as lysine on adipocyte differentiation is poorly understood especially in ruminant animals. Therefore, the aim of the present study was to elucidate the influence of lysine on adipocyte differentiation and adipogenic genes in cultured bovine preadipocytes. The preadipocytes were treated with different concentrations of lysine (40, 160, 320 mg/L) or troglitazone (10 μM) for 2 days and then subsequently cultured in differentiation medium until day 6. Expression levels of C/EBPα were significantly higher (p

The current study was conducted to investigate the effect of early feed restriction (FR, 8 to 14 d of age) on growth performance and immune function in broiler chickens. Birds were fed corresponding diets from 3 to 35 d of age, which consisted of three phases: starter (3 to 7 d of age), FR (8 to 14 d of age) and re-alimentation (15 to 35 d of age) phases. During the FR period, each group of birds was fed the basal diet ad libitum (CON), 85% (EN85) and 70% (EN70) of lower calorie diet ad libitum (qualitative FR), and 85% (FI85) and 70% (FI70) of voluntary intake on a daily basis (quantitative FR). As a result, there was no statistical difference in weight gain, feed intake and feed conversion ratio between the CON and quantitative or qualitative FR groups during the entire (3 to 35 d) periods. In particular, the EN85 group resulted in a significant (p

Cidec is a lipid droplet-associated protein, which inhibits lipolysis, leading to the accumulation of triglycerides in adipocytes. However, the transcriptional regulation of Cidec in adipocyte remains unknown. In the present study we investigated that the mouse Cidec transcript is regulated by PPARgamma2. After the differentiation of adipocyte, the expression pattern of Cidec was similar to that of PPARgamma2. In the presence of a PPARgamma agonist, the level of Cidec mRNA was highly increased. In addition, putative PPRE sites were identified in the Cidec promoter. By chromatin immunoprecipitation assay and reporter assay, we observed the binding of PPARgamma2 to the promoter of Cidec. Gel shift assay and the mutagenesis study were showed that the -219/-207 region of the Cidec promoter could function as a PPRE of the Cidec promoter. These results suggest that PPARgamma2 is required for the transcriptional activity of Cidec during adipogenesis, which could be contributed to understand the molecular mechanism of lipid droplet formation in adipocytes.

Resistin is an adipokine whose physiologic role in obesity, type II diabetes mellitus, and inflammatory diseases has been a subject of debate because while it is expressed in adipocytes and adipose tissue in mouse, it is expressed in leukocytes, such as macrophages, in human. In the present study, we attempt to define the effect of resistin on human dendritic cells (DCs) derived from CD14 + monocytes. When DCs were stimulated with lipoteichoic acid (LTA) and treated with various concentrations of resistin, antigen-uptake process and the endocytic capacity of DCs were decreased. It is intriguing that resistin attenuated cytokine production in LTA-primed DCs. Consequently, T cell activity was reduced when lymphocytes were mixed with Staphylococcus aureus -primed autologous DCs treated with resistin compared to S. aureus -primed DCs without resistin. Our results suggest that resistin interferes with the efficacy of immune responses activated by Gram-positive bacterial infection in human DCs.

