The 1.23 kb DNA fragment encoding the early protein EP0 of pseudorabies virus (PRV) Ea strain was amplified by PCR technique and cloned into pBluescriptII sk+.Three sequencing plasmids containing the partial fragment of the EP0 gene were constructed and the sequences were obtained by Sanger's sequencing technique. Compared with PRV InFh strain, there were multipile site-mutations and a deleted-mutation in the EP0 gene of PRV strain Ea，and the diversity of amino acid residues also existed.Then, the EP0 gene was inserted into an expression vector, pET-28a, fused into the downstream of the 6ΧHis-Tag in frame, to yield the expression plasmid pETEP0. After induction by IPTG, a high expression of fusion protein was obtained, SDS-PAGE analysis and Western blotting showed that the fusion protein was 62kD and the protein was specific to antisera against PRV Ea strain. This indicated that the EP0 gene be expressed in BL21(DE3) and the expression products have immuno-genicity.

Adult ; Chondrosarcoma ; metabolism ; pathology ; Collagen Type II ; metabolism ; Diagnosis, Differential ; Femoral Neoplasms ; metabolism ; pathology ; Giant Cells ; pathology ; Humans ; Male ; Osteoclasts ; pathology ; Osteosarcoma ; metabolism ; pathology ; S100 Proteins ; metabolism ; Vimentin ; metabolism

The degQ gene, amplified from Bacillus subtilis by PCR, was cloned to pUBS (sucrose induced secretion vector). After transformed into DB403, recombination named DB403(pUBSD) was formed. The results of the fermentation showed that degQ gene enhanced the expression of B. subtilis fibrin enzyme. The activity of the enzyme was increased to 2.2 times as the original one. In this article, the effects of different conditions, such as different kinds of sugar, different concentration of sucrose and different induced time were also be investigated and compared.

OBJECTIVETo explore the correlation between syndrome types of late-onset acne female patients and constitutions of Chinese medicine (CM).

METHODSA questionnaire was performed in 365 late-onset acne female patients and 135 healthy subjects (as the control) using Professor WANG Qi's. methods and Standards for Chinese Medical Constitutions Classification.

RESULTSTheir CM constitutions were sequenced as damp-heat constitution, yin-deficiency constitution, balanced constitution, yang-deficiency constitution, blood-stasis constitution, qi-stagnation constitution, qi-deficiency constitution, phlegm-damp constitution, inherited special constitution, with statistical difference when compared with those of the control group ( χ2 = 85.206, P < 0.01). In the 365 female late-onset acne patients, 114 (31.23%) were with Chongren imbalance syndrome, 108 (29.59%) were with blood stasis or coagulated phlegm syndrome, 83 (22.74%) were with dampness heat syndrome, and 60 (16.44%) were with wind heat syndrome. There was statistical difference in CM constitution distributions among different CM syndrome types (χ2 = 105.671, P < 0.01). The distribution of CM medical constitutions was different between the two groups. Biased constitutions were often seen in the patient group, while balanced constitution was often seen in the control group. Binary Logistic regression analysis indicated that influencing factors covered sweet food, light diet, roasted food, coffee, stress, work pressure, and family pressure. Of them light diet was one protective factor, while the rest were adverse factors.

CONCLUSIONThe etiology and syndrome types of female late-onset acne female patients were associated with CM constitution.

Acne Vulgaris ; epidemiology ; Body Constitution ; Female ; Humans ; Medicine, Chinese Traditional ; Surveys and Questionnaires ; Syndrome ; Yang Deficiency ; Yin Deficiency

