Deletion analysis of SMN1 and NAIP genes in Southern Chinese children with spinal muscular atrophy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1, SMA2, and SMA3. The survival of motor neuron (SMN) gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was performed to detect the deletion of both exon 7 and exon 8 of SMN1 and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients and 76% (13/17) of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients. Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NAIP deletion. The findings of homozygous deletions of exon 7 and/or exon 8 of SMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion of SMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NAIP gene may be a modifying factor for disease severity of SMA1. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA.

Correlation between infection of different glycoprotein B genotypes of human cytomegalovirus and human chronic periodontitis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the correlation between infection of different human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes and human chronic periodontitis.</p><p><b>METHODS</b>A nested-polymerase chain reaction (nPCR) was employed to detect HCMV gB gene in the subgingival plaque samples from 65 chronic periodontitis patients and in the gingival crevicular fluid samples from 24 periodontally healthy control. The amplification fragments of gB gene were further genotyped by restriction fragment length polymorphism (RFLP). The correlation among infection with the different HCMV genotypes and the severity of periodontal lesion were evaluated.</p><p><b>RESULTS</b>In terms of teeth examined, the prevalence of HCMV (59.23%, 154/260) in the chronic periodontitis lesions was significantly higher than that of HCMV (32.29%, 31/96) in the periodontally healthy control (P < 0.01). Of the HCMV DNA positive samples from the chronic periodontitis lesions, 11.7% (18/154) was genotyped as gB I, 80.5% (124/154) as gB II, and 7.8% (12/154) as gB I and gB II co-infection, and of the HCMV DNA positive samples from the periodontal healthy control, 45.2% (14/31) was genotyped as gB I, 38.7% (12/31) as gB II, and 16.1% (5/31) as gB I and gB II co-infection. The gB II genotype was more dominant among the chronic periodontitis lesions compared with that among the periodontally healthy control (P < 0.01). In chronic periodontitis, no statistical significance could be found between infection of different HCMV gB genotypes and the different clinical parameters of CAL, PD and GI (P > 0.05).</p><p><b>CONCLUSIONS</b>Subgingival infection with HCMV is closely associated with chronic periodontitis. Infection of HCMV may not correlate directly with severity of periodontitis. However, gB II may be the dominant genotype of HCMV, which is associated with the chronic periodontitis.</p>

Anti-inflammation and anti-oxidation effects of recombinant human superoxide dismutase on acute lung injury induced by meconium aspiration in infant rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the anti-inflammation and anti-oxidation effects of recombinant human CuZn superoxide dismutase(rhSOD) on acute lung injury (ALI) induced by meconium aspiration in rats.</p><p><b>METHODS</b>1 ml/kg of 20% human newborn meconium suspension was intratracheally (IT) administrated to induce the model of ALI in 32 male Sprage-Dawley rats, and the animals were then randomized to 4 groups: 3 treatment groups with IT administration of 5, 10 and 20 mg/kg rhSOD dissolved in 1 ml/kg saline and the control group with IT administration of 1 ml/kg saline. The animals were killed after 24 h of treatments. The measurements included lung tissue wet/dry ratio, broncho-alveolar lavage fluid (BALF) protein, BALF protein/plasma protein (pulmonary permeability index, PPI),lung myeloperoxidase (MPO) and superoxide dismutase (SOD) activity, nitric oxide (NO) and 8-isoprostane levels. Lung injury score was also evaluated.</p><p><b>RESULTS</b>Compared with the control group, pulmonary MPO activity, NO and 8-isoprostane levels were significantly decreased and SOD activity was markedly increased in all rhSOD treatment groups (P<0.05 or 0.01). Compared with the rhSOD 5 mg/kg group, pulmonary 8-isoprostane level was further low in the rhSOD 20 mg/kg group(P=0.01). Lung injury score was decreased in rhSOD 20 mg/kg group (P<0.05). But there were no statistically differences in lung wet/dry, BALF protein and PPI among all groups.</p><p><b>CONCLUSION</b>The results suggest that a single IT dose of 5,10 or 20 mg/kg rhSOD can prevent lung damages in rats with ALI following meconium aspiration.</p>

