HSV-1即刻早期基因的5′UTR对报告基因mRNA翻译的影响

mRNA疫苗的5′非翻译区(5’untranslated regions,5′UTR)对其表达起着重要作用,报告基因增强绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)用于检测5′UTR调控mRNA表达已经是一个较为成熟的实验方法.为研究单纯疱疹病毒1型(herpes simplex virus 1,HSV-1)即刻早期基因的5′UTR作为报告基因EGFP mRNA的5′UTR对其翻译的影响,本研究将4个即刻早期基因的5’UTR以及表达能力较强的人β珠蛋白基因(humanβGlobin,hBg)5′UTR插入报告基因EGFP 5’端,通过观察绿色荧光强度研究不同即刻早期基因5′UTR对mRNA翻译的影响.结果表明:经mRNA转染试剂和纳米脂质体颗粒分别递送进293T细胞和小鼠体内;RL2的5′UTR作为报告基因EGFP 5′UTR的表达水平与hBg 5′UTR相近,RS1、US1、US12表达水平明显弱于hBg 5′UTR.因此,RL2基因的5′UTR有潜力作为mRNA疫苗的5′UTR.

Predominant mucosal IL-8 mRNA expression in non-cagA Thais is risk for gastric cancer

AIM: To study gastric mucosal interleukine-8 (IL-8) mRNA expression, the cytotoxin-associated gene A (cagA) mutation, and serum pepsinogen (PG)?I/II ratio related risk in Thai gastric cancer.METHODS: There were consent 134 Thai non-cancer volunteers who underwent endoscopic narrow band imaging examination, and 86 Thais advance gastric cancer patients who underwent endoscopic mucosal biopsies and gastric surgery. Tissue samples were taken by endoscopy with 3 points biopsies. The serum PG?I, II, and Helicobacter pylori (H. pylori) immunoglobulin G (IgG) antibody for H. pylori were tested by enzyme-linked immunosorbent assay technique. The histopathology description of gastric cancer and non-cancer with H. pylori detection was defined with modified Sydney Score System. Gastric mucosal tissue H. pylori DNA was extracted and genotyped for cagA mutation. Tissue IL-8 and cyclooxygenase-2 (COX-2) mRNA expression were conducted by real time relative quantitation polymerase chain reaction. From 17 Japanese advance gastric cancer and 12 benign gastric tissue samples, all were tested for genetic expression with same methods as well as Thai gastric mucosal tissue samples. The multivariate analysis was used for the risk study. Correlation and standardized t-test were done for quantitative data, P value < 0.05 was considered as a statistically significant.RESULTS: There is a high non cagA gene of 86.8 per cent in Thai gastric cancer although there are high yields of the East Asian type in the positive cagA. The H. pylori infection prevalence in this study is reported by combined histopathology and H. pylori IgG antibody test with 77.1% and 97.4% of sensitivity and specificity, respectively. The serum PG?I/II ratio in gastric cancer is significantly lower than in the non-cancer group, P = 0.045. The serum PG?I/II ratio of less than 3.0 and IL-8 mRNA expression ≥ 100 or log10 ≥ 2 are significant cut off risk differences between Thai cancer and non-cancer, P = 0.03 and P < 0.001, respectively. There is a significantly lower PGI/II ratio in Japanese than that in Thai gastric cancer, P = 0.026. Serum PG?I/II ratio at cut off less than 3.0 and IL-8 mRNA expression Raw RQ > 100 or log10 > 2 are significantly difference between Thai cancer group when compared to non-cancer group, P = 0.013 and P < 0.001, respectively. In the correlation study, low PG?I/II ratio does not associate with chronic atrophic gastritis severity score in Thais non-cancer cases. However, there is a trend, but not significant convert correlation between IL-8 mRNA expression level and low PG?I/II ratio in Thai positive H. pylori infection. The high expression of IL-8 gene demonstrates a poorer prognosis by stage and histology.CONCLUSION:Predominant gastric mucosal IL-8 mRNA expression level, H. pylori infection, and low PG?I/II ratio are relative risks for Thai gastric cancer without correlation with cagA mutation.