Acid-labile subunit(ALS)는 85-kDa 크기의 당단백질로서 7.5-kDa의 insulin-like growth factor(IGF) 및 40~45-kDa IGF-binding protein-3와 결합하여 150-kDa ternary complex를 형성하는 혈장단백질이다. 선행연구에서 본 연구진은 reverse transcription-polymerase chain reaction(RT-PCR) 방법으로 돼지(porcine) ALS(pALS)의 coding sequence를 합성하여 plasmid vector에 삽입시켜 ‘expression construct’를 제작한 바 있다. 그러나 본 expression construct의 pALS coding sequence에는 PCR error로 추정되는 원인으로 말미암아 2개의 bases에서 mis-sense mutation이 일어난 것이 발견되었다. 본 연구에서는 ‘site-directed mutagenesis’ 방법으로 pALS의 올바른 coding sequence를 합성하여 ‘insert DNA’의 마지막 codon 다음에 ‘His-tag’ sequence가 위치한 pET- 28a(+) plasmid expression vector에 삽입하였다. 본 expression construct는 E. coli BL21(DE3) 세포에서 ‘induction’ 시켰고, 발현된 유전자재조합(recombinant) peptide는 Ni-affinity chromato- graphy로 정제하였다. 이렇게 affinity chro- matography로 정제된 peptide는 SDS-PAGE에서 66kDa 위치에 single band를 나타냄으로써 recombinant pALS의 예상된 질량과 일치하였다. 이상의 결과는 본 연구에서 recombinant pALS peptide가 성공적으로 발현정제되었음을 시사한다. 【Acid-labile subunit(ALS) is a 85-kDa glycosylated plasma protein which forms a 150-kDa ternary complex with 7.5-kDa insulin-like growth factor(IGF) and 40~45-kDa IGF-binding protein-3. In a previous study, the present authors prepared a porcine ALS(pALS) expression construct by inserting a pALS coding sequence into a plasmid vector following synthesis of the sequence by reverse transcription-polymerase chain reaction(RT-PCR). The expression construct, however, was subsequently found to have a mis-sense mutation at two bases of the pALS coding sequence which is presumed to have occurred through a PCR error. In the present study, the correct coding sequence was synthesized by the site-directed mutagenesis and inserted into the pET-28a(+) plasmid expression vector containing the His-tag sequence flanking the last codon of the insert DNA. After induction of the expression construct in E. coli BL21(DE3) cells, the resulting presumptive recombinant peptide was purified by the Ni-affinity chromatography. Upon SDS- PAGE, the affinity-purified peptide was resolved as a single band at a 66-kDa position which is consistent with the expected molecular mass of the presumptive recombinant pALS. Collectively, results indicate that a recombinant pALS peptide was successfully expressed and purified in the present study.】

Adipocyte-specific secretory factor (ADSF)/resistin, a hormone, is a small cysteine-rich protein secreted from adipose tissue and has been implicated in modulating adipogenesis in humans and rodents. The objective of this study was to clone a gene encoding ADSF/resistin and to characterize its function in Korean Native Cattle (Hanwoo). The coding sequence was 330 base pairs and it encoded a protein of 109 amino acids. An NCBI BLAST-search revealed the cloned cDNA fragment shared significant homology (82%) with the cDNA encoding the human ADSF/resistin. The nucleotide sequence homology of the Hanwoo sequence was 73% and 64% for the rat and mouse, respectively. A 654 bp ADSF/resistin gene promoter was cloned and putative binding sites of transcription factors were identified. Tissue distribution of ADSF mRNA was examined in liver, skeletal muscles (tenderloin, biceps femoris), subcutaneous fat, and perirenal fat by RT-PCR. ADSF mRNAs were detected in fat tissues but not in liver and muscles, suggesting that ADSF/resistin expression may be induced during adipogenesis. Although, the physiological function of ADSF/resistin in the cow remains to be determined, these data indicate ADSF is related to the adipocyte phenotype and may have a possibly regulatory role in adipocyte function.

Myostatin is a negative regulator of skeletal muscle growth and a loss of functional myostatin protein increases muscle hypertrophy and hyperplasia in cattle. The present study was conducted to investigate whether maternal passive immunization against myostatin would improve growth performance in chickens. A complete broiler myostatin cDNA was cloned and it was expressed into two transcripts as 1,128 bp and 985 bp by alternative splicing. A conjugated mature myostatin (350 bp) was used to induce autoimmunization and maternal passively immunized chickens was used for the experiment. It was confirmed that there was a maternal passive immunization against myostatin at zero weeks of age, but its effect was reduced by 6 weeks of age. The auto-immunized groups showed smaller body weights than those of control group during the growing period and the difference was getting bigger with time until 6 weeks of age. These results suggest that passive autoimmunization against myostatin used in this study is not potent enough to stimulate growth performance in chickens. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 7 : 1017-1021)

Endoglucanase A from Clostridium thermocellum which is resistant to pancreatic proteinase was selected out of numbers cellulases then were expressed in lactobacilli. Recombinant lactobacilli expression vector, pSD1, harboring the endoglucanase gene from C. thermocellum under the control of its own promoter, was constructed. Both L. bulgaricus and L. plantarum were electrotransformed with pSD1. The endoglucanase activities of 0.120 and 0.144 U/ml were found in culture media of L. bulgaricus and L. plantarum containing pSD1, respectively. In vitro survival characteristics of the transformed lactobacilli were tested. Both L. bulgaricus and L. plantarum showed a similar resistance to low pH 3. Moreover, L. plantarum was bile-salt resistant in the presence of 0.3 and 1% oxgall. L. bulgaricus and L. plantarum showed a rather homogenous resistant pattern against the tested antibiotics. Both of the strains were resistant to amikacin, gentamicin, streptomycin, kanamycin, and colistin.