Determination of six major human herpes viruses in cerebrospinal fluid and blood of children with consensus primers / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To identify 6 major human herpesviruses with consensus primers and to explore its clinical application.</p><p><b>METHODS</b>Based on the highly-homogeneous regions of DNA polymerase gene in human herpesviruses,Two pairs of primer were synthesized. One pair was designed to amplify herpes simplex virus type 1, type 2, Epstein-Barr virus and cytomegalovirus; and another was used to amplify varicella-zoster virus or human herpesvirus 6. Virus species identification was performed by restriction enzyme digestion with BamH I and BstU I. Thirty-eight CSF specimens of clinically diagnosed viral encephalitis,and 49 blood specimens from 27 confirmed cases and 22 clinically diagnosed ones were tested for herpes virus DNA using the PCR-RFLP assay with these primers.</p><p><b>RESULTS</b>Thirteen out of 38 CSF specimens (34.2%) were herpes virus positive. All blood specimens from 27 confirmed cases showed positive results, while for 22 clinically diagnosed cases 16 (72.7%) were positive. The types of herpes virus were determined using restriction enzyme digestion with BamH I and BstU I. Two CSF specimens from the patients, who were treated with aciclovir for 2 - 3 days, were still positive for herpes virus DNA by this method. None of the control blood or CSF controls were positive for herpesvirus by PCR.</p><p><b>CONCLUSION</b>The PCR-RFLP method used in this study is a specific, sensitive and practicable one for diagnosis of herpes virus infection.</p>

Early arterial atherosclerosis and level of plasma homocysteine in simply obese children / 中华儿科杂志

<p><b>OBJECTIVE</b>To understand whether hyperhomocysteinemia and early arterial atherosclerosis exist in simply obese children.</p><p><b>METHODS</b>Totally 68 simply obese children (age 6-14 years, mean 10.8 +/- 2.3 years) were enrolled in this study, 50 were male and 18 were female. Body mass index (BMI) of the obese children was equal to or more than 22. The height of the children was (145 +/- 22) cm. Meanwhile, 26 normal children (age 6 - 14 years, mean 10.9 +/- 2.0 years) were selected as control group, 17 of these children were male and 9 were female. Their height was (148.5 +/- 5.8) cm. There were no significant differences in height and age between the obese and the control children. The carotid intimal-medial thickness (IMT), brachial artery flow-mediated vasodilation were examined by Doppler Flow/Dimension System and the liver was examined by B-mode ultrasound imager. Plasma homocysteine was determined by the automated chemiluminescent enzyme immunoassays. Serum lipid concentration was determined by biochemical analytic method. Blood pressure of the right upper limbs was measured. A detailed medical and family history was systematically recorded.</p><p><b>RESULTS</b>BMI was (27.8 +/- 4.5) in the obese children and (16.2 +/- 2.5) in the controls. There was significant difference between two groups (P < 0.01). The obese children had significantly increased values than the controls for the carotid intimal-medial thickness (P < 0.01). Right carotid IMT, right inner-carotid IMT, left carotid IMT and left inner-carotid IMT were respectively (0.54 +/- 0.13) mm, (0.69 +/- 0.14) mm, (0.52 +/- 0.12) mm and (0.67 +/- 0.14) mm in obese children and were respectively (0.45 +/- 0.04) mm, (0.46 +/- 0.04) mm, (0.45 +/- 0.05) mm and (0.46 +/- 0.03) mm in control groups. Conversely, the flow-mediated brachial artery dilation of the obese children was significantly lower than that of the controls [(11.0 +/- 4.3)% vs. (17.5 +/- 4.9)%, P < 0.01]. The obese children had higher level of plasma homocysteine than the controls [(7.9 +/- 2.7) micromol/L vs. (5.6 +/- 2.1) micromol/L, P < 0.01]. Total cholesterol (TC) in the obese children dramatically increased, so did triglyceride concentration (TG), LDL-cholesterol (LDL-ch) and apolipoprotein-B (apo-B). Of the obese children, had fatty liver or the tendency to fatty liver. Six cases of the 68 obese children (8%) had hypertension. Of the 68 obese children, 57 (84%) had the history of consuming excessive food or taking less exercise. Forty-four percent of the obese children (30/68) came from the obese families in which at least one of the parents or grandparents was obese. Twenty-nine percent (20/68) and 22% (15/68) of the obese children respectively came from the families in which at least one of the parents or grandparents suffered from hypertension or coronary heart disease.</p><p><b>CONCLUSION</b>Early arterial atherosclerotic changes existed in simply obese children. Hyperhomocysteinemia may be an important factor of the obesity-induced early arterial atherosclerosis during childhood.</p>