Cloning and mRNA expression of macrophage migration inhibitory factor (MIF) gene of large yellow croaker (P seudosciaena crocea)

Mammalian macrophage migration inhibitory factor （MIF） plays an important role as an indispensable mediator in the pathogenesis of inflammatory disease like septicemia, but little is known about the role of MIF homologue in fish septicemia. The authors have cloned the MIF homologue in large yellow croaker Pseudosciaena crocea （LycMIF） using RACE approach. The full-length cDNA of LycMIF was 634 bases and contained an ORF of 345 bases encoding a protein of 115 amino acid residues. As demonstrated by RT-PCR and QRT-PCR assay, MIF mRNAs were constitutively expressed in 11 selected tissues and were abundant in brain and liver. Moreover, the LycMIF transcripts in the liver and head kidney were responsive to bacteria infection and could be significantly up-regulated. Our results provide the first direct evidence that fish MIF was implicated in pathogenesis of fish vibrosis and play an important role in response to bacteria infection.

奶牛妊娠中后期胎盘mRNA差异表达基因分析

奶牛的繁殖问题是制约我国奶业快速发展的瓶颈。为了研究奶牛妊娠中后期胎盘差异基因表达情况,探索mRNA在奶牛妊娠过程中的调控作用,本研究以奶牛妊娠中后期胎盘、非妊娠对照组子宫为对象,使用RNA-seq测序技术进行转录组分析。结果发现,与非妊娠对照组相比,妊娠中期和后期胎盘分别有4333和4354个基因的表达出现显著变化,其中上调的基因分别有2328和2519个,下调的基因分别有2005和1835个;778个差异基因只存在于妊娠中期,757个差异基因只存在于妊娠后期。GO分析结果显示,妊娠中后期差异表达的基因主要在定位、生物粘附、细胞连接、生殖等过程。KEGG分析结果显示,妊娠中后期差异表达的基因在ECM-受体相互作用、细胞粘附分子、细胞周期、粘着斑、PI3KAkt信号通路、癌症的途径以及蛋白质的消化吸收等通路。与非妊娠奶牛相比,妊娠中后期胎盘基因存在明显的差异表达,其中PLET1、CSH1、SULT1E1、PRP1、LPAR2、CSF1R、PDCD1等基因表达明显上调,推测其可能参与奶牛的妊娠调控,在一定程度上影响着妊娠的发生。本研究结果为深入挖掘奶牛妊娠中后期的分子调控机制提供了理论基础。

Loss of heterozygosity and mRNA expression at DCC locus in gastric cancer

AIM To assess the effects of the DCC gene changes on the development and progression of gastric cancer. METHODS The loss of heterozygosity (LOH) and mRNA expression (LOE) of DCC gene was studied in 51 surgical specimens of gastric cancer with PCR based detection. RESULTS LOH was found in 35 3% (18/51) of the specimens and it was more frequently detected in stages Ⅲ and Ⅳ cancer (50 5%) than in stages Ⅰ and Ⅱ (14 3%) ( P <0 05). The occurrence of LOH was not found to be correlated to the histological type, tumor size, invasion depth and lymph node metastasis of gastric cancer. Among the 51 cases, mRNA expression of DCC gene was studied in 26 cases of which LOE was found in 30 8% (8/26). LOE was not significantly correlated to LOH and other clinicopathological parameters. CONCLUSION LOH and LOE of DCC gene are frequently encountered in gastric cancer and LOH of DCC gene is a late event and associated with progression of gastric cancer.

Overexpression of sterol carrier protein-2 mRNA in patients with cholesterol gallstones

BACKGROUND: Hypersecretion of biliary cholesterol is believed to be one of the important causes of lithogenic bile. Sterol carrier protein-2 ( SCP2 ) participates in choles- terol trafficking and metabolism and may play a key role in cholesterol gallstone formation. This study was undertaken to investigate the expression of liver SCP2 mRNA in pa- tients with cholesterol gallstone and those patients with non-cholesterol gallstone. METHODS: The expression of liver SCP2 mRNA was studi- ed in 36 patients with cholesterol gallstone and 30 patients with non-cholesterol gallstone by reverse transcription-poly- merase chain reaction (RT-PCR). RESULT: The expression of SCP2 mRNA was increased more significantly in patients with cholesterol gallstone than in patients with non-cholesterol gallstone. CONCLUSION: The SCP2 gene was overexpressed in pa- tients with cholesterol gallstone, indicating that SCP2 may be one of the important causes of cholesterol gallstone.