OBJECTIVE CYP2D is one of the most abundant subfamily of CYPs in the brain, especially in the cerebellum. Brain CYP2D is responsible for the metabolism of endogenous neurotransmitters such as tyramine and serotonin. Our previous studies have shown brain CYP2D can be regulated by exogenous and endogenous substances with tissue- specificity. The purpose of this study is to examine the effects of cerebral CYP2D on the mice behavior and the regulatory mechanism of brain CYP2D by growth hormone. METHODS Mice received the stereotaxic injection with CYP2D inhibitor quinine in deep cerebellar nuclei of cerebellum. The animals were tested with rotarod apparatus, balance beam, water maze, elevated plus maze and open field. The changes in CYP2D22, PPARαand PPARγ in brain regions and liver were assayed in male growth hormone receptor knockout mice, SH-SY5Y cells and HepG2 cells. RESULTS The inhibition of cerebellum CYP2D significantly affected the spatial learning and exploring ability of mice. Compared with WT mice, CYP2D expression was lower in brain regions from GHR(-/- ) male mice; however, hepatic CYP2D level was similar. Pulsatile GH decreased PPARα mRNA level, and increased mRNA levels of CYP2D6 and PPARα in SH- SY5Y cells. In HepG2 cells, pulsatile GH resulted in decreases in PPARα and PPARγ mRNA levels, but not CYP2D6. PPARα inhibitor induced CYP2D6 mRNA and protein by 1.32-fold and 1.43-fold in SH-SY5Y cells. PPARγ inhibitor decreased CYP2D6 mRNA and protein by 74.76% and 40.93%. PPARα agonist decreased the level of CYP2D22 mRNA in liver and cerebellum, while PPARγ agonist rosiglitazone resulted in diametrically increases. The luciferase assay showed that PPARγ actived the CYP2D6 gene promoter while PPARα inhibited its function. Pulsatile GH declined the binding of PPARα with CYP2D6 promoter by 40%, promoted the binding of PPARγ with CYP2D6 promoter by approximate 60%. The levels of brain and liver PPARα expression in male GHR(-/- ) mice is obviously higher than those in WT mice. The level of PPARγ in male GHR(-/- ) mice was decreased in the frontal cortex and hippocampus, while remained stable in the cerebellum and striatum; meanwhile, PPARγ was increased in the liver. CONCLUSION Brain CYP2D may be involved in learning and memory functions of central system. Masculine GH secretion altered the PPARs expression and the binding of PPARs to CYP2D promoter, leading to the elevated brain CYP2D in a tissue- specific manner. Growth hormone may specifically alter the metabolic and synthetic of important endogenous substances in the central nervous system (such as serotonin) through the specific regulation of brain CYP2D expression.

Objective: To evaluate the accuracy of automated classification of ICD-O-3 morphology code from pathology reports by text-mining and support vector machine ( SVM ) , in order to provide basis for automated tumor coding in Chinese. Methods: The tumor report cards of Zhejiang residents from 2017 to 2019 were collected from Chronic Disease Surveillance Information Management System of Zhejiang Province. According to ICD-O-3, the keywords of the pathology reports were extracted, and SVM was used for automatic classification. The classification results were compared with those of 16 professionals with more than two years of experience in tumor coding, and the accuracy rate, recall rate and F-score were calculated for effect evaluation. Results: Totally 83 082 cases from 2017 to 2019 were included and were categorized into 17 morphological classifications, with 52 877 ( 63.65% ) cases of adenocarcinoma, squamous carcinoma and transitional cell carcinoma. A total of 1 090 keywords were enrolled into main corpus. The total F-score, accuracy rate and recall rate are 85.69, 77.20% and 96.27%, respectively. Conclusion Text-mining combined with SVM can improve the efficiency of ICD-O-3 morphology coding; however, the accuracy needs to be further improved.

Objective To investigate the effect of L-arginine on expression of protein kinase C(PKC)mRNA during pulmonary ischemia and reperfusion injury(PIRI)in the rabbits.Methods Single lung ischemia and reperfusion animal model was used in vivo.The rabbits were randomly divided into three groups(n=9,in each),sham operated group (Sham),PIR group(I-R)and PIR+L-arginine group(L-Arg).Changes of several rariables including malondialdehyde (MDA),superoxide dismutase(SOD),malandialde hyde(MDA),nitril oxide(NO),wet to dry ratio of lung tissue weight(W/D)and index of quantitative assessment(IQA)of histolngic lung injury were recorded at 60 minutes after reperfusion in lung tissue.Meanwhile the location and expression of PKC mRNA were observed.Lung tissue was prepared for light microscopic and electron microscopic observation at 60 minutes after reperfusion.Results In comparison with group I-R,PKC mRNA strongly expressed in intima and extima of small pulmonary artery as well as thin-waU vessels (mostly small pulmonary veins).The average optical density values of PKC-?,?and?mRNA in small pulmonary veins in L-Arg group had significance(all P＜0.01);SOD increased while MDA,W/D and IQA decreased at 60 minutes after reperfusion in lung tissue(P＜0.01 and P＜0.05).A morphologically abnormal changes of the lung tissue,were lessen markedly in L-Arg group.Conclusion L-arginine possess notably protective effects on PIRI in rabbits by activating PKC-?,?and?mRNA expression in lung tissue,raising NO level,dropping oxygen free radical level and decreasing lipid peroxidation.

OBJECTIVETo investigate the expression of motilin and its precursor mRNA in normal human thyroid. To compare the expression differences of motilin and it precursor mRNA between normal thyroid and intestines. To study the expression of motilin and its precursor mRNA in human thyroid tumors and their clinical implications.

METHODSRT-PCR, Southern blot and molecular cloning were used to detect motilin transcript expression in human thyroid and mucous membrane of small intestine. Real-time PCR and immunohistochemical techniques were used to quantify motilin precursor mRNA and motilin peptide in thyroid tissue samples including adenoma, medullary carcinoma, follicular carcinoma, papillary carcinoma and nodular goiter.