Effects of IGF-II on promoting proliferation and regulating nitric oxide synthase gene expression in mouse osteoblast-like cell / 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 the effects of insulin-like growth factor II (IGF-II) on promoting cell proliferation, regulating levels of cellular nitric oxide (NO) and mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells.</p><p><b>METHODS</b>Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-II. After the cells were treated with IGF-II at different concentrations for different time duration, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used to examine cell proliferation, and nitrate reductase method was applied to detect NO concentrations in cell culture supernatants and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine transcription levels of cellular iNOS and eNOS mRNAs.</p><p><b>RESULTS</b>After the MC3T3-E1 cells were treated with IGF-II at concentration of 1 ng/ml for 72 h, 10 and 100 ng/ml for 24, 48 and 72 h respectively, all the MTT values increased (P<0.05 or P<0.01) with obvious dosage-time dependent pattern. NO levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-II for 48 h, and with 1, 10 and 100 ng/ml IGF-II for 72 h were remarkably lower than that of the normal control, respectively (P<0.05 or P<0.01). After the cells were treated with 100 ng/ml IGF-II for 48 h cellular iNOS mRNA levels were significantly decreased (P<0.01). But the levels of eNOS mRNA in the cells treated with each of the used IGF-II dosages for different time duration did not show any differences compared with the normal control (P>0.05).</p><p><b>CONCLUSION</b>IGF-II at different concentrations could promote proliferation of mouse MC3T3-E1 cell. This cell proliferation promotion was associated with the low NO levels maintained by IGF-II. Higher concentration of IGF-II could down-regulate iNOS gene expression at the level of transcription but not affect transcription of eNOS mRNA, which might be one of the mechanisms for IGF-II maintenance of the low NO levels in MC3T3-E1 cells.</p>

Effects of insulin-like growth factor II on regulating nitric oxide synthase gene expression in mouse osteoblast-like MC3T3-E1 cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the effects of insulin-like growth factor II (IGF-II) on regulating the levels of nitric oxide (NO) and the mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells.</p><p><b>METHODS</b>Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-II. After the cells were treated with IGF-II at different concentrations for different intervals of time, MTT colorimetry was used for examining the cell proliferation. Nitrate reductase method was applied for detecting the NO concentrations in cell culture supernatants and RT-PCR employed for determining the levels of cellular iNOS and eNOS mRNAs.</p><p><b>RESULTS</b>After the MC3T3-E1 cells were treated with IGF-II at the dosages of 1 microg/L for 72 h, 10 and 100 microg/L for 24, 48 and 72 h respectively, all the MTT values increased markedly (P < 0.05 or P < 0.01). After the cells were treated for 48 and 72 h at the dosage of 100 microg/L IGF-II respectively, the levels of NO in the supernatants of cell cultures and cellular iNOS mRNA decreased significantly (P < 0.01). However, the levels of eNOS mRNA in the cells treated with any of the IGF-II dosages for the different times were stable (P > 0.05).</p><p><b>CONCLUSIONS</b>IGF-II at the dosages of 1 approximately 100 microg/L showed the effects on promoting proliferation, which as probably due to the maintenance of low NO levels. Inducible NOS gene expression at the level of transcription was down regulated in the MC3T3-E1 cell treated with higher dosage of IGF-II (100 microg/L) but eNOS mRNA was not, which might be one of the mechanisms for the maintenance of low NO levels.</p>

Gene Diagnosis of Spinal Muscular Atrophy in Children / 实用儿科临床杂志

Objective To establish a gene diagnosis assay for spinal muscular atrophy(SMA) in children. Method Analysis of the survival motor neuron (SMN) gene in 19 SMA patients and in 21 normal controls were performed by using polymerase chain reaction - fragment length polymorphism (PCR - RFLP) method. Result Deletion of exon 7and 8 in SMNt gene were found in all 19 SMA patients, while no such changes were found in normal controls. Conclusion The SMNt gene exon 7 and 8 examine can be applied to SMA gene diagnosis, and the PCR- RFLP method have higher sensitivity and particularity to the SMA diagnosis.

