Objective To establish an animal model of high-iodine and low-protein in Wistar rats,and to observe the effect of combined excess-iodine and low-protein diet on growth,metabolism and morphological changes in thyroid.Methods According to body weight[(110 ± 10)g] and sex(half male and half female),one hundred and ninety-two Wistar rats,1 month after weaning,were randomly divided into ① normal iodine control group (NI),② 10-fold excess-iodine group (10HI),③ 50-fold excess-iodine group (50HI),④ 100-fold excess-iodine group (100HI),⑤ low-protein control group (LC),⑥ low-protein and l 0-fold excess-iodine group (L10HI),⑦low-protein and 50-fold excess-iodine group (L50HI),⑧ low-protein and 100-fold excess-iodine group(L100HI).Twenty-four rats were in each group,with the experimental period of 6 months.The iodine content of NI and LC groups was 4.65 μg/d; 10HI,50HI and 100HI groups were 46.50,232.50 and 465.00 μg/d,respectively.The animal's body weight,water and feed consumption were recorded weekly.At the end of 60,120,180 days,urine and blood samples were collected from eight rats in each group.Urinary iodine was tested by arseni cerium catalytic spectrophotometry; serum iodine was tested by the method of chloric acid.Histological change of the thyroid gland was observed by transmission electron microscopy and hematoxylin-eosin (HE) staining at the end of 6 months; apoptosis of thyroid was tested by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method.Results At the end of 4,8,16,18,22 and 24 weeks,the differences of body mass of rats among groups were statistically significant(F =4.26,3.75,4.98,4.09,3.28,3.95,all P ＜ 0.05).At the end of 60,120,180 days,the differences of iodine concentration in urine and blood among groups were statistically significantly (H =5.37,6.03,all P ＜ 0.05).Light microscopy showed that thyroid follicular epithelial cells became flattened,and follicles became distended with colloid following increasing of iodine concentration.Electron microscopy showed increased glial vesicles,condensation of nuclear chromatin,karyopyknosis,and karyolysis with increasing of iodine concentration.The differences of apoptotic indexes among groups were statistically significant (F =4.59,P ＜ 0.01).The apoptotic indexes of L50HI and L100HI groups [(21.50 ± 5.20)‰,(26.70 ± 6.40)‰] were higher than those of 50HI and 100HI groups [(11.20 ± 4.30)‰,(19.40 ± 4.80)‰,P ＜ 0.01 or ＜ 0.05].Conclusion Excessiodine and low-protein can cause growth retardation,abnormal iodine metabolism,and thyroid follicular epithelium damage in Wistar rats.

Objective Toinvestigatetheclinicalvalueofhumanneutrophillipocalin(HNL)detectioninthedifferentialdiagnosis of bacterial and viral infections of elderly patients with acute respiratory infection .Methods 142 elderly patients with respiratory infection were divided the bacteria group (96 cases) and the virus group (46 cases) according to their infections ,42 healthy people in the corresponding period were enrolled as the control group .Enzyme-linked immunosorbent assay and highly sensitive dry chemi-cal particles enhanced immune turbidity assay were employed to detect their blood HNL and C-reactive protein(CRP) ,respectively , and virus-specific antibodies detection were performed simultaneously .Results Compared the blood HNL ,CRP levels and their positive rates of patients in bacteria group with those in the virus group ,control group ,respectively ,differences showed statistically significant(P<0 .01) ,while the differences of indicators listed above between the virus group and control group had no statistically significant(P>0 .05) .Antibiotic treatment before and 24 ,48 and 72 hours after ,the concentrations of HNL were (216 .8 ± 64 .1) , (192 .0 ± 41 .2) ,(158 .0 ± 54 .5) and (87 .0 ± 12 .4)μg/L ,respectively ,while those of CRP were (50 .9 ± 40 .9) ,(46 .2 ± 18 .3) , (39 .6 ± 9 .6) and (12 .6 ± 9 .8) mg/L ,respectively .Sensitivity ,specificity ,positive predictive value and negative predictive value of HNL detection were 90 .6% ,90 .9% ,91 .5% and 89 .9% ,respectively ,which were higher than those of CRP (88 .5% ,85 .2% , 86 .7% and 87 .2% ,respectively) ,with statistically significant difference(P<0 .05) .Conclusion NHL detection possesses impor-tant significance in differential diagnosis between bacterial and viral infections of elderly patients with acute respiratory infection .