Correlation of Helicobacter pylori and interleukin-8 mRNA expression in high risk gastric cancer population prediction

AIM:To evaluate(1) the association of the Helicobacter pylori(H.pylori) test and interleukin-8(IL-8) m RNA expression alone and the severity of gastric cancer(GC);(2) the association of both tests were added to patients' characteristics to identifli Thai suspected patients of gastric cancer who would receive the most benefit; and(3) diagnostic value of levels of IL-8 m RNA expression for gastric cancer.METHODS: A cross-sectional analytical study was completed with 220 patients with 86 GC patients who underwent endoscopy with gastric surgery divided into non-metastasis and metastasis groups,and 134 patients with benign lesions who underwent endoscopic examination,at the Gastrointestinal Surgery and Endoscopy Unit,Chiang Mai University Hospital between 2006 and 2010. Of 220 patients,86 cases of diagnosed gastric adenocarcinoma were in an advanced stage and 134 cases were non-cancer patients. RESULTS: The IL-8 m RNA expression showed predominant association with advanced GC when compared to H. pylori infection alone [OR(95%CI); 0.86(0.49-1.53) vs 5.44(3.08-9.62)] when including the patients' characteristics the highest of the area under the receiver operating characteristic curves(Au ROC) of the model were males older than 40 years of age [AuROC(95%CI); 0.81(0.75-0.86)]. However,preliminary testing for diagnostic indices of four cut-off points of IL-8 m RNA expression to predict the severity of GC cases found an increasing suboptimal trend from the likelihood ratio of positive to differentiate the severity in the GC group. The IL-8 mRNA expression showed a predominant association with GC when compared to H. pylori infection,especially in males older than 40 years of age who may benefit most from this test. CONCLUSION:The future research of IL-8 mRNA expression to predict severity in the gastric cancer group should be warranted.

The mRNA Expression and Methylation Status in Imprinting Control Region of H19 Gene Between Cattle-Yak and Their Parents

The H19 gene, which is imprinted with preferential expression from the maternal allele, was one of the first identified imprinting genes in mammals. Recent studies revealed that correct imprinting of the H19 gene plays a vital role in human spermatogenesis. To investigate whether imprinting defects were associated with the hybrid sterility of male cattle-yak, the methylation patterns of the H19 imprinting control region （ICR） and H19 mRNA expression in the testes of cattle-yak, yak, and cattle were examined. The results showed that the 3rd CCCTC-binding factor （CTCF） site of the H19 ICR was significantly hypomethylated in the testes of cattle-yak compared with yak or cattle. As expected, H19 was expressed at a significantly higher level in cattle-yak than in yak or cattle. These results suggest that imprinting defects of the CTCF- binding site in the HI9 ICR were possibly associated with disturbed spermatogenesis of male cattle-yak. Thus, we propose that disorders in H19 imprinting, resulting in an increased H19 mRNA expression, might contribute to the sterility of F1 male hybrids between cattle and yak.

cDNA cloning and mRNA expression of the translationally controlled tumor protein(TCTP)gene from Japanese sea perch(Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

Relationships of growth and mortality to enzymatic activity,and the relative mRNA expression of cultured scallops Patinopecten yessoensis in the Yellow Sea, China

