Isolation, Identification and Tissue Expression Profile Analysis of One Novel Differentially Expressed Sequence Tag in the Longissimus dorsi Muscle from Meishan, Meishan × Large White Hybrid and Large White Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differential display technique was performed to investigate the differences of gene expression in the Longissimus dorsi tissue from Meishan, Meishan × Large White hybrid and Large White pigs with nine 3'-end anchored primers in combination with ten 5'-end arbitrary primers and nearly 3000 reproducible bands were examined. One novel expressed sequence tag (EST4, GenBank accession number: AY553914) that was differentially expressed in Meishan, Meishan× Large White hybrid and Large White pigs was isolated from the Longissimus dorsi muscle tissue and identified through semi-quantitative RT-PCR. BLAST analysis revealed that the 350 bp long EST (EST4) was not homologous to any of the known porcine genes. Tissue expression profile analyses showed that the EST4 was expressed in most of tissues.LIU Yong-gang, Ph D candidate

Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

Protein tyrosine phosphatases（PTPs） are crucial regulators of signal transduction. Among them, PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts. Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting anti- serum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.

Porcine NF-κB p65 Subunit:Molecular Characterization,Tissue Expression and Transcriptional Profile in Porcine Epidemic Diarrhea Virus-infected IPEC-J2 Cells

The p65 protein is a functional subunit of NF-κB family and exhibits a crucial role in host immune and inflammatory responses,apoptosis and tumor proliferation if improperly-regulated.Given its ubiquitous association with nearly all the animal cells and its pleotropic functions,the gene encoding NF-κB p65 subunit was cloned and sequenced from porcine kidney(PK-15)cells.The gene was 1662 bp in length,encoded a 553-amino acid protein and contained the prototypical NF-κB functional domains.Real-time quantitative RT-PCR and Western blot were used to characterize the transcription and expression levels of the p65 in different pig tissues.The results indicated that the p65 gene and protein were both broadly expressed in pig tissues,but most highly expressed in the intestine-associated lymph nodes and the lungs.To localize the recombinant protein in intestinal porcine epithelial cells(IPEC-J2),the gene was subcloned into the vector pEGFP(pEGFP-p65).Using fluorescence microscopy,the protein was found confined to the cytoplasm in normal cells;however,during porcine epidemic diarrhea virus(PEDV)infection,mRNA and protein expression were significantly up-regulated and the protein exhibited an overt tendency for nuclear translocalization consistent with a regulatory role in antiviral innate immunity.

Tissue expression and immunolocalization of cellular repressor of E1A-stimulated gene in postinfarction dysfunctional myocardium

Background Cellular Repressor of E1A-stimu-lated gene(CREG) is widely expressed in adult tissues such as the brain,heart,lung,liver,intestine and kidney in mice.It is not known whether tissue CREG is decreased in the common setting of myocardial infarction which may lead to heart failure.We studied the expression and protein localization of CREG and its main receptor(IFR2R) in a mouse model of myocardial infarction.Methods Male mice were randomized to proximal left anterior descending ligation.The animals were killed on day 1,3,7,14,and 28 after ligation to examine gene expression and protein production of CREG and IGF2R from the infarct,peri-infarct,and contralateral zones of infarcted heart.Results There was decreased CREG mRNA production throughout the myocardium at dav 1,and the expression gradually increased at day 28 after myocardial infarction.The decreased expression of this glycoprotein was not confined strictly to the infarct or peri-infarct zones but also expressed by cardiac myocytes within the myocardium in the contralateral normal zone.Levels of CREG protein in the infarct and peri-infarct zones declined to 1/3- to 1/2-fold of normal levels and declined to 1/2- to 2/3- fold in the contralateral zone.Finally,the expression of the IGF2R mRNA transcripts was downregulated at day 3 and 7 after ligation in the infarct and peri-infarct zones,suggesting that the signal transduction pathways necessary for CREG in the heart remain intact as CREG biosynthesis decreases. Conclusions CREG is constantly present in a model of large myocardial infarction and is decreased at the early stage within the myocardium.The decreased expression of this glycoprotein is not only confined strictly to the infarct or periinfarct zone but also is expressed by cardiac myocytes within the myocardium contralateral to the infarct.Therefore CREG production decreased due to myocardial stress response to injury.

