0.05). Conclusion Preproghrelin-Leu72Met is not significantly associated with T2DM and DN in Shanghai Han populations,while T2DM with AA genotype is characterized by significant declination in urine microalbumin when compared with CA and CC genotypes.Leu72Met polymorphism(C→A)may postpone the development of microalbuminuria in T2DM subjects.

Four small GTPase genes which may be relative to sclerotial development were firstly cloned from medicinal fungus Polyporus umbellatus using rapid amplification of cDNA end PCR (RACE) method. The results showed that full-length cDNA of PuRhoA was 698 bp contained 585 bp ORF, which was predicted to encode a 194 amino acid protein with a molecular weight of 21.75 kD with an isoelectric point (pI) of 6.44; the full length cDNA of PuRhoA2 was 837 bp in length and encoded a 194 amino acid protein with a molecular weight of 21.75 kD and an isoelectric point (pI) of 6.33; the full length cDNA of Puypt1 was 896 bp in length and encoded a 204-aa protein with a molecular weight of 22.556 kD and an isoelectric point (pI) of 5.75; the full length cDNA of PuRas was 803 bp in length and encoded a 212-aa protein with a molecular weight of 23.821 kD and an isoelectric point (pI) of 5.2. There are fani acyl transferase enzyme catalytic site and myrcene-transferase enzyme catalytic site in PuRhoA1 while the PuRhoA2 only possess myrcene-transferase enzyme catalytic site. Puypt1 contains the Rab1-Ypt1 conserved domain of small GTPase family and PuRas contains the fani acyl transferase enzyme catalytic site. According to the phylogenetic analysis all these four small GTPase clustered with basidiomycete group. Quantitative real-time PCR analysis revealed that Puypt1, PuRas and PuRhoA1 transcripts were significantly higher in the beginning of sclerotial formation than that in the mycelia, whereas the transcripts levels of PuRhoA2 gene were particularly lower in sclerotia than that in mycelia, suggesting that these four genes might be involved in P umbellatus selerotial development.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; Fungal Proteins ; genetics ; GTP Phosphohydrolases ; genetics ; Genes, Fungal ; Mycelium ; Phylogeny ; Polyporus ; enzymology ; genetics ; Real-Time Polymerase Chain Reaction

Objective To investigate the percentages of Th17 and CD4+CD25+FoxP3+ regulatory T(Tr) cells and the levels of related cytokines IL-6,IL-23,IL-17 and TGF-β in serum of patients with anlylosing spondylitis(AS).Methods Forty patients with AS and 37 age-matched healthy donors were studied.Flow cytometry Was used to analyze the percentages of blood Th17 and CD4+CD25+FoxP3+Tr cells.The levels of serum IL-6,IL-23,IL-17 and TGF-β were assayed by enzyme-linked immunosorbent assay ( ELISA).Results The proportion of Th17 cells in AS group was significantly higher than those in normal group [ (1.02±0.34)％ vs (0.68±0.29)％,P＜0.05) ],and the proportion of CD4+CD25+FoxP3+ cells was lower in AS group comparing with normal group [(3.77±0.81)％ vs (4.69±1.23)％,P＜0.05)].Meanwhile,serum levels of IL-6,IL-23 and IL-17 were significantly higher in AS group than those in normal group [ (6,15±2.71) ng/L vs (3.31±1.65) ng/L; (9.44±3.12) ng/ml vs (5.82±2.61) ng/ml;(10.53±4.97) ng/L vs (6.78±3.26) ng/L,all P＜0.01 ].In contrast,TGF-β level was decreased in AS group compares with the normal group [ ( 4.76±2.15) ng/ml vs (5.16±2.02) ng/ml,P＞0.05 ],but the difference was not significant.No associations of serum eytokine levels with clinical and laboratory parameters were found in AS.Conclusion The abnormality Th17 cells and Tr cells and their related cytokines IL-6,IL-23,IL-17 and TGF-β changes in patients with AS,which may be involved in immunological pathogenesis of AS.

