Objective To develop a murine model for infection by Cladosporium carrioni. Methods A total of 72 ICR mice were equally divided into 4 groups, group A (healthy mice inoculated by C. Carrioni suspension of 1 × 108 cfu conidia mL-1, group B (immune-suppressed mice inoculated by C. Carrioni sus pension of 1 × 108 cfu conidia mL-1), group C (immune-suppressed mice inoculated by C. Carrioni suspen-sion of 1 × 106 cfu conidia mL-1), group D (healthy mice inoculated by sodium chloride solution). C. Car-rioni suspension or sodium chloride solution was subcutaneously inoculated into foot pads of mice. On day 7, 30 and 60 after inoculation, 6 mice were killed in each group followed by the measurement of thickness of foot pads, pathology and mycology of skin samples taken from foot pads. Results In group A, B and C, there were swelling, blackening, ulceration and crusts at the inoculation site of all mice, with a morbidity of 100%. The thickness of foot pads in group A on day 30 was significantly higher than that on day 7 (2.40 ± 0.45 mm vs 2.85 ± 0.47 mm, P < 0.05), but lower than that on day 60 (1.64 ± 0.13 ram, P < 0.05). In group B, increased thickness of foot pads was observed on day 30 compared with that on day 7 and day 60 (2.19 ± 0.27 mm vs 1.80 ± 0.21 mm and 1.86 + 0.22 mm, respectively, both P < 0.05), which was the case with group C (1.98 ± 0.06 nun vs 1.51 ± 0.11 mm and 1.82 ± 0.09 mm, respectively, both P < 0.05). No significant changes occurred to the thickness of foot pads in group D from day 7 to day 60 (P > 0.05). Pathological changes in group A, B and C included necrosis, abscess and chronic granuloma formation; dark brown sclerotic bodies were observed on HE and PAS staining as well as on direct microscopy; cultures of tissue samples grew Cladosporium carrionii. The mice in group D remained uninfected. Conclusion Mouse model for chromoblastomycosis may be established by subcutaneous inoculation of Cladosporium carrionii suspension into foot pads of healthy or immuno-suppressed mice.

Objective To investigate the effect of sulforaphane on 1-methyl-4-phenylpyridinium (MPP +)-induced cell viability loss in cultured PC12 cells and to explore the possible mechanism.Methods MPP + induced damage in PC12 cells was prepared as oxidative damage model.Sulforaphane (0.5,1.O,2.5,5.0 and 10.0 μmol/L) was added in each group cell growth medium.Subsequent experiments were divided into 4 groups:(A) normal control group,(B) sulforaphane group,(C) MPP+ injury group,(D)sulforaphane pretreatment + MPP+ injury group.Cell viability was detected by MTT assay,and the sulforaphane pretreatment PC12 cell viability was observed in different concentrations.Flow cytometry was used to detect changes in the rate of apoptosis in different packet PC12 cells,and protein expression levels of nuclear factor erythroid2-related factor 2 (Nrf2),heme oxygenase (HO-1) and human NAD (P) H dehydrogenase,quinone 1 (NQO1) were detected by Western blot when the PC12 cells were incubated with sulforaphane (2.5 μmol/L) and (or) MPP+ (500 μmol/L) for 24 h in vitro.Results Compared to control group (cell survival rate was 98.70％),the survival percents of PC12 cells were significantly decreased in MPP+-treated group (58.16％).A significant difference was showed between group A and C (F =21.83,P ＜ 0.05),and the cell survival rate in group D was significantly improved.Compared to control group,the rate of apoptosis in MPP+ injury group was increased,and the rate of apoptosis after pretreatment of the sulforaphane was significantly reduced.Compared to MPP+ injury group,the levels of Nrf2,HO-1 and NQO1 protein expression were significantly increased in sulforaphane pretreatment group.Conclusion Sulforaphane have a protective effect against MPP+-induced PC12 cell model damage,and the protective effect may be achieved by activating the Nrf2-antioxidant response element pathway.

