Electroacupuncture Promotes Angiogenesis in Mice with Cerebral Ischemia by Inhibiting miR-7.

OBJECTIVE: To observe the angiogenesis effect of electroacupuncture (EA) at Shuigou acupoint (GV 26) in the treatment of cerebral ischemia, and explore the value of miRNA-7 (miR-7) in it. METHODS: First, 48 mice were randomly divided into sham operation, middle cerebral artery occlusion (MCAO) model, and EA treatment groups. Then 9 mice were divided into carrier control group, miR-7 knockout group and miR-7 overexpression group (n=3 each group). Finally, 20 mice were divided into model and carrier control group, model and miR-7 knockout group, EA treatment and carrier control group and EA treatment and miR-7 overexpression group, with 3-6 mice in each group. The MCAO model was established in the MCAO and EA groups. Neurological deficit score and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to evaluate the severity of cerebral ischemia. Hematoxylin-eosin staining was used to describe basic pathological changes. Immunohistochemistry was used to quantify cerebral microvessel density. Real-time PCR and Western blot were used to detect the expression of miR-7 and its downstream target genes Krüppel-like factor 4/vascular endothelial growth factor (KLF4/VEGF) and angiopoietin-2 (ANG-2) in the ischemic cerebral cortex. RESULTS: After EA, neurological deficit scores and infarction volumes decreased, and the density of cerebral microvessels increased. In the MCAO group, miR-7 expression was higher than that in the sham group (P<0.01). After EA at GV 26, miR-7 expression decreased (P<0.01) and the expression of downstream target genes KLF4/VEGF and ANG-2 increased as compared with the MCAO group (P<0.01). After EA combined with overexpression of miR-7, the expression of downstream target genes KLF4/VEGF and ANG-2 decreased compared to the control EA group (P<0.01). After miR-7 knockdown, the expression of KLF4/VEGF and ANG-2 increased (P<0.05 or P<0.01). CONCLUSIONS: EA could promote angiogenesis in MCAO mice likely by inhibiting the expression of miR-7 and relieving inhibition of downstream target genes KLF4/VEGF and ANG-2.

Characterization of membrane occupation and recognition nexus repeat containing 3, meiosis expressed gene 1 binding partner, in mouse male germ cells.

Mammalian spermatogenesis is a well-organized process of cell development and differentiation. Meiosis expressed gene 1 (MEIG1) plays an essential role in the regulation of spermiogenesis. To explore potential mechanisms of MEIG1's action, a yeast two-hybrid screen was conducted, and several potential binding partners were identified; one of them was membrane occupation and recognition nexus repeat containing 3 (MORN3). MORN3 mRNA is only abundant in mouse testis. In the testis, Morn3 mRNA is highly expressed in the spermiogenesis stage. Specific anti-MORN3 polyclonal antibody was generated against N-terminus of the full-length MORN3 protein, and MORN3 expression and localization was examined in vitro and in vivo. In transfected Chinese hamster ovary cells, the antibody specifically crossed-reacted the full-length MORN3 protein, and immunofluorescence staining revealed that MORN3 was localized throughout the cytoplasm. Among multiple mouse tissues, about 25 kDa protein, was identified only in the testis. The protein was highly expressed after day 20 of birth. Immunofluorescence staining on mixed testicular cells isolated from adult wild-type mice demonstrated that MORN3 was expressed in the acrosome in germ cells throughout spermiogenesis. The protein was also present in the manchette of elongating spermatids. The total MORN3 expression and acrosome localization were not changed in the Meig 1-deficient mice. However, its expression in manchette was dramatically reduced in the mutant mice. Our studies suggest that MORN3 is another regulator for spermatogenesis, probably together with MEIG1.

Urinary S-phenylmercapturic acid as a key biomarker for measuring occupational exposure to low concentrations of benzene in Chinese workers: a pilot study.

