Construction of Saccharomyces cerevisiae whole-cell biocatalyst system for conversion miltiradiene / 药学学报

Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases (SmCPR1) from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.

Methylation analysis of CpG island DNA of FMR1 gene in the fragile X syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a method of methylation-sensitive restriction enzymes based quantitative PCR (MSRE-qPCR) for analysis of CpG island DNA of FMR1 gene, and to assess its value for molecular diagnosis of fragile X syndrome.</p><p><b>METHODS</b>Thirty boys with mental retardation and abnormal repeats of 5'(CGG)n in the FMR1 gene and 20 mothers were analyzed by conventional PCR screening. Eag I was used to digest genomic DNA, and qPCR was performed to amplify CpG island in the FMR1 gene using both undigested and digested templates. Raw Ct values were obtained through quantitative PCR amplification. The degree of CpG island methylation was calculated by 2 - U+0394 U+0394 Ct. The result of MSRE-qPCR was verified by Southern blotting. 30 healthy females and 30 healthy males were used as controls to optimize the established MSRE-qPCR method.</p><p><b>RESULTS</b>The ranges of 2 - U+0394 U+0394 Ct value for normal methylation, partial methylation and full methylation were determined. Among the 30 patients, 3 were found to have partial methylation of CpG island of the FMR1 gene, and 27 were found to have full methylation (3/30 results were verified by Southern blotting). Only 7 mothers were found abnormal methylation of CpG island of FMR1 gene, whilst the remaining 13 mothers were normal.</p><p><b>CONCLUSION</b>MSRE-qPCR is a quick and reliable method for quantitative analysis of CpG island methylation status in FMR1 gene, which may provide a new strategy for the diagnosis of fragile X syndrome.</p>

Estimation of PMI using late postmortem phenomena in the basis of 49 cases / 法医学杂志

OBJECTIVE@#To discuss the influencing factors of using late postmortem phenomena to estimate PMI and to provide experience for an accurate estimation.@*METHODS@#Forty-nine corpses of late postmortem were collected in Shaoxing City, Zhuji area from 2004 to 2011. The related factors were analyzed including season, scene, estimated PMI, exact PMI, cause of death and main factors effected PMI, etc.@*RESULTS@#Of all 49 cases, 20 corpses were outdoor, 11 were indoor and 18 were in water. Thirty-seven cases were successful to estimate PMI and 12 cases were unsuccessful. The main factors affected PMI were infection, poisoning, human destruction and high-pressure electric shock, etc.@*CONCLUSION@#In general, PMI can be correctly estimated by late postmortem phenomenon. When the cases included infection, poisoning and human destruction, we should estimate PMI with the comprehensive analysis.

Maternal-neonatal vitamin D status and related factors / 中华儿科杂志

<p><b>OBJECTIVE</b>To determine the relationship between maternal and neonatal vitamin D status and related factors.</p><p><b>METHOD</b>Serum 25-(OH)D levels were measured by ELISA in 499 pregnant women at 30 - 37 weeks gestation and in cord blood of their infants born at term (37 - 42 wk gestation) in Southeastern China at 28.9°N latitude. One-way analysis of variance (ANOVA) was used to explore maternal and neonatal vitamin D levels by season. Pearson linear and linear regression of partial correlation was used to analyze the relationship between maternal and neonatal 25-(OH) D levels. The multiple factors related to maternal vitamin D status was assessed by binary logistic regression.</p><p><b>RESULT</b>The levels of serum 25-(OH)D were (33.0 ± 13.4) nmol/L in mothers and (31.0 ± 12.5) nmol/L in their newborns. Serum 25-(OH)D < 50 nmol/L was shown in 88.8% of mothers and 91.2% of their neonates. Both maternal and neonatal 25-(OH)D levels varied with season (Ps = 0.000). Vitamin D level was the lowest in spring, with the 25-(OH)D concentration < 50 nmol/L in 98.6% of mothers and 99.3% of their neonates. The highest vitamin D level was presented in fall, but there were still 64.0% of mothers and 75.0% of neonates with 25-(OH)D < 50 nmol/L. Except for season, calcium-vitamin D supplement and intake of egg ≥ 600 g per week during pregnancy benefited to improve maternal vitamin D level [25-(OH)D ≥ 50 nmol/L] [OR = 2.3 (95%CI:1.0, 5.3), 3.4 (95%CI:1.2, 9.9) respectively]. There was a positive correlation between maternal and neonatal 25-(OH)D measures in the sample as a whole (r = 0.45, P = 0.000, N = 499), the correlation was of no statistical significance when maternal serum 25-(OH)D was ≤ 25 nmol/L.</p><p><b>CONCLUSION</b>Hypovitaminosis D was common in late pregnant mothers and their newborns in southeastern China, especially in spring. Vitamin D supplement and intake of vitamin D-rich food were beneficial to improvement of maternal vitamin D level. There was a moderate and positive correlation between maternal and neonatal 25-(OH)D concentrations in this population. The correlation was lost when maternal serum 25-(OH)D ≤ 25 nmol/L.</p>