Objective To develop a murine model of chromomycosis by using Cladosporium carrioni and explore the pathogenicity of Cladosporium carrio nii in mice. Methods The suspension of Cladosporium carrioni was inoculated to two groups of mice, the immunocompetent mice and the immunosuppressed mice, b y intraperitoneal route using 1 ml inoculum containing 108 conidia/mL. All mice were sacrificed 30 days after inoculation, and then macroscopic examination, his topathology and fungal culture were performed. Results The morbidity in both g roups was 100% according to the dark brown hyphae and sclerotic bodies found in histopathologic examination and fungal culture. Macroscopic examination found th at the adhesion among the internal organs in immunocompetent mice was more sever e than that in immunosuppressed mice. Histopathologic sections showed that necro sis and inflammatory infiltration in immunocompetent mice were more obvious than those in immunosuppressed mice. Conclusions The virulence of Cladosporium car rionii strains is strong enough to construct experimental murine model of chromo mycosis, and animal passage of the strains is unnecessary. This murine model cou ld be used to study the pathogenesis of chromomycosis.

OBJECTIVE: To explore new hallmarks affecting the prognosis of patients with laryngeal squamous cell carcinoma (LSCC) via investigating the expression of CyclinE and p27 in LSCC tissues. METHOD: The expression of CyclinE and p27 was detected via Elivision immunohistochemical staining in 160 LSCC tissues and 20 normal laryngeal tissues (NLT). The relationship between CyclinE/ p27 and LSCC/ NLT was analyzed via Log-rank analysis. The relationship of CyclinE and p27 protein was statistically analyzed by spearman correlation analysis. The relationship between CyclinE/p27 and clinical-pathology-factors of patients with LSCC, such as age, gender, tumor site, diameter, differentiation, lymph node metastasis and PTNM stage were analyzed by Chi-square test. The relationship between clinical-pathology-factors, CyclinE, p27 and overall survival time of patients with LSCC was analyzed via Cox multiplicity and Kaplan-Meier survival analysis. A significant difference was recognized by P<0.05. RESULT: In LSCC the positive rates of CyclinE and p27 protein was 62.50% and 41.25% respectively (P<0.05). In NLT the positive rates of CyclinE and p27 protein was 35% and 70% respectively (P<0.05). The expression of CyclinE or p27 protein was closely correlated with lymph node metastasis, PTNM stage of patients with LSCC (P<0.05). The expression of CyclinE and p27 had no significant correlations with patients' gender, age and tumor site, diameter differentiation (P>0.05 for all). A negative correlation was found between the expression of CyclinE and p27 protein, r= -0.767(P<0.05). Kaplan-Meier survival analysis showed that the overall survival rate of patients with LSCC was 36.9% (P<0.05). The 5-year survival rate in positive group of CyclinE was 8%, in negative group was 80% (P<0.05). On the contrary, the 5-year survival rate of patients with LSCC in positive group of p27 protein was 77.27%, the rate was 5.32% in negative group (P<0.05). Cox multivariate regression analysis revealed that lymph node metastasis, PTNM stage, CyclinE and p27 were independent risk factors of prognosis for patients with LSCC. CONCLUSION It is the molecular basis underlying the development and invasion/ metastasis of LSCC that activation of CyclinE gene accompanying inactivation of p27 gene. It is very important of co-detecting CyclinE and p27 protein to predict the prognosis of patients with LSCC.
Carcinoma, Squamous Cell ; metabolism ; pathology ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Female ; Head and Neck Neoplasms ; metabolism ; pathology ; Humans ; Laryngeal Mucosa ; metabolism ; pathology ; Laryngeal Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Oncogene Proteins ; metabolism ; Prognosis ; Squamous Cell Carcinoma of Head and Neck ; Survival Rate

Objective To use the fluorescence PCR‐melting curve method to detect CYP2C9 and VKORC1 gene polymorphism in Xinjiang Hui population ,to analyze their gene distribution and gene mutation frequency ,and to evaluate the clinical applicability of the fluorescence PCR‐melting curve method .Methods The fluorescence PCR‐melting curve method and sequencing method were adopted to contrastively detect CYP2C9*2 ,CYP2C9*3 and VKORC1(‐1639G/A)gene polymorphism .Results Among detected 228 Xinjiang Hui individuals ,199 cases of CYP2C9*1/*1 ,2 cases of CYP2C9*1/*2 ,26 cases of CYP2C9*1/*3 and only 1 case of CYP2C9*3/*3 were detected ,no case of CYP2C9*2/*2 and CYP2C9*2/*3 was detected .Two kinds of allele G and A were detected for VKORC1(‐1639G/A) ,in which VKORC1‐1639G/G type was detected in 2 cases ,VKORC1‐1639G/A type was detected in 39 cases and VKORC1‐1639A/A type was detected in187 cases ,compared with the sequencing method ,the results of the fluorescence PCR‐melting curve method were completely consistent .Conclusion Xinjiang Hui population also has CYP2C9 gene *2 ,*3 loci and VKORC1 gene(‐1639G/A) locus polymorphism ,their occurrence frequency has a certain difference with Xingjiang Uygur and other regional populations ,the adopted fluorescence PCR‐melting curve method used in the gene polymorphism detection can meet clinical detection requirements .

