组织内部门运作的变革优化

通过对某些影响组织因素的讨论 ,指出规模的发展有着由小到大再到小的趋势 ,探讨了规模与寿命的关系。对部门进行了新的划分 ,分析了部门变革优化的动力、阻力及变革的成本和变革的误区 。

Generation of Antibodies Against DMRT1 and DMRT4 of Oreochromis aurea and Analysis of Their Expression Profile in Oreochromis aurea Tissues

Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing （Doublesex/Mab-3 DNA-binding motif） gene, is highly conserved across species. Vertebrate DMRT1 （DM-related transcription factor 1） expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 （DM-related transcription factor 4） and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.

Expression of TRAIL and TRAIL receptors in normal and malignant tissues

TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, is a member of the TNF family of proteins.Tumour cells were initially found to have increased sensitivity to TRAIL compared with normal cells, raising hopes thatTRAIL would prove useful as an anti-tumor agent. The production of reliable monoclonal antibodies against TRAIL andits receptors that can stain fixed specimens will allow a thorough analysis of their expression on normal and malignanttissues. Here we report the generation of monoclonal antibodies against TRAIL and its four membrane-bound receptors(TR1–4), which have been used to stain a range of normal and malignant cells, as routinely fixed specimens. Low levelsof TRAIL expression were found to be limited mostly to smooth muscle in lung and spleen as well as glial cells in thecerebellum and follicular cells in the thyroid. Expression of the TRAIL decoy receptors (TR3 and 4) was not aswidespread as indicated by Northern blotting, suggesting that they may be less important for the control of TRAILcytotoxicity than previously thought. TR1 and TR2 expression increases significantly in a number of malignant tissues,but in some common malignancies their expression was low, or patchy, which may limit the therapeutic role of TRAIL.Taken together, we have a panel of monoclonal antibodies that will allow a better assessment of the normal role ofTRAIL and allow assessment of biopsy material, possibly allowing the identification of tumors that may be amenable toTRAIL therapy.

Molecular cloning,tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding

Mucins are important components of mucus, which form a natural, physical, biochemical and semipermeable mucosal layer on the epidermis offish gills, skin, and the gastrointestinal tract. As the first step towards characterizing the function of Muc2, we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp, and analyzed its tissue-specific expression pattern by quantitative real-time PCR （qPCR）. The obtained sequence comprised 41 bp 5＇-untranslated region （5＇-UTR）, 2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains （VWD-C8-TIL-VWD-C8） and had high homology with mucins from other vertebrates. Among four candidate reference genes （β-Actin, RP113a, RPII, 18S） for the qPCR, RPII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine, in the order （highest to lowest） middle-intestine 〉 fore-intestine 〉 hind-intestine. Muc2 was expressed relatively poorly in other organs （brain, liver, kidney, spleen, skin and gill）. Furthermore, after 20-days of starvation, M. amblycephala Muc2 expressions after refeeding for 0 h, 3 h, 16 h, 3 d, and 10 d were significantly decreased in the three intestinal segments （P〈0.05） at 16 h, and were then upregulated to near the initial level at 10 d.

Cloning and characterization of a novel gene encoding a putative seven-span transmembrane protein localized in endoplasmic reticulum

Objective: To clone and analyze the structure of a novel gene, named EST 1 (endoplasmic reticulum localized seven span transmembrane protein 1) and to analyze the expression pattern and intracellular location of EST 1. Methods: The cDNA library was screened to isolate novel cDNA fragment. The structure of novel gene was analysed by computer software. Expression of EST 1 was analyzed by dot blot and Northern blotting. Intracellular localization was observed after EST 1 enhanced green fluorescence protein (EGFP) fusion gene was transfected into mammalian cells. Results: The full length cDNA of mouse EST 1 was 1 802 bp, with a 1 293 bp open reading frame encoding 431 amino acids. It was predicated that protein encoded by EST 1 contained a signal peptide sequence at the N terminus, seven putative transmembrane domains, and an ER retaining signal at the C terminus. EST 1 EGFP fusion protein showed an ER like intracellular distribution in mammalian cells. Expression pattern analysis showed that EST 1 is expressed in all tissues examined. Conclusion: EST 1 is encoding a putative seven span transmembrane protein localized in endoplasmic reticulum. EST 1 was expressed in all tissues examined, suggesting an essential function of EST 1 in cells.