ObjectiveThe aim of the present study was to investigate the incorporation of plasmid DNA (pDNA) onto a coronary stent by chemo-immuno-conjugation for achieving site-specific gene delivery.MethodsA gene eluting stent was fabricated by reacting with polyallylamine bisphosphonate (PAA-BP) to introduce amine reactive groups on the surface.Then an anti-DNA antibody was chemically coupled and pDNA was immunologically tethered on the stent surface.Radioactive-labeled antibody was used to evaluate binding capacity and stability.ResultsThe presence of amine groups on the modified stent surface was confirmed by XPS and AFM analysis.The isotope label assay indicated that the amount of antibody chemically linked on the stents was 15-fold higher than that of the control stent and its retention time was also significantly longer.ConclusionThe results suggested that a large amount of reactive amine groups were introduced on the PAA-BP modified 316L coronary stent surface.This study provide a potential metal surface modification method that could facilitate coupling and tethering of biological molecules such as anti-DNA antibody and plasmid DNA (pDNA) to achieve sustained and highly localized gene delivery for substrate-mediated gene transfection.

Aged, 80 and over ; Antibodies, Monoclonal ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Diagnosis, Differential ; Granuloma ; metabolism ; pathology ; Humans ; Keratins ; immunology ; Male ; Mucin-1 ; metabolism ; Skin Neoplasms ; metabolism ; pathology

Aged ; Carcinoid Tumor ; pathology ; Humans ; Laryngeal Neoplasms ; pathology ; Male ; Neoplasm Metastasis

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

OBJECTIVETo analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique.

METHODFungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on β-tubulin gene were used to identify the mycobiota.

RESULTAccording to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples.

CONCLUSIONPCR-SSCP based on β-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.

The optimum condition of shaking-flask p roducing enzyme were the tempe rature 26℃,initial pH 6 4,fermentation period 19 hours,medium volume 15mL m e dium/300mL Flask.soymilk-clotting enzyme was obtained from ammonium sulfate p r ecipitation.The optimum temperature and pH for the soymilk-clotting activity wa s 70℃and 5 8.The enzyme was easy to lose activity in acid or alkaline circumst a nce.About 60% of the original activity remained after 1 hour at 60℃.Ca 2+ ,Fe 2+ , Mg 2+ ,Na +increased the clotting activity,whereas Zn 2+ ,Al 3+ ca use inhibition.

BACKGROUND AND PURPOSE: The purposes of this study were to standardize and validate a Korean version of the Insomnia Severity Index (ISI-K), and to evaluate its clinical usefulness. METHODS: We translated the ISI into Korean and then translated it back into English to check its accuracy. The 614 patients with sleep disorders who were enrolled in this study comprised 169 with primary insomnia, 133 with comorbid insomnia, and 312 with obstructive sleep apnea. All subjects underwent one night of polysomnography (PSG) and completed the Korean versions of both the Pittsburgh Sleep Quality Index (PSQI-K) and the Epworth Sleepiness Scale, as well as the ISI-K. The ISI-K was compared to these sleep scales and various PSG sleep parameters. RESULTS: The internal consistency the ISI-K total score was confirmed by a Cronbach's alpha of 0.92, and the item-to-total-score correlations (item-total correlations) ranged from 0.65 to 0.84, suggesting adequate reliability. The correlation between the ISI-K total score and PSQI-K was 0.84, which suggested adequate convergent validity. Low-to-moderate correlations were obtained between the ISI-K total score and PSG-defined sleep parameters: 0.22 for sleep onset latency, 0.38 for wake after sleep onset, and 0.46 for sleep efficiency. A cutoff score of 15.5 on the ISI-K was optimal for discriminating patients with insomnia. The test-retest scores over a 4-week interval with 34 subjects yielded a correlation coefficient of 0.86, suggesting excellent temporal stability. CONCLUSIONS: The findings of this study show that the ISI-K is a reliable and valid instrument for assessing the severity of insomnia in a Korean population.
Humans ; Polysomnography ; Sleep Apnea, Obstructive ; Sleep Wake Disorders ; Sleep Initiation and Maintenance Disorders* ; Weights and Measures