AIMTo investigate the activation of the nuclear factor of activated T cells (NFAT) and its function in the corticosterone (CORT)-induced apoptosis of rat Leydig cells.

METHODSNFAT in rat Leydig cells was detected by Western blotting and immunohistochemical staining. Cyclosporin A (CsA) was used to evaluate potential involvement of NFAT in the CORT-induced apoptosis of Leydig cells. Intracellular Ca(2+) was monitored in CORT-treated Leydig cells using Fluo-3/AM. After the Leydig cells were incubated with either CORT or CORT plus CsA for 12 h, the levels of NFAT2 in the nuclei and in the cytoplasm were measured by semi-quantitative Western blotting. The role of NFAT2 in CORT-induced Leydig cell apoptosis was further evaluated by observing the effects of NFAT2 overexpression and the inhibition of NFAT2 activation by CsA on FasL expression and apoptosis.

RESULTSWe found that NFAT2 was the predominant isoform in Leydig cells. CsA blocked the CORT-induced apoptosis of the Leydig cells. The intracellular Ca(2+) level in the Leydig cells was significantly increased after the CORT treatment. The CORT increased the level of NFAT2 in the nuclei and decreased its level in the cytoplasm. CsA blocked the CORT-induced nuclear translocation of NFAT2 in the Leydig cells. Both CORT-induced apoptosis and FasL expression in the rat Leydig cells were enhanced by the overexpression of NFAT2 and antagonized by CsA.

CONCLUSIONNFAT2 was activated in CORT-induced Leydig cell apoptosis. The effects of NFAT2 overexpression and the inhibition of NFAT2 activation suggest that NFAT2 may potentially play a pro-apoptotic role in CORT-induced Leydig cell apoptosis through the up-regulation of FasL.

Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Nucleus ; metabolism ; Corticosterone ; pharmacology ; Cytoplasm ; metabolism ; Immunohistochemistry ; Kinetics ; Leydig Cells ; cytology ; drug effects ; physiology ; Male ; NFATC Transcription Factors ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective To examine the prevalence ofHelicobacter pylori in Shanghai and relevant risk factors, evaluate the resistance proifle ofH. pylori isolates to antibiotics used in ifrst-line therapy in two hospitals in Shanghai.MethodsH. pylori were isolated from the biopsy samples of gastric mucosa collected from September 2013 to January 2015. Antimicrobial susceptibility test was performed by E-test method for 131H. pylori strains to 4 antibiotics, clarithromycin, metronidazole, amoxicillin and tetracycline. Results A total of 955 patients receiving gastroscopy were enrolled. And 248 (26.0%)H. pylori strains were isolated from the biopsy samples of gastric mucosa. Overall, 14.5%, 64.1%, 0 and 0.8% of the strains were resistant to clarithromycin, metronidazole, amoxicillin and tetracycline, respectively. Resistance to two drugs was found in 10.7%(14/131) of the strains, and majority (92.8%, 13/14) of which were resistant to clarithromycin and metronidazole.Conclusions The prevalence ofH. pylori in gastric mucosa is rather lower compared with the data reported previously. It is associated with the sex, age and clinical outcome of patients, however, antibiotic resistance profile is not related to these factors.H. pylori eradication therapy should be individualized according to the results of susceptibility test in Shanghai.

AIM AND METHODSTo investigate the role of mitogen-activated protein kinase phosphatase-1 (MKP-1) in the regulation of cells proliferation, the expression of MKP-1 and extracellular signal-regulated kinase-1 (ERK-1) in heart and aorta of spontaneous hypertensive rat (SHR) and WKY were studied. We also investigated the effect of MKP-1 genes,which were transfected into vascular smooth muscle cells (VSMC) using the classical calcium phosphate coprecipitation technique, on the incorporation of 3H-TdR in VSMC stimulated by angiotensin II (Ang II).