Effect of Coriolus versicolor polysaccharide B on membrane glycosaminoglycans and cellular glutathione changes in RAW264.7 macrophages exposed to angiotensin II / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of Coriolus versicolor polysaccharide B (CVP-B) on increased membrane glycosaminoglycans (GAG) expression and intracellular glutathione (GSH) of RAW264.7 macrophages exposed to angiotensin II (Ang II).</p><p><b>METHODS</b>The plasma membrane of RAW264.7 macrophages exposed to Ang II treatment was isolated by ultracentrifugation, and the membrane GAG expression was analyzed using 1, 9-dimethylmethylene blue (DMMB) spectrophotometric assay for sulfated GAG. The intracellular reduced GSH was determined using fluorophotometry.</p><p><b>RESULTS</b>The GAG content in the macrophage membranes increased by up to 54% following cell exposure to 1.0 micromol/L Ang II, whereas in presence of 1.0 micromol;/L Ang II, CVP-B at 1, 10, and 50 microg/ml decreased the GAG content by 13%, 43% (P<0.01), and 52% (P<0.01), respectively. The macrophage GSH activity decreased by 69% following incubation with 1.0 micromol;/L Ang II for 24 h, and CVP-B treatment at 1, 10, and 50 microg/ml in presence of 1.0 micromol;/L Ang II resulted in significant increment of GSH activity by 31%(P<0.05), 104% (P<0.01), and 168% (P<0.01), respectively.</p><p><b>CONCLUSION</b>These data provide the first evidence that CVP-B inhibits elevated GAG expression in RAW264.7 macrophage membrane induced by Ang II.</p>

Study on the Social Adaptation of Chinese Children with Down Syndrome

PURPOSE: To evaluate social adjustment and related factors among Chinese children with Down syndrome (DS). PATIENTS AND METHODS: A structured interview and Peabody Picture Vocabulary Test (PPVT) were conducted with a group of 36 DS children with a mean age of 106.28 months, a group of 30 normally-developing children matched for mental age (MA) and a group of 40 normally-developing children matched for chronological age (CA). Mean scores of social adjustment were compared between the three groups, and partial correlations and stepwise multiple regression models were used to further explore related factors. RESULTS: There was no difference between the DS group and the MA group in terms of communication skills. However, the DS group scored much better than the MA group in self-dependence, locomotion, work skills, socialization and self-management. Children in the CA group achieved significantly higher scores in all aspects of social adjustment than the DS children. Partial correlations indicate a relationship between social adjustment and the PPVT raw score and also between social adjustment and age (significant r ranging between 0.24 and 0.92). A stepwise linear regression analysis showed that family structure was the main predictor of social adjustment. Newborn history was also a predictor of work skills, communication, socialization and self-management. Parental education was found to account for 8% of self-dependence. Maternal education explained 6% of the variation in locomotion. CONCLUSION: Although limited by the small sample size, these results indicate that Chinese DS children have better social adjustment skills when compared to their mental-age-matched normally-developing peers, but that the Chinese DS children showed aspects of adaptive development that differed from Western DS children. Analyses of factors related to social adjustment suggest that effective early intervention may improve social adaptability.