OBJECTIVE: This study analyzed the level of urinary S-phenylmercapturic acid (U-SPMA) for low benzene exposure in a group of Chinese shoe-making workers. METHODS: Urinary samples from 55 workers exposed to benzene at levels lower than 10 parts per million (ppm) were collected at postshift. U-SPMA level was determined using high-performance liquid chromatography/mass spectrography (HPLC/MS) method. RESULTS: Good linearity of U-SPMA was observed within the range from 10 to 320 µg/L (r = 0.9994). Concentration of airborne benzene ranged from 0.71 to 32.17 mg/m³, and three segments were divided with different levels of exposure (≤6.0, 6.0 to 10.0, 10 to 32.5 mg/m³), the median U-SPMA concentrations were 49.55, 102.15, and 335.69 µg/g Cr, respectively. CONCLUSION: A good linear correlation was found between U-SPMA levels and airborne benzene concentrations. The selected method could be applied for detecting other working conditions in China.

[Construction of a recombinant plasmid of BC022687 and identification of its expression and localization in CHO cells].

OBJECTIVE: To construct a mammalian expression plasmid of the BC022687 gene and investigate the expression and localization of the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The BC022687 coding sequence was amplified by polymerase chain reaction (PCR) and subcloned into the pEGFP-C1 vector carrying the gene of green fluorescence protein (GFP). After the target region was sequenced, the recombinant plasmid was transfected into CHO cells, and its expression in the CHO cells was determined by Western blot. The localization of GFP-tagged BC022687 in the CHO cells was observed by laser scanning confocal microscopy. RESULTS: BC022687 was successfully cloned into the mammalian expression vector pEGFP-C1, with the restriction fragment length of 950 bp. The expression of the fusion protein was confirmed, with the relative molecular weight of 64 000. The GFP-tagged BC022687 protein was mainly localized in the cytoplasm, and also presented in the centrioles in the transfected CHO cells. CONCLUSION: The successful construction of the plasmid expressing BC022687 in CHO cells has laid a foundation for further studies on the role of this protein in ciliogenesis.

[A cross-sectional study about the effects of residential exposure to power transmission line on human nervous system symptoms and hematology].

OBJECTIVE: To investigate the possible effects on nervous system and health condition under the exposure to electromagnetic field. METHODS: Take the resident around the power transmission line as the objects and were divided into 3 groups by the distance from the power transmission line 20 m, 100 m and 500 m, respectively. Some living conditions and health conditions were recorded by face-to-face the questionnaire survey, and Hematological indices of each groups were examined including IgG, IgM, leukocyte formulae, erythrocyte, hemoglobin and platelet. RESULTS: There was no significant difference in each group, according exposure of daily life, such as drinking and smoking (P > 0.05). Compared with the each distance groups, it was presented significant difference between the distance from the power transmission line and the incidence of headache or dizziness, insomnia and easy weary and so on (P < 0.05). In hematology aspect, with the horizontal distance from the power transmission line decreasing, PLT level of residents was reductive and the difference was statistically significant (P < 0.001), whereas leukocyte formulae, erythrocyte, hemoglobin, IgG and IgM had no significant difference among each group (P < 0.05). CONCLUSION: Closely exposure to electromagnetic field may induce headache and so on and decrease the level of PLT.

Trans, trans-muconic acid as a biomarker of occupational exposure to high-level benzene in China.

OBJECTIVE: The work aimed to study the potential correlation between the high-level benzene exposure and its urinary metabolites S-phenylmercapturic acid (SPMA) and trans, trans-muconic acid (t,t-MA) in Chinese shoe-making workers. METHODS: Individual benzene-exposed levels were determined by gas chromatography analysis, urinary t,t-MA, and urinary SPMA were determined by high performance liquid chromatography-an ultraviolet detector and liquid chromatography/electrospray tandem mass spectrometry method, respectively. RESULTS: The concentration of benzene ranged from 2.57 to 146.11 mg/m³. And the correlation between benzene and t,t-MA was significantly higher than that of SPMA at the postshift, for example, the correlation coefficient was 0.905 and 0.537 for t,t-MA and SPMA, respectively. Moreover, The relative internal exposure index of t,t-MA (0.28 mg/g Cr: mg/m³) was more similar to the data supplied by American Conference of Governmental Industrial Hygienists compared to the index of SPMA (0.025 mg/g Cr:mg/m³). CONCLUSIONS: t,t-MA appeared to be a more specific biomarker than SPMA at high-level benzene exposure.

[Establishment of biological limit value of urinary S-phenylmercapturic acid for occupational exposure to benzene].