The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli

The Clock gene,a key molecule in circadian systems,is widely distributed in the animal kingdom. We isolated a 936-bp partial c DNA sequence of the C lock gene( Pva- clock) from the darkbarbel catfish P elteobagrus vachelli that exhibited high identity with C lock genes of other species of fish and animals(65%–88%). The putative domains included a basic helix-loop-helix(b HLH) domain and two period-ARNT-single-minded(PAS) domains,which were also similar to those in other species of fish and animals. P va- Clock was primarily expressed in the brain,and was detected in all of the peripheral tissues sampled. Additionally,the pattern of P va- Clock expression over a 24-h period exhibited a circadian rhythm in the brain,liver and intestine,with the acrophase at zeitgeber time 21:35,23:00,and 23:23,respectively. Our results provide insight into the function of the molecular C lock of P. vachelli.

Cloning the sterol carrier protein 2 genes of Japanese toad （Bufo japonicus formosus） and Chinese toad （Bufo gargarizans） and its tissue expression analysis

In this study,to clarify the bioactive polypeptides included in the skins and secretions of Bufo,we screened the Japanese toad(Bufo japonicus formosus) skin cDNA library by colony polymerase chain reaction(PCR),and obtained a transcript of 1 075 bp consisting of 1 37 bp 5′ untranslated region(UTR),515 bp 3′ UTR and a 423 bp open reading frame(ORF) encoding a polypeptide of 140 amino acid residues(GenBank accession number: KF359945).Homolog analysis showed a 70%–96% homology with sterol carrier protein-2(SCP-2) present in other animals,which is implicated in lipid metabolism of other organisms.The gene SCP-2 of Chinese toad(B.gargarizans) was cloned from a first strand cDNA of Bufo skin(GenBank accession number: KF381341) via PCR,whose encoding polypeptide has only one amino acid difference from that of Japanese toad.Tissue distribution analysis showed that SCP-2 expressed in all organs tested,though in the liver and spleen it manifested lower expression than in other organs.These findings might indicate SCP-2 being one of the active ingredients in toad skin.These findings may in turn have implications for further drug development from traditional Chinese medicine sources.

Molecular cloning and expression analysis of FTZ-F1 in the GIFT tilapia,Oreochromis niloticus

The FTZ-F1 genes encode orphan receptors of the nuclear receptor superfamily and in mammals have been found to play important roles in the proper development of the adrenal-gonadal axis and sex-determination.We isolated the homologue of FTZ-F1 in genetically improved farmed tilapia(gfFTZ-F1).The full-length cDNA was isolated from the ovary,which included an open reading frame encoding a predicted protein of 486 amino acids.Sequence,tissue distribution and phylogenic analysis of the FTZ-F1 showed that the gfFTZ-F1 belonged to SF-1/Ad4BP group and that gfFTZ-F1 transcripts were only expressed in the gonads and kidney but not in other tissues.Likewise our data suggests that the gfFTZ-F1 gene may share similar functions with other fish and mammalian counterparts,though further study is needed to make any definitive conclusions.

Cloning,tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

Pituitary adenylate cyclase activating polypeptide （PACAP） has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide （PRP） from the brain of largemouth bass （Micropterus salmoides） and used real-time quantitative PCR to detect PRP- PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing： a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAR Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-Iong and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PA CAP- long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch （dph）. The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP- PA CAP acts as an important factor in appetite regulation in largemouth bass.

Cloning and sequence analysis of cold inducible RNA-binding protein cDNA from testis tissue in BALB/C mice

The effect of cold stress on the gene expression of cold-inducible RNA-binding protein （CIRP） in the intravital animals has not been reported till now. Compared with their organism cells, there were much more complicated regulatory mechanisms for cold stress response in the organisms. The BALB/C mice with cold treatment were used as experimental animals for this study. The cDNA of CIRP was firstly cloned from the testis tissues of the BALB/C mice treated by cold stress The results indicated that CIRP in the organisms could be induced at low temperature and may protect the organisms from the cold damage. The amino acid sequence deduced via cDNA clone was 100%, 99.4%, 95.5%, 67.4%, 76.9%, 79.1% and 58.4% identical with that of CIRP in mice, rats, human, bullfrog, Xenopus and axolotl cells, respectively. These results showed that the CIRP was highly conserved in the evolution process and may be involved in various physiological functions. Therefore, this study will establish a systematic model for experiments and provide a new foundation for exploring the molecular mechanisms of human and other animals under cold stress.

Cloning and analysing of 5' flanking region of Xenopus organizer gene noggin

Xenopus organizer specific gene Noggin possessesnearly all the characterestic properties of the action of or-ganizer to specify the embryonic body axis. To analyzehow the maternal inherited factors cofltrol its expressionpattern, we cloned the 5’ regulatory region of noggn gene.The 1.5 kb upstream sequense could direct reporter geneto express in vivo and data from deletion analysis indi-cated that a 229 base pair fragmet is essential for acti-vating noggn expression. We further demonstrated thatthe response elements within this regulatory region wereindeed under the control of growth factor activin and Wntsignaling pathway components.