Influence of different DNA extractions on the identification of streptococcus sanguis group by arbitrary primed polymerase chain reaction / 浙江大学学报·医学版

OBJECTIVE: To assess the influence of different DNA extractions on the identification of streptococcus sanguis group (SSG) species by arbitrary primed polymerase chain reaction (AP-PCR). METHODS: AP-PCR was used to distinguish SSG species by designing 25bp arbitrary primer 5'AAG AGA GGA GCT AGC TCT TCT TGG A 3', and the genomic DNA was extracted by 3 methods. RESULTS: There were great differences in the main band of DNA polymorphism among SSG species. The similar band could be got from the different DNA extractions in the same species. CONCLUSION: Different DNA extractions have no influence on the identification of SSG.

Effect of adriamycin on sarcoplasmic reticulum Ca2+ -ATPase activity in cardiomyocyte of rabbits / 浙江大学学报·医学版

OBJECTIVE: To evaluate influences of regular-dose of adriamycin (ADR) on heart function and sarcoplasmic reticulum (SR) Ca2+ -ATPase in cardiomyocyte of rabbits. METHODS: Nine rabbits received intraveneous injection of ADR (2mg/kg) once a week for 8 weeks, the rabbits injected with saline were used as control group. Cardiac output (CO), blood pressure (BP), mean artery pressure (MAP), left ventricular systolic pressure( LVSP), left ventricular end-diastolic pressure (LVEDP), calcium in cardiomyocyte (MyoCa2+) of rabbits and SR Ca2+ -ATPase activity were examinated 3 weeks after the final injection. RESULTS: CO, LVSP and SR Ca2+ -ATPase activity were significantly decreased in ADR treated group compared with the control group. Conversely, LVEDP and MyoCa2+ were significantly increased in ADR treated rabbits. CONCLUSION: Heart function can be decreased by regular-dose of ADR in injection. Calcium overload in cardiomyocyte and decrease of SR Ca2+ -ATPase activity is important physiopathologic mechanism in ADR-induced impairment of heart.

Pathogen and spectrum of disease in infants with human cytomegalovirus infection / 中华传染病杂志

Objective To investigate the correlation between pathogens and spectrum of disease in infants with human cytomegalovirus(HCMV) active infection.Methods A total of 378 cases of HCMV infection diagnosed by the identification of HCMV IgM or PP65 antigen of HCMV.HCMV gB genotyping was carried out by nested PCR and restriction fragment length polymorphism(RFLP) in 107 cases.The results of pathogen,spectrum of disease and clinic feature were analyzed.Results In all 378 infant patients with HCMV,27.78% were systemic infection and 72.22% involved just single organ.Hepatitis,HCMV inclusion disease,thrombocytopenic purpura,pneumonia were pre- dominant with 33.07%,27.78%,13.49%,6.35% respectively.The rate of HCMV inclusion dis ease in infants younger than 2 weeks was higher than in those aged from 3 12 weeks(P ~ 0.05) and children older than 12 weeks(P＜0.01).Infants with higher rate of PP65 antigen positive cells were apt to systemic infection than those with lower rate of PP65 positive cells(P＜0.01).Infants,who were positive by detections of all three methods,were apt to systemic infection than others(P＜0.01). Moreover,infants positive of IgM and PP65 antigen were apt to systemic infection than those just positive by one of the two methods(P＜0.01).The result of gB genotype analysis in 107 cases showed 53 cases of gBⅠ,20 of gBⅡ.18 of gBⅢ.7 of gBⅠ+gBⅡ,5 of gBⅠ+gBⅢand 4 of gBⅡ+gBⅢ,and gBⅣwas not found.Conclusion HCMV could infect multiple organs and have some different clinic features.Combination of different methods can increase the sensitivity to detect the pathogen.The gBⅠgenotype is most prevalent in these infants.