Objective To investigate the relationship between the expression level of platelet membrane glycoprotein?3(GPⅢa,CD61)and the severity of disease in patients with hemorrhagic fever with renal syndrome(HFRS).Methods One hundred and four patients with HFRS and 30 healthy individuals were recruited.The percentage of CD61 positive platelets and the mean fluores- cence intensities(MFI)of platelet membrane glycoprotein?3 were determined by flow cytometry (FCM).The 104 patients studied were divided into three groups based on their expression levels of platelet membrane glycoprotein?3 at oligurie phase.Clinical data and laboratory parameters in different groups were compared and analyzed.Results The expression levels of CD61 in patients with HFRS were significantly higher than those in control group,although no significant difference in the percentage of CD61 positive platelets between patients with HFRS and controls was detected.The MFI of CD61 expression in patients with HFRS at fever phase,oliguric phase and polyuric phase was 19.75?2.57,17.46?1.48 and 15.55?0.60,respectively,which was significantly higher than that in control group(3 20?0.12).The expression level of CD61 in patients with HFRS at oliguric phase was negatively correlated with platelet count and serum albumin(r=-0.637 and-0.695,respec- tively)and positively correlated with white blood cell count,blood urea nitrogen,serum creatinine and alanine aminotransferase(r=0.945,0.904,0.956 and 0.891,respectively).When the patients were compared according to the expression levels of CD61,it was indicated that the higher the expression level of CD61,the higher the incidence of uremia,hypoalbuminemia,abnormal liver func- tion and leukocytosis.Conclusions The expression levels of platelet membrane glycoprotein?3 in patients with HFRS are different in different clinical phases and are significantly correlated with the severity of the disease in the patients.It suggests that the expression levels of platelet?3 integrin are dramatically increased in patients with HFRS,which may be an indicator for the severity of disease and be helpful for monitoring the state of the patients' diseases and evaluating the severity of the disease.

ObjectiveTo investigate the current status of Kaschin-Beck disease in Shandong province, and to provide a scientific basis for decision-making in controlling the disease. Methods According to the National Monitoring Program of Kaschin-Beck disease requirements, historical serious villages of Kaschin-Beck disease in Qingzhou of Shandong province were selected annually; children aged 7 to 16 were chosen to receive clinical examination and children aged 7 to 12 were taken X-ray examination. Clinical and X-ray diagnosis was carried out according to the Diagnostic Criteria of Kashin Beck Disease(GB 16003-1995). Results From 1996 to 2010, in 53 diseased villages, three thousand three hundred and eighteen school children aged 7 to 16 were clinically diagnosed, and child Kaschin-Beck disease of degree Ⅰ and above were not detected; three thousand and ninety-one school children aged 7 to 12 were examined by X-ray, forty cases were found positive, and the total positive rate was 1.29％(40/3091 ). The year with the highest positive rate was 2002, and the rate was 3.49％(13/372) ; the positive rate was 0 in 1996 and 2008. The difference of the X-ray positive rate between each year was statistically significant(x2 =31.54, P ＜ 0.01 ). ConclusionsChild Kashin-Beck disease in Qingzhou is basically under control.Since etiology of Kashin-Beck disease is still unclear, surveillance of the disease still needs to be strengthened.