Vitamin D receptor expression in human bone tissue and dose-dependent activation in resorbing osteoclasts

The effects of vitamin D on osteoblast mineralization are well documented. Reports of the effects of vitamin D on osteoclasts, however, are conflicting, showing both inhibition and stimulation. Finding that resorbing osteoclasts in human bone express vitamin D receptor （VDR）, we examined their response to different concentrations of 25-hydroxy vitamin D3 [25（OH）D3] （100 or 500 nmol·L^-1） and 1,25-dihydroxy vitamin D3 [1,25（OH）2D3] （0.1 or 0.5 nmol·L^-1） metabolites in cell cultures. Specifically, CD14＋ monocytes were cultured in charcoal-stripped serum in the presence of receptor activator of nuclear factor kappa-B ligand （RANKL） and macrophage colony-stimulating factor （M-CSF）. Tartrate-resistant acid phosphatase （TRAP） histochemical staining assays and dentine resorption analysis were used to identify the size and number of osteoclast cells, number of nuclei per cell and resorption activity. The expression of VDR was detected in human bone tissue （ex vivo） by immunohistochemistry and in vitro cell cultures by western blotting. Quantitative reverse transcription-PCR （qRT-PCR） was used to determine the level of expression of vitamin D-related genes in response to vitamin D metabolites. VDR-related genes during osteoclastogenesis, shown by qRT-PCR, was stimulated in response to 500 nmol·L^-1 of 25（OH）D3 and 0.1-0.5 nmol·L^-1 of 1,25（OH）2D3, upregulating cytochrome P450 family 27 subfamily B member I （CYP27B1） and cytochrome P450 family 24 subfamily A member I （CYP24A1）. Osteoclast fusion transcripts transmembrane 7 subfamily member 4 （tm7sf4） and nuclear factor of activated T-cell cytoplasmic 1 （nfatcl） where downregulated in response to vitamin D metabolites. Osteoclast number and resorption activity were also increased. Both 25（OH）D3 and 1,25（OH）2D3 reduced osteoclast size and number when co-treated with RANKL and M-CSF. The evidence for VDR expression in resorbing osteoclasts in vivo and low-dose effects of 1,25（OH）2D3 on osteoclasts in vitro may therefore provide insight into the effects of clinical vitamin D treatments, further providing a counterpoint to the high-dose effects reported from in vitro experiments.

Interventional effect of hirudin on the expression of microtubule-associated protein 2 in peripheral tissue of hematom of model rats with acute intracerebral hemorrhage