12 pages, 7 figures., Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5' deletions as well as site-specific mutations of the FAS promoter. We show that the -332 and -65 E-boxes are bound by USF in both fasted and refed mice, while the -150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the -150 SRE or the -65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the -150 SRE but, unexpectedly, of the -65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the -150 SRE and that USF binding to the -65 E-box is also required for SREBP binding and activation of the FAS promoter., The work was supported by NIH grant DK36264 to H.S.S.

In an attempt to isolate novel molecules that may play a regulatory role in adipocyte differentiation, we devised an experimental strategy to identify adipose tissue-specific genes by modifying cDNA microarray technique. We used genefilter membranes containing approximately 15,000 rat non-redundant EST clones of which 4,000 EST were representative clones of known genes and 11,000 ESTs were uncharacterized clones. A series of hybridization of genefilter membranes with cDNA probes prepared from various rat tissues and nucleic acids sequence analysis allowed us to identify two adipose-tissue specific genes, adipocyte-specific secretory factor (ADSF) and H-rev107. Verification of tissue-specific expression patterns of these two genes by Northern blot analysis showed that ADSF mRNA is exclusive expressed in adipose tissue and the H-rev107 mRNA is predominantly expressed in adipose tissue. Further analysis of gene expression of ADSF and H-rev107 during 3T3-L1 adipocyte differentiation revealed that the ADSF and H-rev107 gene expression patterns are closely associated with the adipocyte differentiation program, indicating their possible role in the regulation of adipose tissue development. Overall, we demonstrated an application of modified cDNA microarray technique in molecular cloning, resulting in identification of two novel adipose tissue-specific genes. This technique will also be used as a useful tool in identifying novel genes expressed in a tissue-specific manner. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 12 : 1837-1841)

Reversine has been reported to reverse differentiation of lineage-committed cells to mesenchymal stem cells (MSCs), which then enables them to be differentiated into other various lineages. Both adipocytes and osteoblasts are known to originate from common MSCs, and the balance between adipogenesis and osteogenesis in MSCs is reported to modulate the progression of various human diseases, such as obesity and osteoporosis. However, the role of reversine in modulating the adipogenic potential of lineage-committed preadipocytes and their plasticity to osteogenesis is unclear. Here we report that reversine has an anti-adipogenic function in 3T3-L1 preadipocytes in vitro and alters cell morphology and viability. The transforming growth factor-β (TGF-β) pathway appears to be required for the anti-adipogenic effect of reversine, due to reversine-induced expression of genes involved in TGF-β pathway and reversal of reversine-inhibited adipogenesis by inhibition of TGF-β pathway. We show that treatment with reversine transformed 3T3-L1 preadipocytes into MSC-like cells, as evidenced by the expression of MSCs marker genes. This, in turn, allowed differentiation of lineage-committed 3T3-L1 preadipocytes to osteoblasts under the osteogenic condition in vitro. Collectively, these findings reveal a new function of reversine in reversing lineage-committed preadipocytes to osteogenesis in vitro, and provide new insights into adipose tissue-based regeneration of osteoblasts.

The transcription of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis, is dramatically induced by fasting/refeeding and insulin. We reported that upstream stimulatory factor binding to the −65 E-box is required for induction of the FAS transcription by insulin in 3T3-L1 adipocytes. On the other hand, we recently found that two upstream 5′ regions are required for induction in vivo by fasting/refeeding and insulin; one at −278 to −131 albeit at a low level, and the other at −444 to −278 with an E-box at −332 where upstream stimulatory factor functions for maximal induction. Here, we generated double transgenic mice carrying the chloramphenicol acetyltransferase reporter driven by the various 5′ deletions of the FAS promoter region and a truncated active form of the sterol regulatory element (SRE) binding protein (SREBP)-1a. We found that SREBP participates in the nutritional regulation of the FAS promoter and that the region between −278 and −131 bp is required for SREBP function. We demonstrate that SREBP binds the −150 canonical SRE present between −278 and −131, and SREBP can function through the −150 SRE in cultured cells. These in vivo and in vitro results indicate that SREBP is involved in the nutritional induction of the FAS promoter via the −278/−131 region and that the −150 SRE is the target sequence.