Objective To find humoral protein markers to develop new experimental diagnostic methods for tuberculous pleurisy. Methods Proteomes of 7 d and 14 d culture filtrate of Mycobacterium tuberculosis growing in Middlebrook 7H9, serum and pleural effusion from five patients with tuberculous pleurisy were detected by surface-enhanced laser desorptionionization time-of-flight massspectrometry (SELDI-TOF-MS). And the data were analyzed with descriptive statistics method to observed the protein components all owned by three kinds of proteome. Results From protein species, proteins of all culture filtrate were far more than that of pleural effusion and serum while proteins of pleural effusion in four cases were more than that of serum. The kinds of common proteins between culture filtrate and pleural effusion, between culture filtrate and serum, between serum and pleural effusion, among culture filtrate and pleural effusion and serum were different. But the protein of relative molecular mass of 2 660 depending on the ratio of mass to charge existed in all samples of culture filtrate, pleural effusion and serum. Conclusion The protein of relative molecular mass of 2 660 possess the latent quality as a specific humoral protein marker for tuberculous pleurisy. But it is essential that must be further confirmed among large samples.

Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997.
Alcohol Oxidoreductases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Bacteriophages ; genetics ; Benzoquinones ; metabolism ; Blotting, Southern ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Genetic Vectors ; genetics ; Lactams, Macrocyclic ; metabolism ; Multigene Family ; genetics ; physiology ; Polymerase Chain Reaction ; Streptomyces ; genetics ; metabolism

OBJECTIVETo construct the sodium channel gene SCN5A-delQKP1507-1509 mutation associated with congenital long QT syndrome, and its eukaryotic expression vector, and to examine the expression of mutation protein in human embryonic kidney (HEK) 293 cells.

METHODSEukaryotic expression vector PEGFP-delQKP-hH1 for SCN5A-delQKP1507-1509 mutation was constructed by rapid site-directed mutagenesis. HEK293 cells were transfected with the wild or mutant vector using lipofectamine, and then subjected to confocal microscopy. The transfected cells were immunostained to visualize intracellular expression of the mutant molecules.

RESULTSDirect sequence and electrophoresis analysis revealed 9 basic group absences at position 1507-1509. The delQKP1507-1509 mutation eukaryotic expression vector was expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both wild type and mutant molecules on the plasma membrane and there was no difference in the amount of protein, which suggested that the mutant delQKP1507-1509 did not impair normal protein expression in HEK293 cells.

CONCLUSIONSSuccessful construction of mutant SCN5AdelQKP1507-1509 eukaryotic expression vector and expression of SCN5A protein in HEK293 cells provides a basis for further study on the functional effects of congenital long QT syndrome as a cause of SCN5A mutation.

Blotting, Western ; HEK293 Cells ; Humans ; Long QT Syndrome ; congenital ; genetics ; Mutagenesis, Site-Directed ; NAV1.5 Voltage-Gated Sodium Channel ; analysis ; genetics ; physiology

OBJECTIVETo observe the role of Panax notoginosides (PNS) in up-regulation of GATA family transcription factors, and explore intracellular signal pathway of PNS in the proliferation of hematopoietic cells.

METHODSHuman bone marrow cells were incubated with different concentrations of PNS for colony-forming assay. Human cell lines HL-60, K562, CHRF-288 and Meg-01 were incubated with PNS (10 mg/L) for 14 days. The cell nuclear proteins were extracted and analyzed by Western blot with antibodies against GATA-1, GATA-2. Electrophoretic mobility shift assay (EMSA) and antibody gel supershift assay was performed using (32)P labeled GATA consensus oligonucleotide which contains binding site for GATA transcription factors.

RESULTSPNS could promote the proliferation of CFU-GM and CFU-E and induce the expression of GATA-1, GATA-2. The nuclear proteins of both GATA-1 and GATA-2 in K562, CHRF-288 and Meg-01 cells treated by PNS were increased by (1.5 - 2.8) and (2.0 - 3.1)-fold over untreated cells respectively. GATA binding activity initiated by PNS was apparently elevated to form higher density band of GATA-DNA complex. While there was no detectable change in HL-60 cells before and after PNS treatment. The predominant GATA binding complex was mainly attributable to both GATA-1 and GATA-2 proteins being in phosphorylated status.