Analysis of blood lead levels of preschool children in Zhejiang Province with historical comparison / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To know the blood lead levels (BLL) of preschool children in Zhejiang Province between 1997 to 2003.</p><p><b>METHODS</b>The BLL of preschool children was investigated in this province with cluster sampling in 2003, and compared with the results with that of 1997. The BLL was determined with graphite oven atom absorption spectrography method.</p><p><b>RESULT</b>The average BLL of 2013 preschool children in 2003 was (0.34 +/- 0.13) micromol/L, similar with that of 1997 [(0.35 +/- 0.26)micromol/L (P >0.05)]. There were 274 children with BLL >or= 0.483 micromol/L, and the rate of lead poisoning was 13.61 %, lower than that of 1997 (when there were 448 children with BLL >or= 0.483 micromol/L, and the rate of lead poisoning was 23.84%). In 2003, the rates of lead poisoning among preschool children of Hangzhou, Shangyu, Zhoushan and Changshan were 7.69%, 15.37%, 10.87% and 20.15 %, respectively. There were significant differences among the four areas (P < 0.01). In comparison with the results of 1997, the lead poisoning rates of Hangzhou and Shangyu children decreased significantly, while that of Changshan children increased significantly (P <0.01), and that of Zhoushan children didn't change significantly. The highest rate of lead poisoning was found in children of 4-6 years group as 6 years ago.</p><p><b>CONCLUSION</b>The rate of lead poisoning among preschool children has decreased significantly now in Zhejiang Province. But the condition of BLL in preschool children is still far from optimistic. The environmental lead pollution in remote mountain areas and island areas should be highly concerned by the government.</p>

Study on the neurotoxic effects of low-level lead exposure in rats / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To investigate effects of developmental lead exposure on nitric oxide synthase (NOS) activity in different brain regions and on N-methyl-D-aspartate (NMDA) receptor mRNA expression in the hippocampus of rats. On the basis of these observations, we explored possible mechanisms by which lead exposure leads to impaired learning and memorizing abilities in children.</p><p><b>METHODS</b>A series of rat animal models exposed to low levels of lead during the developing period was established (drinking water containing 0.025%, 0.05% and 0.075% lead acetate). NOS activities in the hippocampus, the cerebral cortex, the cerebellum and the brain stem were determined with fluorescence measurement and levels of mRNA expression of the NMDA receptor 2A (NR2A) subunit and NMDA receptor 2B (NR2B) subunit in the rat hippocampus were measured with Retro-translation (RT-PCR).</p><p><b>RESULTS</b>There were no differences in the body weight of rat pups between any of the groups at any given time (P>0.05). The blood lead level of Pb-exposed rat pups showed a systematic pattern of change: at 14 d of age, it was lower than that at 7 d of age, then rising to the peak level at 21 d and finally falling to lower levels at 28 d. The hippocampal NOS activities of lead-exposed groups were all lower than that of the control group on the 21st and 28th day (P<0.01). NOS activities in the cerebellum of lead-exposed groups were all lower than that of the control group on the 21st and 28th day (P<0.001) and the NOS activity of the 0.025% group was significantly lower than that of the 0.05% and 0.075% groups on the 28th day (P<0.05). NOS activity in the cerebral cortex of the 0.075% group was significantly lower than that of the control, 0.025% and 0.05% groups on the four day spans (P<0.001). There was no significant difference of NOS activity in the brain stem between any lead-exposed group and the control group on the four day spans. In the 0.05% and the 0.075% groups, the level of NR2A mRNA expression was higher than that in the control group at 7 d and 14 d of age (P<0.05). In the 0.025% group, the level of NR2A was found to be higher than that in the control group at 7 d of age only (P<0.05). No significant differences were found for the levels of NR2B mRNA expression between any of the groups at any given time.</p><p><b>CONCLUSIONS</b>NOS activity in the hippocampus, the cerebral cortex and the cerebellum are inhibited by lead exposure. The degree of the inhibitory effect depends on the time span of exposure and the lead concentration. Developmental low-level lead exposure was found to raise the level of NR2A mRNA expression in the hippocampus of rats. Developmental low-level lead exposure does not affect the level of NR2B mRNA expression in the hippocampus.</p>

