Background Ultraviolet irradiation promotes cellular apoptosis by affecting the mitochondrial transmembrane potential,including human lens epithelial cells (LECs).Gene associated with retinoid-interferoninduced mortality-19 (GRIM-19),a cell death regulatory protein,is essential for the assembly and function of mitochondrial complex Ⅰ.However,whether LECs apoptosis induced by ultraviolet irradiation is related to GRIM-19 is still unclear.Objective The purpose of this study was to investigate the relationship between the apoptosis of human LECs caused by ultraviolet with GRIM-19 expression in vitro.Methods Human LEC line(SRA01/04)was cultured in α-MEM containing 10％ fetal bovine serum.The cells were exposed to ultraviolet ray at doses of 0,30,60,90,120 or 150 mJ/cm2 when cell growth reached the logarithmic phase and 80％ confluency.The rate of apoptosis of the cells was assayed using flow cytometry,and the level of expression and relative amount of GRIM-19 protein (GRIM-19/β-actin) were detected by Western blot.The relationship between apoptosis and the GRIM-19/β-actin value among the different treatment groups was compared using One-way ANOVA,and the correlation of LECs apoptosis rate and GRIM-19 expression level was assessed by Pearson linear analysis.Results A significant difference was found in the apoptosis rate among the different treatment groups(F=149.32,P＜0.01).Compared with the 0 mJ/cm2 ultraviolet irradiation group,the apoptosis rate of LECs was significantly increased in the 60,90,120 and 150 mJ/cm2 ultraviolet irradiation groups (q =17.02,-25.20,-29.41,-8.61,P ＜ 0.01).The expression of the GRIM-19 protein in the LECs suspension was enhanced by ultraviolet irradiation at 60,90,120 and 150 mJ/cm2.The relative expression of the GRIM-19 protein (GRIM-19/β-actin) was significantly different among the various groups (F=6.87,P＜0.05),and the GRIM-19/β-actin values in the 60,90,120,150 mJ/cm2 ultraviolet irradiation groups were elevated in comparison with the un-irradiated group(2.01±0.76,2.98± 1.80,3.97± 1.61,2.42± 1.28 vs.0.56±0.23),which showed statistically significant differences (q =4.12,-5.04,-7.09,-3.85,P ＜ 0.01).In addition,a positive correlation was seen between the rate of apoptosis and the expression of the GRIM-19 protein(r=0.71,P＜0.01).Conclusions GRIM-19 is expressed in normal human LECs.The apoptosis of human LECs accompanies the up-regulation of GRIM-19.The expression of GRIM-19 in LECs increases with ultraviolet irradiation in a doseindependent manner.

Background Epithelial-myofibroblast transition (EMT) of human lens epithelial cells (LECs) induced by transforming growth factor-β2 (TGF-β2) is the main mechanism in the pathogenesis of posterior capsular opacification(PCO).Seeking an effective drug capable of inhibiting this process is important for the prevention and treatment of PCO.Objective The purpose of this study was to investigate the inhibitory effect of rapamycin (RAPA)on the proliferation of human LECs and TGF-β2-induced EMT.Methods Human LEC strain(SRA01/04)was cultured in DMEM with high glucose and 10％ fetal bovine serum.The cells were consequently cultured in serumfree DMEM with 5 mg/L TGF-β2,TGF-β2+10 mg/L RAPA,TGF-β2 + 100 mg/L RAPA,TGF-β2 + 1000 mg/L RAPA or TGF-β2 +10 000 mg/L RAPA for 72 hours,and SRA01/04 cultured in serum-free DMEM were used as control.The proliferation rate(A490)of SRA01/04 in the different groups was detected using the MTT assay and the rate of inhibition of RAPA was calculated.The expressions of the α-smooth muscle actin(α-SMA) and E-cadherin(E-cad)mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The changes in the expression of α-SMA and E-cad in SRA01/04 were evaluated by Western blot 24,48 and 72 hours after TGF-β2 +400 mg/L RAPA treatment.Results The A490 value of SRA01/04 was 0.680±0.020,0.550±0.013,0.480±0.014,0.400±0.011 and 0.200±0.019 in the control group,TGF-β2 group,TGF-β2 + 10 mg/LRAPA group,TGF-β2 + 100 mg/L RAPA group,TGF-β2 + 1000 mg/L RAPA and TGF-β2 + 10 000 mg/L RAPA group,respectively,showing a gradually declining trend in SRA01/04 rate of proliferation with increasing RAPA concentrations (F =101.920,P =0.000).RT-PCR and Western blot assay showed that the relative expression levels of α-SMA mRNA (α-SMA mRNA/β-actin mRNA)and protein (α-SMA/β-actin)in the cells were significantly increased in the TGF-β2 treatment group.However,with exposure to RAPA,the relative expression levels of α-SMA mRNA and protein were significantly lowered with increasing RAPA concentrations,but the expression levels of E-cad mRNA and protein were raised (α-SMA mRNA:F =294.660,P =0.000 ; α-SMA protein:F =346.950,P =0.000 ; E-cad mRNA:F =264.250,P =0.000 ; E-cad protein:F =317.327,P =0.000).In addition,after exposure to 400 mg/L RAPA,the expression levels of α-SMA protein gradually reduced and those of E-cad protein gradually increased with increasing treatment durations,showing significant differences among the different time points (α-SMA:F =693.864,P =0.000 ;E-cad:F=369.286,P =0.000).Conclusions RAPA can inhibit the proliferation of SRA01/04 in vitro and arrest EMT of SRA01/04 induced by TGF-β2 in a dose-and time-dependent manner.