BACKGROUND： It is suspected that dissociation, destruction or synthetic disorder of microtubule-associated protein 2 （MAP-2） may participate in secondary injury of intracerebral hemorrhage （ICH）, and the reason may be related to thrombin in high concentration after ICH; therefore, the mechanism should be studied further. OBJECTIVE： To explore the effect of hirudin on expression of MAP-2 in peripheral tissue of hematom after ICH and changes of water content in brain tissue and analyze pathogenesis of thrombin in secondary injury after ICH. DESIGN ： Completely randomized grouping design and controlled animal study SEn-ING ： Department of Neurology, the First Affiliated Hospital of Jilin University MATERIALS ： The experiment was carried out in the Neurological Laboratory of the First Affiliated Hospital of Jilin University from April 2003 to April 2004. A number of 80 healthy Wistar rats, of both genders, aged 3-4 months, weighing 250-350 g, were randomly divided into 8 groups： normal control group, 6-hour ICH group, 1-day ICH group, 2-day ICH group, 3-day ICH group, 7-day ICH group, 3-day hirudin group and 7-day hirudin group with 10 in each group. Five rats from each group were selected to measure their water content, and the others were undertaken immunohistochemical stain. Hirudin was produced by Sigma Company, USA, and MAP-2 rabbit-rat polyclonal antibody was provided by Fuzhou Maixin Biotechnology Company Limited. METHODS： ① Model establishing and grouping intervention： Rats in simple ICH group were collected their blood from tails and then inserted with 50 μL non-anticoagulant auto-arterial blood into the cauda of the putamen in right brain within 5 minutes. Rats in hirudin groups were inserted with 10 U hirudin （which was diluted with saline to 20 μL） into local hematom regions within 5 minutes, and the needle was pulled out after 10 minutes. Rats in normal control group were untouched. ② Water content in peripheral tissue of hematom： Based on the ratio between dry weight and wet weight, brain tissue at bleeding side and in right frontal lobe was selected to measure dry and wet weights so as to calculate the water content [（wet weight - dry weight） /wet weight] × 100%.③ Positive expression of MAP-2： Based on immunohistochemical stain, positive MAP-2 cells were regarded as neurons and they were buffy morphological. Positive rate of MAP-2 was calculated, i.e., percentage of positive cells in each sight to total cells in all sights. ④ Statistical analysis： Data among groups were compared with one-way analysis of variance, averages were compared with SNK-q test by each other, and relation between water content and MAP-2 was analyzed with linear regression technique. MAIN OUTCOME MEASURES： Changes of water content and MAP-2 expression in peripheral tissue of hematorn at various time points after ICH and intervention of hirudin. RESULTS： All 80 rats were involved in the final analysis. ①Water content： Water content was increased at day 1, reached peak at day 3 and decreased at day 7. It was （72.31±0.32）%, （77.42±0.53）%, （78.44±0.28）%, （74.10±0.13）%, （74.85±0.51）% and （70.07±0.36）%, respectively in 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was higher than that in normal control group （63.85±0.41, q=-4.684 3 to -7.262 0, P〈 0.05）; that in 2-day and 3-day ICH groups was higher than that in 7-day ICH group （q=-3.053 4, -3.727 0, P 〈 0.05）; and that in 3-day and 7-day ICH groups was higher than that in hirudin groups at the same time points （q=-2.965 6, -2.726 4, P 〈 0.05）. ②Positive expression of MAP-2： Positive expression of MAP-2 was decreased at 6 hours after ICH, reached the lowest value at day 3 and increased at day 7. Positive rate was （78.60±0.42）%, （60.56±0.74）%, （44.60±0.26）%, （25.45±0.85）%, （32.55±0.64）%, （37.69＋0.76）%, （41.75±0.68）%, respectively in 6-hour, 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was lower than that in normal control group [（96.50±0.33）%, q= -3.074 5 to -8.128 5, P 〈 0.05]. In addition, positive cells of MAP-2 disappeared plentifully at 3-7 days after ICH, stain of positive cells were light, and only stain of plasma was positive. That in 3-day and 7-day hirudin groups was higher than that in ICH groups at the same time points （q= -3.391 8, -2.967 9, P 〈 0.05）. Moreover, positive cells of MAP-2 was formed slightly but deeply stained. ③ Results of linear regression： Water content was negatively related to MAP-2 changes at 7 days after ICH （r= -0.894 9, P〈 0.01）, i.e., water content was increased with decrease of MAP-2 expression. CONCLUSION ： The deterioration of MAP-2 may be involved in the pathogenesis of thrombin within the first week after ICH, and the local administration of hirudin can protect neurons.