The bottom aquaculture of yesso scallop (Patinopecten yessoensis) has developed rapidly in the north of Yellow Sea, China, but not without accompanying productivity problems. We, therefore, conducted field surveys to investigate factors related to growth and mortality rates of bottom-cultured scallops after release. Specifically, we focused on the effects of total antioxidant capacity (TAOC) and three key enzymes involved in antioxidant and metabolic function:glutamic pyruvic transaminase (GPT), lactic dehydrogenase (LDH), and superoxide dismutase (SOD). Across summer and winter, we also measured the relative mRNA expression of GPT and SOD to understand their seasonal variation in the scallops, along with how such variation correlated to growth and mortality. Results show that bottom cultured scallops experienced mass mortality during the first six months post-release. During winter (December), scallops grew more rapidly and suffered less mortality than in summer (August). The observed lower performance probably resulted from less advantageous environmental factors during the summer seasons, such as high temperature and low dissolved oxygen. These environmental stressors enhance protein consumption while decreasing energetic resources in scallops. Furthermore, scallops in summer exhibited high antioxidant levels that probably competed for energy with process integral to growth and survival. These negative factors combined to elevate mortality rates. In conclusion, we provided evidence suggesting correlations between metabolic/antioxidant activity and growth and mortality of bottom-cultured yesso scallops. These correlations implied us an accurate method to estimate the performance of bottom culture system. Suggestions about innovative aquaculture techniques were also discussed in the study. Our results might provide a possible guideline to the improvement of bottom culture techniques for this commercially valuable seafood species.

大鼠孕期暴露DBP对子代睾丸mRNA差异表达的影响

目的探讨大鼠孕期暴露DBP对子代睾丸mRNA差异表达的影响。方法选取清洁级SD成年鼠,雌雄比例12交配,确定怀孕后行DBP染毒,试验组灌胃剂量为500 mg/kg,同时设置对照组,每组8只;试验组老鼠染毒至哺乳期结束,饲养子代雄鼠60 d,解剖取睾丸组织,采用二代高通量测序技术对睾丸样本进行基因差异表达分析及筛选,对差异表达基因进行GO富集分析和KEGG富集分析。结果差异表达的基因共有1421个,其中,上调基因722个,下调基因699个。筛选出的差异表达基因GO功能富集分析显示,生物过程(Biological Process BP)中,差异表达基因与老化、细胞增殖、炎症反应等密切相关;细胞成分(Cellular Component CC)中,差异表达基因主要富集在内质网膜、细胞膜等部分;分子功能(Molecular Function MF)中,差异表达基因主要与钙离子结合、趋化因子活性等密切相关。KEGG富集结果显示,孕期暴露DBP对子代雄鼠的影响集中分布在TNF信号传导途径、类固醇生成、细胞黏附分子(CAMs)、醛固酮合成和分泌等通路。结论大鼠孕期DBP暴露可致雄性子代睾丸mRNA差异表达,影响生殖系统的通路主要有TNF信号传导途径、类固醇生成、细胞黏附分子(CAMs)、醛固酮合成和分泌等。

Effects of Dietary Lysine on Growth Performance, Serum Concentrations of Insulin-Like Growth Factor-I (IGF-I) and IGF-I mRNA Expression in Growing Rabbits

The effects of dietary lysine on production performance,serum concentrations of metabolites,growth hormone （GH）,insulin-like growth factor-I （IGF-I） and IGF-I mRNA expression in growing rabbits were examined.One hundred weaned New Zealand rabbits were allocated to individual cages and randomly offered a diet containing 5.5 （L1）,6.5 （L2）,7.5 （L3）,8.5 （L4）,or 9.5 g （L5） lysine per kg diet.The results showed that the average daily gain （ADG） of the rabbits from L3,L4 or L5 was higher than those from L1 or L2 （P 〈 0.05）.The feed gain ratio （F/G） in the rabbits from L4 or L5 was lower than those from L1 or L2 （P 〈 0.05）.Dietary lysine did not affect serum concentrations of total protein （TP）,glucose,insulin （INS）,and growth hormone （GH） （P 〉 0.05）.The quadratic effects of lysine on the serum urea nitrogen （SUN） concentration was detected （P = 0.035）.Serum IGF-I concentrations had a trend to increase quadratically with the increasing dietary lysine （P = 0.07）.A significant correlation was found between serum IGF-I concentrations （x,ng mL-1） and ADG （y,g kg-1）： y = -0.017x2 ＋ 1.984x ＋ 20.87 （R2 = 0.8982,P = 0.003）.The relative abundance of hepatic and muscular IGF-I mRNA tended to increase with increasing dietary lysine levels （P = 0.053 and 0.082,respectively）.Providing the diets mainly consisted of corn,wheat bran and peanut vine,the most appropriate dietary lysine level for growing meat rabbits from weaning to 70 d old was found to be 8.5 g kg-1,and IGF-I may be an important factor controlling growth of weaned rabbits.