CONCLUSIONPNS can induce the synthesis of transcription factors GATA-1 and GATA-2 and enhance their DNA binding activity, which could play a role in the up-regulation of the expression genes related to proliferation and differentiation in hematopoietic cells.

Blotting, Western ; Bone Marrow Cells ; drug effects ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; GATA1 Transcription Factor ; metabolism ; GATA2 Transcription Factor ; metabolism ; Ginsenosides ; pharmacology ; HL-60 Cells ; Humans ; K562 Cells ; Panax ; chemistry ; Up-Regulation ; drug effects

Resistin, a newly discovered peptide hormone mainly secreted by adipose tissues, is present at high levels in serum of obese mice and may be a potential link between obesity and insulin resistance in rodents. However, some studies of rat and mouse models have associated insulin resistance and obesity with decreased resistin expression. In humans, no relationship between resistin level and insulin resistance or adiposity was observed. This suggests that additional studies are necessary to determine the specific role of resistin in the regulation of energy metabolism and adipogenesis. In the present study, we investigated the effect of resistin in vivo on glucose and lipid metabolism by over-expressing resistin in mice by intramuscular injection of a recombinant eukaryotic expression vector pcDNA3.1-Retn encoding porcine resistin gene. After injection, serum resistin and serum glucose (GLU) levels were significantly increased in the pcDNA3.1-Retn-treated mice; there was an obvious difference in total cholesterol (TC) level between the experiment and the control groups on Day 30. In pcDNA3.1-Retn-treated mice, both free fatty acid (FFA) and high density lipoprotein (HDL) cholesterol levels were markedly lower than those of control, whereas HDL cholesterol and triglyceride (TG) levels did not differ between the two groups. Furthermore, lipase activity was expressly lower on Day 20. Our data suggest that resistin over-expressed in mice might be responsible for insulin resistance and parameters related to glucose and lipid metabolism were changed accordingly.
Animals ; Blood Glucose ; analysis ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Fatty Acids, Nonesterified ; blood ; Glucose ; metabolism ; HeLa Cells ; Humans ; Lipid Metabolism ; Male ; Mice ; Resistin ; blood ; physiology ; Triglycerides ; blood

OBJECTIVETo observe the temporal modification of transcription factor Mef2c by histone acetylases (HATs) P300, PCAF, and SRC1 during cardiogenesis and to provide a basis for investigating the pathogenesis of congenital heart disease.

METHODSThe normal heart tissues from embryonic mice (embryonic days 14.5 and 16.5) and neonatal mice (postnatal days 0.5 and 7) were collected. The binding of P300, PCAF, and SRC1 to Mef2c gene and level of histone H3 acetylation in the promoter region of Mef2c were evaluated by chromatin immunoprecipitation assays. Meanwhile, real-time PCR was used to measure the mRNA expression of Mef2c.

RESULTSP300, PCAF, SRC1 were involved in histone acetylation in the promoter region of Mef2c during cardiogenesis in mice, and binding of P300, PCAF, and SRC1 to the promoter of Mef2c varied significantly in different stages of cardiogenesis (P<0.01). The level of histone H3 acetylation and mRNA expression of Mef2c in the promoter region of Mef2c also varied significantly in different stages of cardiac development (P<0.01). The levels of acetylated H3, Mef2c mRNA, and HATs (P300, PCAF, SRC1) changed over time. They were highest on embryonic day 14.5 (P<0.01), decreased gradually with cardiac development, and were maintained at low levels after birth.

CONCLUSIONSThe mRNA expression of Mef2c varies during cardiogenesis in mice, which indicates that Mef2c plays an important role in the process of cardiac development. Meanwhile, histone acetylation in the promoter region of Mef2c is regulated temporally by HATs P300, PCAF, and SRC1.

Animals ; Female ; Gene Expression Regulation, Developmental ; Heart ; embryology ; Histone Acetyltransferases ; physiology ; MEF2 Transcription Factors ; genetics ; physiology ; Male ; Mice ; Promoter Regions, Genetic ; RNA, Messenger ; analysis