Adult ; Female ; Granulomatosis with Polyangiitis ; complications ; Humans ; Nasal Obstruction ; etiology

AIMTo study the pharmacokinetics of spinosin in rat plasma after oral administration of Suanzaoren extract using sulfamethoxazole (SMZ) as internal standard by RP-HPLC method.

METHODSPlasma samples were deproteined with acetonitrile, followed by evaporation of the acetonitrile to dryness. The residual was then resolved in mobile phase and HPLC separation was achieved on a Hypersil C18 (5 microns, 200 mm x 4.6 mm ID) column at 35 degrees C. The mobile phase consisted of acetonitrile-water-acetic acid (15:85:1) at a flow rate of 0.7 mL.min-1. The UV detection wavelength was set at 334 nm.

RESULTSThe calibration curve was shown to be linear over the range from 18.1 to 903.5 micrograms.L-1 (r2 > or = 0.995). Mean recovery was 94.5%. Within-day and between-day precisions RSD were less than 9.0%. The limit of quantitation was 18.1 micrograms.L-1. The plasma spinosin was stable at -20 degrees C.

CONCLUSIONThe simple, sensitive and accurate HPLC method developed has been applied to determine the pharmacokinetics of spinosin in rat plasma after having taken Suanzaoren extract at a single dose.

Administration, Oral ; Animals ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacokinetics ; Flavonoids ; blood ; isolation & purification ; pharmacokinetics ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Seeds ; chemistry ; Ziziphus ; chemistry

OBJECTIVETo determine the role of 1,25(OH)2D3 on the secondary dentin formation and mineralization of the mice.

METHODSThe differences of the mandible mineralization between the wild-type and 1-alpha-hydroxylase gene knockout mice at 6 weeks old were assessed by hematoxylin-eosin (HE) staining, immunohistochemistry, alkaline phosphatase (ALP) histochemistry staining.

RESULTSThe ratio of caries were increased significantly, while the secondary dentin was reduced significantly, the deposition of type I collagen and osteocalcin on the secondary dentin of occlusion surface was decreased significantly, but the deposition of the Biglycan on the dentin was increased significantly, the active of ALP on the odontoblasts were reduced significantly in 1-alpha-hydroxylase gene knockout mice compared to that in the wild-type littermates.

CONCLUSION1,25(OH)2D3 deficiency lead to a defect in the secondary dentin formation and mineralization and caries of the mice.

Animals ; Dentin ; Dentin, Secondary ; Mandible ; Mice ; Osteocalcin

OBJECTIVETo investigate the distribution characteristics of the single nucleotide polymorphisms (SNPs) in the promoter region of the toll-like receptor 9 gene (TLR9) in Chinese Han children from Zhejiang province, and their associations with asthma susceptibility and phenotypes.

METHODSA case-control study was conducted. A total of 312 asthmatic children aged between 1.9 and 11.6 and 339 age matched healthy controls were enrolled in this study from April 2007 to November 2008. The -1486 C/T in rs187084 and -1237 C/T in rs5743836 loci of the TLR9 gene were genotyped by direct DNA sequencing of the PCR products. Serum levels of IFN gamma, IL-12 and IL-4 were detected by enzyme linked immunosorbent assay.Serum levels of total IgE were detected by chemiluminescence, and serum levels of antigen specific IgE antibodies were detected by fluoroenzymeimmunoassay.

RESULTS(1) The -1486 C/T polymorphism was identified in both groups. The genotype frequencies of TT, TC and CC at -1486 C/T were 41.0%, 44.3%, 14.7% in the healthy controls, and 38.8%, 48.4%, 12.8% in the asthmatic children. The -1237 C/T polymorphism was not detected in the population. (2) There were no statistically significant differences in the allele and genotype frequencies at the -1486 C/T locus between the two groups (P;>0.05). (3) Serum levels of IFN gamma and IL-4 differed significantly among the three genotypes at the -1486 C/T locus in asthmatic children (P<0.01). The CC genotype had the lowest levels of serum IFN gamma and the highest levels of serum IL-4 among the three genotypes. There were no significant differences in these cytokines among the healthy controls (P>0.05). No statistical differences of serum IL-12 were found among the three genotypes in the two groups (P>0.05). (4) There were no significant differences of total IgE (log-transformed) among the three genotypes in the asthmatic children (P>0.05).

CONCLUSIONThe -1237 C/T polymorphism of TLR9 gene was not detected in Chinese Han children in this study. The -1486 C/T polymorphism was associated with the levels of serum IFN gamma and IL-4 in children with asthma. However, there were no correlations between the -1486C/T polymorphism and serum IL-12 levels, total IgE levels or asthmatic susceptibility.