OBJECTIVE: To establish the biological exposure limit values of urinary S-phenylmercapturic acid (SPMA) for assessing occupational exposure to benzene. METHODS: Study participants were selected from 55 workers of benzene exposures below 32.5 mg/m(3). The concentration of personal exposure to benzene was measured by gas chromatography and sampled with personal sampler. The urine samples were collected at the end of work shift and individual internal exposure level was evaluated by determination of SPMA in urine by HPLC/MS method. Comparison of external and internal exposure was assessed by the relative internal exposure (RIE) index. RESULTS: The benzene exposure level ranged from 0.71 to 32.17 mg/m(3) (geometric mean 6.98 mg/m(3), median 7.50 mg/m(3)). The urinary SPMA at the end of the work shift were significantly correlated with benzene exposure, (microg/g Cr) = -8.625 + 18.367X (mg/m(3)), r = 0.8035, (P < 0.01). According to the occupational exposure limit for benzene in China and calculation of regression equation, the expected value of urinary SPMA was 101.58 microg/g Cr. Mean level of biotransformation of per mg/m(3) benzene to urinary SPMA was 18.23 microg/g Cr and the metabolic efficiencies of benzene transformation to urinary SPMA decreased with benzene exposure increased. CONCLUSION: Based on abroad documents and data, biological limit value for occupational exposure to benzene in China is recommended as follows: 100 microg/g Cr (47 micromol/mol Cr) for SPMA in the urine at the end of shift.

[Effect of NS-398 on cyclooxygenase-2 expression and proliferation of HepG2 cells].

OBJECTIVE: To investigate anticancer effect and molecular mechanism of N-[(Cyclohexyloxy)-4-nitrophenyl] methanesulfonamide on HepG2 cells in vitro. METHODS: HepG2 cells were treated with various concentrations (100, 200, 300, 400 micromol/L) of NS-398 [selective for cyclooxygenase 2 (COX-2) inhibition]. Cell growth was measured by MTT method, DNA fragmentation gel analysis was used to analyze the apoptosis cells, DNA ploidy and apoptotic cell percentage were examined by flow cytometry (FCM). PGE2 concentration was measured by radioimmunoassay method. The expressions of COX-2 were also examined by Western blot analysis. RESULTS: NS-398 inhibited HepG2 cell proliferation and induced apoptosis in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased and quiescent G1 phase was accumulated with NS-398 concentration increasing. The IC50 of 24 hours was 300 micromol/L. The release of PGE2 was significantly reduced in HepG2 cells with the values of NS-398 being (0.70 +/- 0.02), (0.48 +/- 0.02), (0.29 +/- 0.01) and (0.18 +/- 0.01) respectively, as compared with control group (0.03 +/- 0.01). NS-398 could inhibit the activity and expression of COX-2, with higher concentration, it can significantly down-regulate the expression of COX-2 (t = 3.736, 1.623, 1.810, 2.587, P < 0.01). CONCLUSION: NS-398 might significantly inhibit the proliferation of HepG2 cells and induce apoptosis. The mechanisms were related with the accumulation of quiescent G1 phase and the inhibition of COX-2 activity.

High-performance liquid chromatographic determination of urinary trans, trans-muconic acid excreted by workers occupationally exposed to benzene.

OBJECTIVE: To investigate the relationship between trans, trans-muconic acid (ttMA) as benzene metabolite of occupational workers and benzene concentration in air. METHODS: A rapid and sensitive high-performance liquid chromatography was developed to determine the level of urinary ttMA. ttMA was extrated from urinary samples in liquid-liquid phase a ODS (2) (5u) column (phi 4.6 mm x 150 mm) and detected at wavelength 264 nm in a UV detector using vanillic acid as an internal standard. The mobile phase was acetaticacid/tetrahydrofuran/methanol/water (v/v, 1:2:10:87). The method was validated with 56 urine samples collected from occupationally benzene-exposed individuals. RESULTS: A correlation coefficient (r = 0.9963) was found for ttMA ranging 0.10-10.00 microg/mL. The limit of detection was 0.10 microg/mL. The recovery and reproducibility were generally over 90%. There was a positive correlation between ttMA and benzene level in air. The equation was Y = 0.859 + 0.108C (before work, r = 0.6200) or Y = 1.980 + 0.179C (after work, r = 0.7930). CONCLUSION: This method can be used to determine and control the level of urinary ttMA in those who are occupationally exposed to benzene.