Electro-acupuncture for STAT3 expression and nuclear translocation in hippocampal tissues of rats following cerebral ischemia/reperfusion

BACKGROUND: It has been found in recent years that STAT3 widely distributes in nervous system, including hippocampal CA1-3 region, dentate gyrus and cerebral neocortex, etc. Ischemic brain injury can cause the release of some cytokines and growth factors, while electro-acupuncture may have multi-level, multi-channel and multi-target protective and interventional effects on ischemic brain injury. OBJECTIVE: To observe the effects of electro-acupuncture on STAT3 expression and nuclear translocation in hippocampal CA1 region of rat models of brain ischemia/reperfusion. DESIGN: Randomized and controlled observation. SETTING: Staff Room of Acupuncture and Moxibustion, Department of Acupuncture and Bone Injury, Hubei College of Traditional Chinese Medicine; Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Seventy-two healthy SD rats, of clean degree and either gender, weighing (200±20) g, were provided by the Experimental Animal Center of Hubei College of Traditional Chinese Medicine. STAT3 monoclonal antibody was purchased from Santa Cruz Company, USA, and G-6805 electro-acupuncture instrument was purchased from Shanghai Medical Electronic Instruments Factory. METHODS: This experiment was carried out in the comprehensive laboratory of Department of Acupuncture and Bone Injury, Hubei College of Traditional Chinese Medicine between September 2005 and February 2006. Seventy-two rats were randomly divided into 4 groups: ① control group(n =6): Untouched. ② Sham-operation group (n =18): Artery was isolated, but without inserting thread bolt.③ Model group (n =24): Rat models of local brain ischemia/reperfusion were established with modified suture occlusion. ④Electro-acupuncture group (n =24): Dazhui and bilateral Neiguan points were selected for electro-acupuncture treatment. No. 28 acupuncture needle of 3.33 cm was used in the treatment. A G-6085 electro-acupuncture instrument with continuous wave, frequency of 120 times/min, intensity of 1 mA, 30 min/time, was used. Acupuncture was conducted firstly at ischemia/reperfusion 3 hours, then once every 12 hours. STAT3 positive nuclear translocation in hippocampal CA1 region of rats was observed with immunohistochemical method at 24, 48 and 72 hours after brain ishcemia/reperfusion, and then STAT3 positive cells were counted. MAIN OUTCOME MEASURES: STAT3 positive cells and nuclear translocation in hippocampal CA1 region of rats in each group. RESULTS: All the 72 rats were involved in the result analysis. ①In the control group and sham-operation group, STAT3 positive cells with light cytoplasm and nucleus were decreased , and nuclear translocation was not found. ② In the model group, STAT3 positive cells were mostly found in the cytoplasm of the hippocampal CA1 region at the ischemic side of rats after ischemia/reperfusion 24 hours. They were significantly more than those in the sham-operation group and control group [(18.00±2.68), (9.00±1.35), (8.00±1.22) cells/ mm2, P < 0.01], but cells with nuclear reaction were fewer; At ischemia/reperfusion 48 and 72 hours, STAT3 positive cells were increased, and they were significantly more than those of sham-operation group [(25.00±3.23), (35.00±3.52) cells/mm2, (13.00±1.93), (12.00±1.24) cells/mm2, P < 0.01]. Positive cells with nuclear reaction were found dark-stained. ③At ischemia/reperfusion 24, 48 and 72 hours, STAT3 positive cells were strongly expressed in hippocampal CA1 region at ischemic side of rats of electro-acupuncture group, and they were significantly more than those of model group [(25±3.52), (50±6.31), (75±8.09) cells/mm2, P < 0.01]. STAT3 positive cells were gradually enhanced with time, and considerable STAT3 nuclear positive reaction cells were found. CONCLUSION: Electro-acupuncture can activate STAT3 protein expression in hippocampal tissue of rats with local brain ischemia/reperfusion, promote STAT3 nuclear translocation and function its neuroprotective effect.