Asthma ; blood ; genetics ; Case-Control Studies ; Child ; China ; Female ; Genetic Predisposition to Disease ; Humans ; Male ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Toll-Like Receptor 9 ; genetics

AIMTo investigate how vasonatrin peptide (VNP) can attenuate the growth-promoting effect of hypoxia in cardiac fibroblasts cultured from neonatal rats.

METHODSThe cultured cardiac fibroblasts were divided randomly into four groups: control group, hypoxia group, hypoxia + VNP group and hypoxia + 8-Bromo-cGMP group. The growth of cardiac myocytes was measured by the means of MTT method. The effect of VNP on the intracellular level of cGMP and PCNA were measured by the means of radioimmunoassay and immunohistochemistry stain respectively.

RESULTSHypoxia (24 h) significantly increased the MTT A490nm value of cardiac fibroblasts (P < 0.05 vs control group). Both VNP (10(-7) mol/L) and 8-Bromo-cGMP (10(-3) mol/L) decreased MTT A490 nm value in cardiac fibroblast (P < 0.05 vs hypoxia group). VNP (10(-7) mol/L) increased the intracellular level of cGMP (P < 0.05 vs control and hypoxia group). Hypoxia (24 h) significantly increased the expression of proliferating cell nuclear antigen (PCNA) in cardiac myocytes (P < 0.05, vs control group), but VNP (10(-7) mol/L) decreased it.

CONCLUSIONVNP can attenuate hypoxia-induced growth-promoting effect in cardiac fibroblasts which is associated with the changes of cGMP and PCNA.

Animals ; Animals, Newborn ; Atrial Natriuretic Factor ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Cyclic GMP ; metabolism ; Myoblasts, Cardiac ; cytology ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Rats, Sprague-Dawley

AIMTo investigate effect and mechanism of vasonatrin peptide (VNP) on Ca2+ activated K+ channels (K(Ca)) of vascular smooth muscle cells (VSMCs) isolated from rat mesentery arteries.

METHODSChanges of K(Ca) induced by VNP were measured by the means of whole cell recording mode of patch clamp, furthermore effects of HS-142-1(0.3 g/L), 8-Br-cGMP and methylene blue (MB) were observed.

RESULTSK(Ca) was significantly enhanced by VNP (10(-6) mol/L), which was mimicked by 8-Br-cGMP(10(-3) mol/L) and blocked completely by HS-142-1 or MB (2 x 10(-5) mol/L).

CONCLUSIONVNP increases K(Ca) of VSMCs isolated from rat mesenteric arteries, by binding with natriuretic peptide guanylate cyclase-coupled receptors and increasing the intracellular level of cGMP in VSMCs.

Animals ; Atrial Natriuretic Factor ; pharmacology ; Male ; Mesenteric Arteries ; cytology ; drug effects ; metabolism ; Muscle, Smooth, Vascular ; metabolism ; physiology ; Potassium Channels, Calcium-Activated ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo examine the activation and cytotoxicity of human peripheral blood T lymphocyte induced in vitro by human 4-1-BB ligand (4-1-BBL) gene transfected into tumor Tca8113 cells.

METHODSThe eukaryotic expression vector pEGFP-h4-1-BBL was transfected into human oral carcinoma cell line Tca8113 by Lipofectamine 2000. The transfected cells were then selected in medium containing G-418, cloned by limited dilution and named as Tca8113-4-1-BBL. Human 4-1-BBL mRNA and protein expression of transfected cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting respectively. The tumor cell vaccines (TCV) were obtained by treatment with mitomycin (MMC). Human peripheral blood mononuclear cells (PBMC) were prepared from lymphoprep, and then stimulated with anti-CD-3 mAb and incubated with non-transfected or transfected TCV-Tca8113 cells, respectively. The proliferation of T cells was evaluated by trypan blue exclusion; the CCK-8 was used to detect the cytotoxic effect of T lymphocytes. Meanwhile, the secretion of interferon-gamma (IFN-gamma) and interleukin (IL)-2 in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe Tca8113 cells transfected by pEGFP-h4-1-BBL could express human 4-1-BBL efficiently. As compared with wild type Tca8113 cells, the transfected Tca8113 cells could markedly promote proliferation, IL-2 and IFN-gamma production and cytotoxic activity of lymphocytes.

CONCLUSIONSThe transfection of human 4-1-BBL gene in Tca8113 cells is effective in enhancing its immunogenicity and inducing antitumor immune response in vitro.