The Expression Levels of Endogenous aFGF mRNA in Microwave Burn Wound Tissues and Its Clinical Significance

The expression levels and changes of endogenous acid fibroblast growth factor （aFGF） in microwave burn wound tissues were detected in order to investigate how to get better therapeutic effects by using the exogenous aFGF for repairing trauma. A burnt-wound animal model was established by NS-FⅡ multifunction spectrum therapeutics equipment, and reverse transcriptase-polymerase chain reaction （RT-PCR） and immunohistochemistry assay were applied to detect the expression levels of endogenous aFGF mRNA in microwave burn wound tissues. The expression level of endogenous aFGF mRNA was significantly increased in the burn wound tissues 12 h after burn, reached the peak at 48 h, and gradually deceased 96 h after burn. The expression of endogenous aFGF mRNA after tissue damage was reversible, and its intensity was in accordance with the repair process of tissue damage, suggesting endogenous aFGF may take part in the cell metabolism and proliferation, and then promote the repair of the burn wound.

Single Nucleotide Polymorphism of CYP3A4 Intron 2 and Its Influence on CYP3A4 mRNA Expression and Liver Enzymatic Activity in Human Liver

Summary： In adult liver, CYP3A4 plays an important role in the metabolism of a wide range of en- dogenous and exogenous compounds. To investigate whether there is a single nucleotide polymorphism （SNP） of CYP3A4 intron 2 in the liver and its effects on the mRNA expression and enzymatic activity of CYP3A4, genomic DNA was extracted from 96 liver tissue samples obtained from patients who had undergone liver surgery. An SNP of CYP3A4 intron 2 was identified by polymerase chain reaction （PCR）-single-strand confirmation polymorphism and DNA sequencing. The mRNA expression of CYP3A4 was determined by the fluorescence quantitative PCR technique. The enzymatic activity of CYP3A4 was measured using erythromycin and testosterone as probe substrates. Twelve patients were found to have the SNP/T4127G CYP3A4 within intron 2. The mRNA levels of CYP3A4 in wild-type and SNP/T4127G samples were 2.62±1.09 and 2.79±1.63, respectively （P〉0.05）. Erythromycin N-demethylase activity in wild-type and SNP/T4127G samples were 121.2±32.8 and 124.7±61.6 nmol·mg^-1min^-1, respectively （P〉0.05）. The activity of testosterone 613-hydroxylase was significantly different between wild-type （648±173 pmol·mg^-1·min^-1） and SNP/T4127G samples （540-4-196 pmol.mg-l-minl; P〈0.05）. In conclusion, the SNP/T4127G of CYP3A4 intron 2 exists in the liver. This SNP does not affect the mRNA expression of CYP3A4 but significantly decreases the hepatic micro- somal testosterone 613-hydroxylase activity of CYP3A4. Furthermore, this study indicates that the ap- propriate selection of probe substrates is very important in studying the relationship between the geno- type and phenotype of CYP3A4.

Progastrin-releasing peptide and gastrin-releasing peptide receptor mRNA expression in non-tumor tissues of the human gastrointestinal tract

AIM： To investigate the expression of gastrin-releasing peptide （GRP） and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology techniques. METHODS： Poly A^＋ mRNA was isolated from total RNA extracts using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA （sscDNA）. PCR amplifications were carried out using genespecific GRP and GRP-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene-specific GRP and GRP-receptor hybridization probes. RESULTS： Expression of GRP and GRP-receptor mRNA was detected at various levels in nearly all segments of the non-tumor specimens analysed, except the gallbladder. In most of the biopsy specimens, coexpression of both GRP and GRP-receptor mRNA appeared to take place. However, expression of GRP mRNA was more prominent than was GRP-receptor mRNA. CONCLUSION： GRP and GRP-receptor mRNAs are expressed throughout the gastrointestinal tract and provides information for the future mapping and determination of its physiological importance in normal and tumor cells.