Effect of ketamine on aquaporin-4 expression and neuronal apoptosis in brain tissues following brain injury in rats

BACKGROUND： Aquaporin-4 （AQP-4） is closely related to the formation of brain edema. Neuronal apoptosis plays an important part in the conversion of swelled neuron following traumatic brain injury. At present, the studies on the protective effect of ketamine on brain have involved in its effect on aquaporin-4 expression and neuronal apoptosis in the brain tissues following brain injury in rats. OBJECTIVE： To observe the effect of ketamine on AQP-4 expression and neuronal apoptosis in the brain tissue following rat brain injury, and analyze the time-dependence of ketamine in the treatment of brain injury.DESIGN： Randomized grouping design, controlled animal tria SETTING ： Department of Anesthesiology, the Medical School Hospital of Qingdao University MATERIALS： Totally 150 rats of clean grade, aged 3 months, were involved and randomized into control group and ketamine-treated group, with 75 rats in each. Each group was divided into 5 subgroups separately at 6,12, 24, 48 and 72 hours after injury, with 15 rats at each time point. Main instruments and reagents： homemade beat machine, ketamine hydrochloride （Hengrui Pharmaceutical Factory, Jiangsu）, rabbit anti-rat AQP-4 polyclonal antibody, SABC immunohistochemical reagent kit and TUNEL reagent kit （Boster Co.,Ltd., Wuhan）. METHODS： This trial was carried out in the Institute of Cerebrovascular Disease, Medical College of Qingdao University during March 2005 to February 2006. A weight-dropping rat model of brain injury was created with Feeney method. The rats in the ketamine-treated group were intraperitoneally administered with 50 g/L ketamine （120 mg/kg） one hour after injury, but ketamine was replaced by normal saline in the control group. In each subgroup, the water content of cerebral hemisphere was measured in 5 rats chosen randomly. The left 10 rats in each subgroup were transcardiacally perfused with ketamine, then the brain tissue was made into paraffin sections and stained by haematoxylin and eosin. Neuronal morphology was observed. AQP-4 expression and neuronal apoptosis were measured with immunohistochemical method and TUNEL method respectively. MAIN OUTCOME MEASURES： Water content in brain tissue, neuronal morphology, the number of AQP-4 positive neurons and TUNEL positive neurons in rats of two groups at each time point after injury. RESULTS： Totally 150 rats entered the stage of result analysis. （1） Water content of brain tissue： The water content of brain tissue at each time point after injury in the ketamine-treated group was lower than that in the control group. There were very significant differences in water content at 12 and 24 hours after injury respectively between ketamine-treated group and control group [（77.34±2.35）% vs. （82.31 ±1.48）%; （78.01 ±2.21 ）% vs. （83.86±2.37）%, t=-4.001 6,4.036 7, both P 〈 0.01]. （2） Neuronal morphology： Pathological changes in traumatic region and peripheral region of injury in the ketamine-treated group were significantly lessened, and necrotic and apoptotic cells in the ketamine-treated group were also significantly reduced as compared with control group. （3） AQP-4 expression： AQP-4 positive neurons at each time point in the ketamine-treated group were significantly less than those in the control group. There were very significant differences in AQP-4 expression at 12 and 24 hours after injury between ketamine-treated group and control group [（34.17±4.74） /visual field vs. （43.42±5.65） /visual field;（40.83±3.17） /visual field vs. （58.88±6.23） /visual field,t=3.966 3,8.165 7, both P〈 0.01]. （4） Neuronal apoptosis： TUNEL positive neurons at each time point in the ketamine-treated group were less than those in the control group. There were very significant differences in the neuronal apoptosis at 12 and 24 hours after injury between ketamine-treated group and control group [（26.25±3.04） /visual field vs. （32.75±4.39） /visual field; （29.33± 4.02） /visual field vs. （39.83±5.61） /visual field,t=-3.849 3,5.169 2,both P 〈 0.01]. CONCLUSION： Ketamine can reduce brain edema, AQP-4 expression and neuronal apoptosis following brain injury in rats, and has obvious therapeutic effect on brain injury, especially at 12 and 24 hours after injury.