4-1BB Ligand ; genetics ; Carcinoma, Squamous Cell ; genetics ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Humans ; Interferon-gamma ; immunology ; Interleukin-2 ; immunology ; Lymphocyte Activation ; Mouth Neoplasms ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

The interaction among collagen, von Willebrand factor (vWF) and glycoprotein Ib axis is the first step in hemostasis and thrombosis, especially under high shear condition. To develop a new remedy of anti-thrombosis, mRNA from endothelial cells was extracted, and reverse transcription PCR was adopted to amplify DNA of interest. After sequencing, recombinant expression vector was constructed. The amplified DNA fragment of vWF domain A3 was inserted into expression vector with 6 x his taq, pET20b(+), the recombinant was transformed into E coli (strain DE3) and induced by IPTG. Recombinant vWF-A3 was designated as a recombinant fragment comprising residues 918 - 1114 of mature vWF subunit. It was purified through Ni-NTA resin column and refolded in Tris buffer containing GSH and GSSG. The results showed that rvWF-A3 was expressed successfully in E coli (strain DE3), accounting for 46% of total bacterial protein with its purity of over 95%. It was identified that rvWF-A3 is capable to bind collagen and inhibit the wild vWF binding to collagen by competition. It is concluded that rvWF-A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.
Cloning, Molecular ; Collagen ; metabolism ; Escherichia coli ; genetics ; Humans ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; von Willebrand Factor ; chemistry ; genetics ; metabolism

OBJECTIVETo construct a convenient, reliable and visual model of hair follicle development to test the hair-inductive potential of follicular cells and investigate the molecular mechanism regulating hair follicle morphogenesis and cycling.

METHODSAn open chamber was transplanted into the nude mice dorsal skin, dermal and epidermal cells isolated from newborn C57BL/6 mice skin were mixed at a specific ratio and then injected into the chamber together, 1 week after transplantation, the chamber was removed, and then, hair formation and regeneration after hair plucking was observed.

RESULTS1 week after cells implantation, the wound was moist without apparent contraction and among that pink and translucent tissue was formed. 2 weeks after implantation, the wound healed completely. 3 weeks after implantation, black hair grew from the skin was observed. 4 weeks after implantation, thick and black hair grew from the skin vertically. Completely developed structure of hair follicle was observed with paraffin section and HE staining. 1 week after plucking, new hair had regrown. The ratio of cell component was varied, whereas the other component was fixed at 1 x 10(7) cells. When the number of epidermal cells was reduced to 1 x 10(6) cells, the efficiency of hair follicle reconstitution was mostly unchanged. On the other hand, the density of newly formed hair was diminished considerably by reducing the number of dermal cells to 5 x 10(6) cells or lower. Neither epidermal cells nor dermal cells transplanted alone formed hair follicle.

CONCLUSIONSNewborn mice skin cells transplanted by chamber method can construct a complete model of hair follicle development, which can be used to test the hair-inductive potential of follicular cells and investigate the molecular mechanism regulating hair follicle morphogenesis and cycling.

Animals ; Cells, Cultured ; Hair ; physiology ; Hair Follicle ; physiology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; Regeneration ; Skin ; cytology

The present work was to investigate the effects of vasonatrin peptide (VNP) on cardiomyocyte protein synthesis induced by moderate hypoxia. In cultured neonatal rat cardiomyocytes, MTT methods, total protein measurement and (3)H-leucine incorporation were used to calculate the cell number and measure the protein synthesis of cardiomyocytes. Furthermore, radioimmunoassay was undertaken to observe the effects of VNP on the intracellular levels of cAMP, cGMP and the concentration of endothelin (ET) in the culture medium. The results showed that both the cell number and protein synthesis decreased with severe hypoxia for 24 h. In contrast, under moderate hypoxia, cardiomyocyte hypertrophy developed; the protein synthesis as evidenced by total protein content and 3H-eucine incorporation increased significantly. VNP reduced cardiomyocyte protein synthesis induced by moderate hypoxia in a dose-dependent manner. Furthermore, VNP increased the intracellular level of cGMP and decreased the concentration of ET in the culture medium under moderate hypoxia, but had no effect on the level of cAMP. These results suggest that VNP inhibits moderate hypoxia-induced protein synthesis in cultured neonatal rat cardiac myocytes. This effect is mediated, at least in part, by an increase in intracellular cGMP, a reduction in synthesis, and/or a release in ET of cardiomyocytes.
Animals ; Animals, Newborn ; Atrial Natriuretic Factor ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Dose-Response Relationship, Drug ; Endothelins ; biosynthesis ; Myocytes, Cardiac ; metabolism ; Protein Biosynthesis ; Rats ; Rats, Sprague-Dawley