PGC-1α differentially regulates the mRNA expression profiles of genes related to myofiber type specificity in chicken

Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)played a prominent role in regulating muscle fiber type transition and composition.However,the role of PGC-1αin chicken muscle has seldom been explored.To investigate the effect of PGC-1αon chicken skeletal muscles in this study,the PGC-1αgene was overexpressed or silenced in chicken primary myoblasts by using lentivirus,and then the effects of the PGC-1αgene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation.The results showed that overexpression of PGC-1αfrom proliferation to differentiation was accompanied by the up-regulated expression of Pax7,MyoD,and CnAα,which was significantly(P<0.01)increased after one day of transfection(1 I).The enhancement of MyoG,MEF2 c,and MyHC SM expression lagged,which was improved significantly(P<0.01)after four days of transfection(1 I3 D).Overexpression of PGC-1αdecreased(P<0.01)the MyHC FWM expression after four days of transfection(1 I3 D),and it had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM during myofiber formation.The effective silence(P<0.01)of PGC-1αby lentivirus mediating short hairpin RNA(shRNA)was detected after four days of transfection(1 I3 D)in cultures,and the lack of its function in chicken primary myoblasts significantly(P<0.01)down-regulated the expression of Pax7,MyoD,CnAα,MyoG,MEF2 c,and MyHC SM,significantly(P<0.01)up-regulated the expression of MyHC FWM,and had no significant impact(P>0.05)on the expression of CnB1,NFATc3,and MyHC FRM.These results indicated that the role of PGC-1αin regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals,which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.

Effects of arginine supplementation on splenocyte cytokine mRNA expression in rats with gut-derived sepsis

AIM： To investigate the effects of arginine （Arg）-enriched diets before sepsis and/or Arg-containing total parenteral nutrition （TPN） after sepsis or both on cytokine mRNA expression levels in splenocytes of rats with gut-derived sepsis. METHODS： Rats were assigned to four experimental groups. Groups 1 and 2 were fed with a semipurified diet, while groups 3 and 4 had part of the casein replaced by Arg which provided 2% of the total calories. After the rats were fed with these diets for 10 d, sepsis was induced by cecal ligation and puncture （CLP）, at the same time an internal jugular vein was cannulated. All rats were maintained on TPN for 3 d. Groups 1 and 3 were infused with conventional TPN, while groups 2 and 4 were supplemented with Arg which provided 2% of the total calories in the TPN solution. All rats were killed 3 d after CLP to examine their splenocyte subpopulation distribution and cytokine expression levels. RESULTS： Plasma interleukin （IL）-2, IL-4, tumor necrosis factor-α （TNF-α） and interferon （IFN-γ） were not detectable 3 d after CL.P. There were no differences in the distributions of CD45Ra＋, CD3＋, CD4＋, and CD8＋ cells in whole blood and splenocytes among the four groups. The splenocyte IL-2 mRNA expression in the Arg-supplemented groups was significantly higher than that in group 1. IL-4 mRNA expression in groups 3 and 4 was significantly higher than that in groups 1 and 2. The mRNA expression of IL-10 and IFN-γ was significantly higher in group 4 than in the other three groups. There was no difference in TNF-α mRNA expression among thefour groups CONCLUSION： The influence of Arg on the whole blood and splenic lymphocyte subpopulation distribution is not obvious. However, Arg administration, especially before and after CLP, significantly enhances the mRNA expression levels of Thl and Th2 cytokines in the spleen of rats with gut-derived sepsis.