IN SILICO EXPRESSION ANALYSIS OF HUMAN NOVEL GENE UBAP1 IN MULTIPLE CANCERS

Objective: To identify the differential expression profile of human novel gene UBAP1, a putative nasopharyngeal neoplasms (NPC) relate gene, in multiple cancers. Methods: We first present an EST approach for electronic Northern in silico to analyse expression patterns of UBAP1 in tumor and normal tissues. Full length cDNA of UBAP1 gene was taken as a “probe” sequence, and a blastn search was performed against human EST Database. The Blastn report can be used to determine the fold differences between the pedigree ESTs in different libraries. Especially, the ESTs corresponding to UBAP1 present in fifteen tumor-derived libraries were compared against their normal counterpart to produce an electronic differential expression profile. Second, the distinct down-regulation of UBAP1 in meningioma, glioma, and colorectal tumor was confirmed by differentially RT-PCR analysis. Results: Database surveys indicated that UBAP1 gene was not only ubiquitously expressed in many normal tissues with various levels but also differentially expressed in different tumor tissues, especially down-regulated in multiple neoplastic tissues such as brain, breast, skin, colon, testis and uterus tumor tissues. Furthermore, differential RT-PCR analysis demonstrated that expression of UBAP1 was down-regulated or absent in 7 of 12 (58%) meningioma samples, 6 of 9 (66%) glioma and 7 of 11 (63%) colorectal tumor tissues respectively. Conclusion: we described a data mining procedure in silico that proved to be useful for the identification of differential expression patterns of UBAP1. These findings could be valuable for the investigation of the mechanism the differential expression of UBAP1 gene and its significance in the progression of multiple cancers.

Characterization,Imprinting Status and Tissue Distribution of Porcine GTL2 Gene

The callipyge （CLPG） phenotype, exhibiting polar overdominance （POD）, is an inherited skeletal muscle hypertrophy described in sheep. The callipyge locus maps to the distal portion of ovine chromosome 18 within the DLKI-GTL2 region and corresponds to human chromosome 14 and mouse chromosome 12. The POD phenomenon is confirmed to the homologous region of swine chromosome 7. In order to clone and investigate the expression of porcine GTL2 gene, DNA and RNA samples from 60-day-old F1 animals, generated with reciprocal crosses between Large White and Meishan breeds and their parents, were used. The authors showed that porcine GTL2 acted as a uoncoding RNA. cDNA samples exhibited maternal expression of the gene in the heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, and fat in pigs, and a unique tissue-specific expression different from that of humans and mice. These results indicated that the gene was conserved in the pig, human, mouse, and bovine. It will be of interest to further study the gene functions in muscle growth and fat deposition.

Characterization of T-complex polypeptide 1 (TCP-1) from the Chilo suppressalis HSP60 family and its expression in response to temperature stress

Many proteins require assistance from molecular chaperones at various stages to attain correctly folded states and functional conformations during protein synthesis. In this study, the gene encoding T-complex polypeptide 1（TCP-1）, which belongs to the heat shock protein 60（HSP60） family, was isolated and characterized from the rice stem borer, Chilo suppressalis, by RACE and q PCR, respectively. The full-length c DNA of Tcp-1 was 2 144 bp and encoded a 1 635-bp ORF; the deduced translational product contained 545 amino acids with 5′-and 3′-UTRs and an isoelectric point of 5.29. Cluster analysis confirmed that the deduced amino acid sequence shared high identity（60–99%） with TCP-1 from other insects. To investigate Tcp-1 expression in response to abiotic stress, q PCR was used to analyze expression levels of Tcp-1 m RNA in C. suppressalis larvae exposed to temperatures ranging from –11 to 43°C. With respect to heat shock, Tcp-1 expression was higher than the control after a 2-h exposure to 30 and 36°C and declined at 39 and 43°C. Difference in Tcp-1 expression was observed at temperatures ranging from –11 to 27°C. q PCR analyses revealed that Tcp-1 expression was the highest in hindgut tissue as compared to heads, epidermis, fat body, foregut, midgut, and malpighian tubules. Our results indicated that Tcp-1 expression was differentially expressed in C. suppressalis tissues, and was impacted by temperature stress.