OBJECTIVETo investigate the frequency of CYP2C19 polymorphisms involved in clopidogrel metabolism in Fujian Han population.

METHODSFrequencies of CYP2C19* 2, CYP2C19*3 and CYP2C19*17 in 1001 unrelated Fujian Han volunteers were determined with polymerase chain reaction-restriction fragment length polymorphism and direct sequencing method.

RESULTSThe frequencies of CYP2C19*2, *3 and *17 were 32.4%, 5.8% and 0.4%, respectively. According to genotyping results, intermediate metabolizers (CYP2C19 *1/*2 or *1/*3) and poor metabolizers (CYP2C19 *2/*2 and *2/*3) respectively accounted for 47.95% and 13.99% of all subjects. Above frequencies were similar to those of Japan, Korea, Singapore, Malaysia, Thailand and Chinese Dai, Mongolian,Li and Hui ethnics (P>0.05), but were significantly different from those of Chinese Kazakh and Uygur ethnics, and people from Iran, Russia, Italy, Poland, Norway, Canada native Indians, Bolivia, Egypt or Tanzania (P<0.05).

CONCLUSIONEthnic/regional diversity exist with regard to the prevalence of CYP2C19 polymorphisms. No significant difference were found between Fujian Han Chinese and Dai, Mongolian, Li and Hui from China or other populations from East and Southeast Asia, but higher frequencies of intermediate metabolizers and poor metabolizers compared with populations of Kazakh and Uygur in China, and people from Europe, South America and Africa.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Aryl Hydrocarbon Hydroxylases ; genetics ; China ; Cytochrome P-450 CYP2C19 ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Ticlopidine ; analogs & derivatives ; metabolism ; Young Adult

OBJECTIVETo investigate the differences in semen quality between samples collected by masturbation in the clinic and at home.

METHODSBased on the WHO guidelines, we analyzed the ejaculates collected by masturbation in the clinic and at home from 342 men under infertility assessment and measured the contents of such biochemical markers in the seminal plasma as neutral α-glucosidase, zinc, and fructose. According to the location of semen collection, we divided the samples into two groups, clinic-collected and home-collected, and analyzed the differences in the semen parameters between the two groups with the SPSS 16.0 software.

RESULTSCompared with the clinic-collected semen, the home-collected samples had significantly higher mean values in semen volume (4.0 vs 4.9%), sperm concentration (41 vs 64 x 10(6)/ml), total sperm count (175 vs 270 x 10(6) per ejaculate), progressive sperm motility (40 vs 52%), total count of progressively motile sperm (82 vs 135 x 10(6) per ejaculate) (all P <0.05). No significant differences were found between the two groups in normal sperm morphology (4.0 vs 5.0%) and the contents of neutral α-glucosidase (26 vs 24 mU per ejaculate), zinc (8.0 vs 8.0 μmol per ejaculate), and fructose (62 vs 60 μmol per ejaculate) (all P >0.05). Abnormal sperm concentration (<20 x 10(6)/ml) was observed in significantly fewer of the home-collected samples than the clinic-collected ones (18% [62/342] vs 30% [103/342], P<0.05), and so was abnormal progressive sperm motility (<32%) (64% [219/342] vs 75% [256/342], P<0.05).

CONCLUSIONOur findings show that semen samples collected by masturbation at home has a higher quality than those collected in the clinic. So the location of semen collection should be taken into consideration in infertility investigation.

Humans ; Infertility, Male ; diagnosis ; Male ; Masturbation ; Semen ; enzymology ; physiology ; Semen Analysis ; methods ; Specimen Handling ; methods ; Sperm Count ; Sperm Motility ; alpha-Glucosidases ; analysis