Guipi decoction effects on brain somatostatin levels and receptor mRNA expression in rats with spleen deficiency

BACKGROUND： Somatostatin is abundant in the hypothalamus, cerebral cortex, limbic system, and mesencephalon. Somatostatin mRNA expression in the brain of rats with spleen deficiency is noticeably reduced, as well as attenuation of cognitive function. OBJECTIVE： To observe the interventional effect of Guipi decoction on somatostatin level and somatostatin receptor 1 （SSTRl） mRNA expression in different encephalic regions of rats with spleen deficiency, and to compare the interventional effects of Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet. DESIGN： A randomized controlled observation. SETTING： Basic Medical College, Beijing University of Traditional Chinese Medicine. MATERIALS： Fifty adult Wistar male rats, of clean grade, weighing （160 ± 10） g, were provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Somatostatin 1 polyclonal anti-rabbit antibody and SSTRl in situ hybridization kit were provided by Department of Neuroanatomy, Shanghai Second Military Medical University of Chinese PLA. The drug for developing rat models of spleen deficiency was composed of Dahuang, Houpu and Zhishi, and prepared at 2：1：1. Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet recipes were made according to previous studies. METHODS： This study was performed at the Basic Medical College, Beijing University of Traditional Chinese Medicine from March 2002 to March 2005. The rats were randomly divided into 5 groups, with 10 rats in each group： normal, model, Guipi decoction, Chaihu Shugan powd.er, and Tianwang Buxin pellet groups. Rat models of the latter 4 groups were developed by methods of purgation with bitter and cold nature drugs, improper diet, and overstrain. The rats received 7.5 g/kg of the drugs each morning and were fasted every other day, but were allowed free access to water at all times. The rats were forced to swim in 25 ℃ water until fatigued. Rats in the normal group were intragastrically administered the same amount of normal saline. Rats in the Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet groups were intragastrically administered 7.5 g/kg Guipi decoction, Chaihu Shugan powder, and Tianwang Buxin pellet, respectively, every afternoon. All rats were treated for 6 weeks. MAIN OUTCOME MEASURES： Somatostatin protein and SSTRI mRNA expression in the ventral nucleus of hypothalamus, hippocampal CAl region, and cortex of prefrontal lobe were determined by immunohistochemistry and in situ hybridization, respectively. RESULTS： Fifty rats were included in the final analysis. In the model group, expression of somatostatin protein and SSTRl mRNA in the ventral nucleus of hypothalamus, hippocampal CAl region, and cortex of prefrontal lobe were significantly less than in the normal group （P 〈 0.01）. Above-mentioned indices were identical in the Chaihu Shugan powder and model groups. However, expression of somatostatin protein and SSTRl mRNA were significantly higher in the Guipi decoction group compared to model group （P 〈 0.01）. In the Tianwang Buxin pellet group, SSTRl mRNA expression in rat ventral nucleus of hypothalamus and somatostatin level in rat hippocampal CAl region and cortex of prefrontal lobe, as well as ventral nucleus of hypothalamus, were significantly higher compared to model group （P 〈 0.01 ）. CONCLUSION： Somatostatin level and SSTRl mRNA expression in rats with spleen deficiency were lower than in normal rats. Guipi decoction and Tianwang Buxin pellet up-regulated somatostatin level and SSTRl mRNA expression.

Expression of cytokine mRNA during immuno-modulation of murine suppressor macrophages

In order to analyze the mechanism of immunomodulation by LPS on murine peritoneal suppressor macrophages, we have, using RNase protection assay,checked the changes of mRNA expression pattern of several cytokine genes during the immuno-modulation.It has been found that, after treating peritoneal suppressor macrophages with LPS, mRNAs of IL-12 p35, IL-12 p40,IL-6 and IFN-γ are newly appeared, while those of IL-1α, IL-1β and IL-1Ra are increased and those of other cytokines, like TGF-β1 and MIF are not changed at all.It seems certain that those cytokines, whose expression is increased by LPS stimulation, may be responsible for the functional changes of suppressor macrophages during immuno-modulation. Among these changes, the appearance of IL-12 mRNA may play a critical role, and, in this regard, the synergetic effect between IFN-γ and LPS on the increase of IL-12 p35 and IL-12 p40 mRNA expression is an interesting finding.

Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.