Effects of sodium arsenite on the expression of microRNA-191 and tissue inhibitor of metalloproteinase 3 in L-02 cells

Objective To investigate the effects of sodium arsenite（NaAsO2）on the expression of microRNA-191（miR-191）and tissue inhibitor of metalloproteinase 3（TIMP-3）in human normal hepatic cells（L-02 cells）.Methods L-02 cells were exposed to different doses of Na2As O2[0（control group）,5,25,50 and 75μmol/L]

Molecular characterization and expression patterns of nuclear androgen receptors in the ovoviviparous black rockfish Sebastes schlegelii

nARs are ligand-activated transcription factors associated with gonadal development and reproductive regulation.In this study,two nuclear androgen receptor genes(ara and arb)were cloned from an ovoviviparous teleost,black rockfish(Sebastes schlegelii).The phylogenetic analysis of nARs showed that nARαand nARβclustered into teleost nARαand nARβ,respectively,but differed from the nARs of tetrapods.Four module domains of the nuclear receptor superfamily are present in the black rockfish nARs,an N-terminal domain(NTD),a DNA-binding domain(DBD),a hinge region(HR)and a ligand-binding domain(LBD).Among the four domains,only the DBD and LBD of nARαand nARβare relatively conserved.Tissue distribution analysis revealed that ara was mainly expressed in the gonad,muscle,intestine and kidney in males and in female,while arb was mainly detected in the gonad,followed by the intestine,kidney and head kidney.During the gonadal development process,the expression levels of ara and arb were significantly decreased from the regenerating to late stages of spermatogenesis and significantly increased in the degeneration stage in the testis(P<0.05).In the ovary,the expression levels of ara and arb were not significantly different in different stages.In situ hybridization revealed that in black rockfish,both ara and arb transcripts were localized to the Sertoli cells of the testis in black rockfish.In general,the present study is the first to characterize and analyse the expression of nuclear androgen receptors,including ara and arb,in black rockfish.

Identification and expression of a novel human testis-specific gene by digital differential display

Background Evidence for the importance of genetic factors in male infertility is accumulating This study was designed to identify a novel testis specific gene related to spermatogenesis by a new strategy of digital differential display (DDD) Methods Based on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene Multi tissue RT PCR was performed to analyse its tissue specific expression The full length cDNA of the new gene was obtained using the BLAST program Sequencing was performed and the result was analysed Semi quantitative RT PCR and Northern blot analyseis of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene The sequence of the opening reading frame was integrated into the pQE 30 vector expressed in Escherichia coil strain M15(pREP4) With IPTG induction, the target protein was detected Results A full length cDNA sequence of the new gene named SPATA12 (GeneBank accession number AY221117) in human testis was identified SPATA12 was 2430 bp in length, located in chromosome 3p21 1 3p21 2 The sequence of the opening reading frame was 676-1248 bp, as was confirmed by RT PCR and sequencing The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20 417 8 and isoelectric point of 5 23 The sequence has no significant homology with any known protein in databases Semi quantitative RT PCR and Northern blot analyses of multiple tissues showed that SPATA12 was expressed significantly in normal human testis The expression recombinant of SPATA12 was constructed and a high level of the histidine tagged fusion protein was obtained Conclusions DDD can be confirmed by SPATA12 as a novel computational biology based approach for identification of the testis specific expression genes SPATA12 may function as a testicular germ cell associated gene that plays some roles in spermatogenesis Moreover, a great amount of SPATA12 protein could be obtained by the gene recombination technique, thus providing a reliable foundation for investigating the biological function of this new protein

A novel cytochrome P450 gene from Catharanthus roseus cell line C_(20)hi:cloning and characterization of expression