RESULTS(1) The expression of motilin and its precursor mRNA in normal human thyroid was primarily in the thyroid C cells. (2) RT-PCR and Southern blot showed that motilin mRNA expressed in human thyroid was identical to that expressed in duodenum with identical sequence deposited in NCBI Genbank of America. (3) Immunohistochemistry, Western blot research and real-time PCR studies showed that motilin and its precursor mRNA were expressed in normal and tumor tissues of human thyroid. Thyroid tumors (acidophilic adenoma, medullary carcinoma, follicular carcinoma, papillary carcinoma and nodular goiter) showed intense and diffuse immunostaining for motilin peptide. Moreover, the expression of motilin and its precursor mRNA in thyroid medullar carcinoma and acidophilic adenoma were significantly higher than those of normal thyroid tissue (P < 0.05). The expression in thyroid follicular and papillary carcinomas were significantly lower than those of normal thyroid tissue (P < 0.05). There was no difference of the expression between nodular goiter and normal thyroid tissue (P > 0.05).

CONCLUSIONSMotilin peptide and its precursor mRNA are expressed in C cells of human thyroid. The sequence of motilin is identical to that expressed in duodenum from NCBI Genbank of America. The expressions of both motilin and its precursor mRNA in thyroid medullary carcinoma and acidophilic adenoma are significantly increased. In contrast, their expressions in thyroid follicular and papillary carcinomas are significantly decreased. Motilin may regulate physiological functions of the thyroid through parafollicular cells. Motilin may be involved in the pathogenesis of medullary carcinoma and acidophilic adenoma of the thyroid.

Adenocarcinoma, Follicular ; genetics ; Adult ; Aged ; Biomarkers, Tumor ; metabolism ; Carcinoma, Medullary ; genetics ; Carcinoma, Papillary ; genetics ; metabolism ; Female ; Humans ; Intestines ; metabolism ; Male ; Middle Aged ; Motilin ; genetics ; metabolism ; Nervous System Neoplasms ; metabolism ; RNA Precursors ; metabolism ; RNA, Messenger ; metabolism ; Thyroid Gland ; metabolism ; Thyroid Neoplasms ; genetics ; metabolism

OBJECTIVETo evaluate the effect of PKC-delta inhibitor Rottlerin on human colon cancer cells and its mechanism.

METHODSHuman colon cancer cell line SW1116 cells were treated with Rottlerin. The transcriptional level of DNA methyltransferase (Dnmt)1, Dnmt3a and Dnmt3b was detected by real-time RT-PCR. Cell cycle distribution was evaluated by flow cytometry (FCM). In addition, cellular morphological changes were examined by light microscopy.

RESULTSPKC-delta inhibitor decreased the expression of Dnmt1, Dnmt3a mRNA, up-regulated APC, p21(WAF1) and p16(INK4A) mRNA. Demonstarted by flow cytometry, Rottlerin increased the percentage of cell cycle G0/G1 phase cell numbers (P = 0.02) and decreased the percentage of cell cycle G2/M phase cell numbers (P = 0.01). Remarkable changes of cellular morphology were observed under light microscope: The volume and cytoplasm of cells treated with Rottlerin were increased. The cell contour was not very clear, and mitotic figures were less frequently seen.

CONCLUSIONPKC-delta inhibitor Rottlerin inhibites cell division and proliferation of the colon cancer SW1116 cells through regulating DNA methylation and blocking the signaling pathway of mitogen-activated protein kinase (MAPK).

Acetophenones ; pharmacology ; Adenomatous Polyposis Coli Protein ; genetics ; Benzopyrans ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Protein Kinase C-delta ; antagonists & inhibitors ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects

Danshen,an efficacious agent for cardiovascular diseases,has been found to play an essential role in kidney injury.In the present study,the effect of Danshen on cisplatin-induced renal dysfunction was investigated in a mouse model.Danshen was administered to mice at a dose of 3 g/kg 4 days before and 3 days after cisplatin treatment.A single intraperitoneal injection of 20 mg/kg cisplatin was used to induce nephrotoxicity.The mice were sacrificed 72 h after cisplatin intoxication.Biochemical parameters including serum creatinine and blood urea nitrogen were analyzed.Histopathological changes of kidney tissues were detected using HE staining.Antioxidant enzymes (GSH-Px and SOD) and peroxidative product (MDA) were detected.Protein expressions of Nrf2 and its target genes including HO-1 and NQO1 were measured by Western blotting.The results showed that pretreatment with Danshen significantly reduced serum creatinine and blood urea nitrogen in the cisplatin-treated mice.Histopathological examination showed that Danshen mitigated the renal damage induced by cisplatin.Moreover,Danshen restored the activities of antioxidant enzymes (GSH-Px and SOD) and normalized the MDA contents in renal tissues.Western blotting revealed that Danshen enhanced the expressions of Nrf2 and its target genes in cisplatin-exposed mice.It was suggested that Danshen protects against the cisplatin-induced renal impairment in the mice,which is potentially associated with the upregulation of Nrf2-mediated signaling pathway.