RESULTS(1) Compared with that of WKY, MKP-1 expression in heart and aorta were significantly decreased by 53% (P < 0.01) and 45% (P < 0.01) in SHR, respectively. While the expression of ERK-1 in heart and aorta of SHR were higher than that of WKY (P < 0.01). The ratio of ERK-1/MKP-1 in heart and aorta of SHR were significantly higher than that of WKY. (2) 3H-TdR incorporation in VSMC stimulated by Ang II (10(-7) mol/L) was increased by 207% (P < 0.01), compared with control group. In the transfected cells with wild MKP-1 gene, Ang II-induced incorporation of 3H-TdR lowered 63%, compared with untransfected cells (P < 0.05). There were no marked inhibitive role between mutant MKP-1-transfected cells and blank vector-transfected cells in response to Ang II, compared with Ang II group (P > 0.05).

CONCLUSIONThese results showed that the expression of ERK-1 in heart and aorta isolated from SHR, which stimulated proliferation and hypertrophy of cells, is higher than that of MKP-1 which dephosphorylates and inactivated ERK-1. In addition, MKP-1 significantly inhibits Ang II-stimulated proliferation of VSMC.

Angiotensin II ; pharmacology ; Animals ; Aorta ; cytology ; enzymology ; Cell Proliferation ; Cells, Cultured ; Dual Specificity Phosphatase 1 ; metabolism ; Heart ; Hypertension ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocardium ; cytology ; enzymology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY

This study was purposed to construct the recombinant hF9 minigene and its stable nonsense mutant cell lines, and to investigate its significance. Minigene hF9 was cloned into the mammalian expression vector pCMV-Tag3B; a nonsense mutant containing a premature termination codon (PTC) in the 121(st) amino acid residue was obtained by PCR site-directed mutagenesis; minigene hF9 and nonsense mutant were respectively transfected into HepG2 cells with G418 treatment to get stable HepG2-WT and HepG2-N cell lines. The results confirmed that the minigene hF9 and nonsense mutant were constructed successfully. The gene of interest was amplified by RT-PCR from the stable cell lines, and the minigene hF9 was expressed in the stable cell lines. It is concluded that the recombinant hF9 minigene and its stable nonsense mutant cell lines are constructed successfully. The cell lines can be used to screen the drugs treating the nonsense mutation-caused hemophilia according to PTC read-through approaches.
Cell Line, Tumor ; Codon, Nonsense ; Factor IX ; genetics ; Genetic Vectors ; Hemophilia B ; genetics ; Humans ; Recombinant Proteins ; genetics

OBJECTIVETo evaluate the influence on the synaptic protein expression in different brain regions of ICR mice after lambda-cyhalothrin (LCT) exposure during postnatal period.

METHODSTwo male and 4 female healthy ICR mice were put in one cage. It was set as pregnancy if vaginal plug was founded. Offspring were divided into 5 groups randomly, and exposed to LCT (0.01% DMSO solution) at the doses of 0.1, 1.0 and 10.0 mg/kg by intragastric rout every other day from postnatal days (PND) 5 to PND13, control animals were treated with normal saline or DMSO by the same route. The brains were removed from pups on PND 14, the synaptic protein expression levels in cortex, hippocampus and striatum were measured by western blot.

RESULTSGFAP levels of cortex and hippocampus in the LCT exposure group increased with doses, as compared with control group (P < 0.05), while Tuj protein expression did not change significantly in the various brain regions of ICR mice. GAP-43 protein expression levels in the LCT exposed mouse hippocampus and in female ICR mouse cortex increased with doses, as compared with control group (P < 0.05). Presynaptic protein (Synapsin I) expression levels did not change obviously in various brain regions. However, postsynaptic density protein 95 (PSD95) expression levels of the hippocampus and striatum in male offspring of 10.0 mg/kg LCT group, of cortex of female LCT groups, and of female offspring in all exposure groups, of striatum, in 1.0 or 10.0 mg/kg LCT exposure groups significantly decreased (P < 0.05).

CONCLUSIONSEarly postnatal exposure to LCT affects synaptic protein expression. These effects may ultimately affect the construction of synaptic connections.

Animals ; Animals, Newborn ; Brain ; drug effects ; metabolism ; Corpus Striatum ; drug effects ; metabolism ; Female ; Hippocampus ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Nitriles ; toxicity ; Pyrethrins ; toxicity ; Synapsins ; metabolism