An expressed sequence tag(EST)obtained from a subtractive-suppression hybridization cDNA library constructed using Catharanthus roseus cell line C_(20)hi and its parental cell line C_(20)D was used to clone a ful-length cytochrome P450 cDNA of cyp71d1.The encoded polypeptide contained 507 amino acids with 39-56% identity to other CYP7ID subfamily members at the.amino acid level.Expression characteristics of cyp71d1 were determined using semi-quantitative RT-PCR.The cyp71d1 transcript was expressed in all three cell lines with the highest level in the cell line C_(20)hi.In the mature C.roseus plant,the cyp71d1 cDNA was highly expressed in petals,roots and stems,but very weakly expressed in young leaves.Its transcription level increased with the development of flowers.2,4-D could down-regulate the transcription of cyp71d1,as did KT,but only to a minor degree.Neither light nor yeast elicitor could induce the transcription of cyp71d1.

Effects of CeCl_3 and LaCl_3 on callus and root induction and the physical response of tobacco tissue culture

La3＋ and Ce3＋ have positive effects on plant growth and production. Although it is well known that rare earth elements promote cell growth. The biological effects of La^（3＋） and Ce^（3＋） on callus, shoot and root induction in tobacco are still unclear. The relationships among callus induction, rooting, enzyme activities and stomatal characteristics in tobacco are unknown. The objectives of this study were to identify the relationships between the induction of calluses, shoots, roots, stomata and enzyme activities. The induction percentages of calluses, buds, roots were recorded at 5,10,15, 20 and 25 days after La^（3＋） and Ce^（3＋） treatments. Peroxidase isoenzyme activity was determined by electrophoresis. The characteristics of the stomata were observed under an optical microscope. Our results show that low concentrations of Ce^（3＋）（〈15 mg/L） result in increases in the induction percentages of calluses,buds and roots, but La^（3＋）（〉5 mg/L） inhibits the induction of calluses, buds and roots. There are more peroxidase isoenzyme bands in Ce^（3＋） treatments than in La^（3＋） treatments. This is consistent with the induction percentages of calluses,buds and roots in Ce^（3＋） and La^（3＋） treatments. High enzyme activities may promote the induction of calluses, buds and roots. The stomata area and stomata number of leaves are significantly different between La^（3＋） treatments and Ce^（3＋） treatments. La^（3＋） improves the stomata area and number. Based on these results, we speculate that La^（3＋） may promote the development of the photosynthetic system. Ce^（3＋）may promote tobacco growth and rooting by improving enzyme activities.

Molecular cloning and functional identification of sterol C24-methyltransferase gene from Tripterygium wilfordii

Sterol C24-methyltransferase(SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from Tripterygium wilfordii(Tw SMT1). Tw SMT1(Gen Bank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 k Da protein. The polypeptide encoded by the SMT1 cDNA was expressed and purified as a recombinant protein from Escherichia coli(E. coli) and showed SMT activity. The expression of Tw SMT1 was highly up-regulated in T. wilfordii cell suspension cultures treated with methyl jasmonate(Me JA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs(leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterolbiosynthesis of T. wilfordii and will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of T. wilfordii.

Identification of three members of the Bmp family from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to a high fat diet

In this study,three members of the Bmp family were cloned and characterized in yellow catfish Pelteobagrus fulvidraco,including Bmp2a,Bmp4 and Bmp9.The predicted amino acid sequences of P.fulvidraco Bmp2a,Bmp4 and Bmp9 showed the characteristic domains of the Bmp family,including an N-terminal signal peptide,Arg-X-XArg site,TGF-β family signature and seven conserved cysteines,indicating that function is likely to have been conserved during evolution.mRNAs of the three Bmp genes had a variable level of expression in tissues.Compared to the control diet,a high fat diet tended to down-regulate the mRNA expression of Bmp2a,Bmp4 and Bmp9 in mesenteric fat,liver and ovary,while it tended to up-regulate their mRNA levels in muscle and kidney.The responses to dietary lipid status and the potential role in lipid metabolism have not previously been reported and reinforces the idea of their multiple functions.Our findings provide the first data about the potential role of the Bmp family in lipid metabolism in teleost.