Treatment and follow-up of children with transient congenital hypothyroidism / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To study the clinical therapy and prognosis in children with transient congenital hypothyroidism (CH).</p><p><b>METHODS</b>Fifty-seven children with CH diagnosed after neonatal screening were treated with low-dosage levothyroxine (L-T4). Follow-up evaluation included the determination of TT3, TT4 and TSH serum levels and the assessment of thyroid gland morphology, bone age, growth development and development quotients (DQ). A full check-up was performed at age 2, when the affected children first discontinued the L-T4 treatment for 1 month, and one year later. Development quotients were compared with a control group of 29 healthy peers.</p><p><b>RESULTS</b>The initial L-T4 dosage administered was 3.21-5.81 microg/(kg.d) with an average of (16.25+/-3.87) microg/d. Mean duration of therapy was (28.09+/-9.56) months. No significant difference was found between study group and control group in the DQ test (average score (106.58+/-14.40) vs (102.4+/-8.6), P>0.05) and 96.49% of the CH children achieved a test score above 85. Bone age, 99mTc scans and ultrasonographic findings were all normal, and evaluation of physical development was normal too, as were the serum levels of TT3, TT4 and TSH after one year of follow-up.</p><p><b>CONCLUSION</b>A L-T4 dosage of 3.21-5.81 microg/(kg.d) was found sufficient for the treatment of transient CH. The treated children showed satisfactory overall mental and physical development at age 2. So it is possible for CH children to stop taking medicine if their laboratory findings and physical development are all normal after regular treatment and 2-3 years of follow-up.</p>

Effects of lead exposure on nitric oxide synthase activity in different brain regions of developmental rat / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe the influence of lead exposure on the activity of nitric oxide synthase (NOS) in different brain regions of rat.</p><p><b>METHODS</b>By establishing a series of rat models exposed to different low levels of lead (drinking water containing 0.025%, 0.050%, 0.075% of lead acetate) during developing period, NOS activities in hippocampus, cerebellum, cerebral cortex and brain stem were studied.</p><p><b>RESULTS</b>On the 21st day after birth, NOS activities in hippocampus of three levels of lead exposed groups [(1.53 +/- 0.20), (1.66 +/- 0.23), (1.88 +/- 0.32) U/mg pro respectively], and in cerebellum [(0.87 +/- 0.24), (0.85 +/- 0.09), (0.91 +/- 0.18) U/mg pro respectively] were significantly lower than those of control group [(2.36 +/- 0.18), (1.41 +/- 0.18) U/mg pro, respectively, P < 0.01]. NOS activities in cerebral cortex of 0.075% group [at 7, 14, 21 d of age [(1.29 +/- 0.14), (1.03 +/- 0.15), (0.69 +/- 0.10) U/mg pro] were significantly lower than those in control group [(2.54 +/- 0.31), (1.64 +/- 0.22), (1.24 +/- 0.14) U/mg pro respectively], and 0.025% group [(2.42 +/- 0.19), (1.59 +/- 0.17), (1.27 +/- 0.12) U/mg pro respectively], and 0.050% group [(2.56 +/- 0.53), (1.77 +/- 0.19), (1.24 +/- 0.10) U/mg pro respectively, P < 0.05]. There were no significant differences among control, 0.025%, and 0.050% groups (P > 0.05). Lead exposure had no influence on NOS activity in brain stem at the same age (P > 0.05).</p><p><b>CONCLUSION</b>NOS activities in hippocampus, cerebellum and cerebral cortex were inhibited by low level lead exposure and the degree of the effect was related to Pb exposure time and/or level of Pb exposed.</p>

Inactivation Kinetics of Bacillus coagulans Spore by the Combination of High Hydrostatic Pressure and Moderate Heat / 微生物学通报

The combination effect of high hydrostatic pressure and moderate heat on the inactivation kinetics of Bacillus coagulans spore in phosphate buffer and UHT(Ultra High Temperature)whole milk was investigated.The pressure come-up time and corresponding log-reduction of spore inactivation were considered during pressure-thermal treatment.Bacillus coagulans spore had a much higher resistance to pressure in UHT whole milk than in phosphate buffer.Survival data were modeled using the linear,Weibull and log-logistic models to obtain relevant kinetic parameters.The tailing phenomenon occurred in all survival curves,indicating the linear model was not adequate for describing these curves.The log-logistic model produced best fits to survival curves,following by Weibull model.