Cloning and screening of scarless healing-related gene(s) in rabbit skin

Background: Over the past years, scientists have been working on the mechanisms of the scarless healing. The remarkable phenotypic differences between fetal and adult healing may lead us to find out their characteristics in genetics, which represent potentially important mechanisms to explain the differences in the quality of wound repair observed in fetus versus adult tissues. Methods: Middle laparotomy and hysterotomy were performed on pregnant rabbits on 20-day gestation to expose the fetal back, and longitudinal incision which penetrated full skin was made on the back of fetus. The trauma fetus skin was harvested at 12 h post-operation (FT), the fetus control (FC) and trauma adult skin (AT) were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. An improved suppression subtractive hybridization (SSH) method was applied to analyze the samples. Having taken one of the three samples as Tester respectively, the other two together as Drivers, one forward and two reverse hybridization products were gotten. Having amplified by selective PCR, the products were inserted into vector, and then transferred into E.coli HB101. The colonies were screened by electrophoresis, reverse Northern afterwards, and the positive clones were sequenced. BLAST in NCBI was performed to compare and analyze the positive clones (expressed sequence Tag, ESTs). Results: Totally 298 clones were gotten and 61 positive clones were obtained after screening. The 61 selected positive clones were sequenced and 54 sequences were goten. Conclusion: Instead of traditional SSH, an improved SSH with 2 Drivers was applied in the experiment. The improved program is reasonable and correct in both theory and practice.

Immunohistochemical Study of Hexose Transporters GLUT-2 and GLUT-5 in Birds Gastrointestinal Tract

The hexoses glucose, galactose and fructose serve as important dietary energy sources in animals and glucose plays a central role in energy homeostasis within eucaryotic cells. As relatively little is known about patterns of hexose transporters expression in birds gastrointestinal tract, the aim of the study was to examine glucose transporters 2 （GLUT-2） and glucose transporters 5 （GLUT-5） expression in stomach and duodenal epithelium of two different species--broilers （Gallus gallus domesticus） and ostriches （Struthio camelus var. domesticus） chicken. Materials from the two parts of gastrointestinal tract were collected from six 7-day-old female broilers and six 7-day-old female ostriches. Specimens were fixed with 10% formalin, embedded into paraffin, cut into 7 lain thick slices, followed by immunohistochemical staining with polyclonal primary antibodies rabbit anti-GLUT-2 and rabbit anti-GLUT-5; the staining was carried out according to the manufacturers guidelines （IHC kit, Abcam, UK）. The investigation showed that the staining for both antibodies was more intensive in the epithelial cells of stomach and duodenum of the 7-day-old broilers. In duodenal epithelium, goblet cells and brush border membranes were stained in both species, however the signal was stronger for GLUT-5 than GLUT-2. Staining for GLUT-2 and GLUT-5 occurred in different parts of gastrointestinal tract of 7-day-old ostriches, but was weaker compared to 7-day-old broilers, which showed that the gastrointestinal tract of 7-day-old female broilers was more developed for transportation ofhexoses than 7-day-old female ostriches.

Clinical pathology of nodal micrometasteses in non-small cell lung cancer

Objective： To explore whether the conventional pathologic stages of some non-small cell lung cancer （NSCLC） patients were underestimated. Methods： 195 lymph node samples were taken from 25 NSCLC patients during the operations. Firstly, each resulting tissue block was processed for routine paraffin embedding. Then the 6- 10 serial sections were chosen, each 5/am thick, from every paraffin block of the lymph node. Finally, the first and the second last sections of each lymph node were stained by hematoxylin eosin （HE）, and the other serial sections were used for the immunohistochemical （IHC） staining examination with the monoclonal antibody against cyokeratin 19. Results： With HE staining, 30 of the 195 regional lymph nodes revealed dominant nodal metastases, and none showed micrometastases. IHC staining was performed on 135 lymph nodes that were identified as free of metastases by HE staining, 31 showed micrometastases; none showed gross nodal metastases. There was a significant difference between HE staining staging and IHC staining staging （P〈0.05）. Conclusion： Conventional HE staining can accurately detect gross nodal metastases in the lymph nodes of NSCLC patients, but is unfit for detecting lymph nodal micrometastases. IHC staining analysis can significantly facilitate the detection of occult micrometastatic tumor cells in lymph nodes, and its assessment of nodal micrometastases can provide a refinement of TNM stage for NSCLC patients. Our results provide a rationale for extensive lymph nodes sampling