Objective To optimize the short tandem repeats(STR)which link closely to survival motor neuron(SMN)and have redundant polymorphism information contents,and to use these STR in the prenatal diagnosis of spinal muscular atrophy(SMA).Methods Eleven STR loci(D5S435,D5F153, DSF151,D5S637,D5S1413,D5S125,D5S464,D5S1556,DSF149,D5S351,MAP1B-5')were amplified by PCR.Then the PCR products were detected by polyacrylamide gel electrophoresis(PAGE)and analyzed by silver staining.STR loci were evaluated and optimized by their PIC values.PCR-PAGE and gene scan were combined to make genetic link analysis for SMA families based on the optimized STR.Results Three STR loci(D5S435,DSF149 and D5S351)were selected with 8,19 and 18 polymorphic fragments detected respectively in 100 normal individuals.Their PIC values were 0.84,0.91 and 0.92 respectively.Four carriers and 2 normal individuals were detected from 6 SMA families with linkage analysis by using the 3 STR.Conclusion This genetic diagnosis system based on the 3 STR loci can provide rapid prenatal diagnosis for SMA families,can eliminate maternal blood contamination,and also can discriminate carriers from normal individuals in the fetuses,which makes the prenatal diagnosis system of SMA perfect.

Objective To prepare the survival motor neuron(SMN)polyclonal antibody and explore the localization of SMN protein in transfected cells and its expression in skeletal muscles of patients with spinal muscular atrophy(SMA).Methods A prokaryotic expressional plasmid named pET-28? (+)/SMN was constructed and SMN-His fusion protein was induced.The fusion protein was used to immunize New Zealadd rabbits to prepare SMN polyclonal antibody.A eukaryotic expressional plasmid named pcDNA3.1/myc-HisB-SMN was constructed and used to transfect CHO cells.Skeletal muscles were collected from 3 patients with bone fracture who were regarded as normal controls, and 3 SMA patients of type Ⅰ, 3 of type Ⅱ and 3 of type Ⅲ who were ascertained by genetic analysis.Western-blotting and immunofluorescence stain were applied to study the expression of SMN in transfected CHO cells and skeletal muscles of normal individuals and SMA patients.Results Correct pET-28a(+)/SMN prokaryotic expressive plasmid was constructed and SMN-His fusion protein was obtained from E coli BL21 transformed with pET-28a(+)/SMN.Then, rabbit anti-human full-length SMN polyclonal antibody of high specificity and sensitivity was obtained from rabbits immunized by SMN-His fusion protein.SMN proteins were shown diffusedly locating in the cytoplasm and nucleus of CHO cells transfected with pcDNA3.1/myc-HisB-SMN plasmid and mainly accumulating around the nucleus.The results of Western-blotting were as follows:the average ratio of SMN band density to glyceraldehyde phosphate dehydrogenase(GAPDH)band density (SMN/GAPDH)is 0.619 in skeletal muscles from normal controls, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type Ⅲ and Ⅱ were 0.347 and 0.340 respectively, which were lower than that of normal controls.However, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type I was only 0.079, which was quite lower than that of normal controls.Conclusions The rabbit anti-human full-length SMN polyclonal antibody is of high specificity and sensitivity, which makes the basis for the research of SMN function and SMA pathogenesis.There may be a correlation between the SMN level in skeletal muscle and the severity of disease.

Objective To observe the effects of different doses of Kiwi fruit essence on fibroblast growth factor 2(FGF2)and extracellular regulatory protein kinase 1/2(ERK1/2)in lung tissue of rats with pulmonary fibrosis.Methods SD rats were divided into blank control group(0.9％NaCl),model group(0.9％NaCl),positive control group(5 mg·kg-1 prednesone acetate),experimental group-L,-M,-H(60,120,180 mg·kg-1 Kiwi fruit essence),except the blank control group was injected with 0.9％NaCl at one time.The other groups were injected 5 mg·kg-1 bleomycin A5 into the trachea to construct the rat pulmonary fibrosis model.The expressions of FGF2 and ERK1/2 proteins and mRNA were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction.Results The relative expression levels of FGF2 protein in blank control group,model group,positive control group and experimental group-L,-M,-H groups on the day 28 were 0.07±0.01,0.63±0.15,0.14±0.02,0.16±0.01,0.26±0.02 and 0.44±0.03,respectively;the relative expression levels of ERK1/2 protein on day 28 were 0.06±0.00,0.41±0.05,0.09±0.00,0.10±0.00,0.14±0.02,0.21±0.02,respectively;the relative expression levels of FGF2 mRNA on day 28 were 0.98±0.05,9.32±1.03,2.21±0.32,2.87±0.44,5.32±0.21,6.37±0.66,respectively;the relative expression levels of ERK1/2 mRNA on day 28 were 1.00±0.01,9.22±0.85,1.93±0.63,2.36±0.54,5.34±0.44 and 6.85±1.09,respectively.The above indexes in the model group were significantly different from those in the blank control group(all P＜0.05).The above indexes in the positive control group and the experimental-L,-M,-H groups were significantly different from those in the model group(all P＜0.05).Conclusion Kiwi essence may inhibit pulmonary fibrosis by regulating the expression of FGF2 and ERK1/2.

OBJECTIVETo evaluate the technique and outcome of arthroscopic single-bundle reconstruction of posterior cruciate ligament (PCL) with quadrupled hamstring tendon.

METHODSFrom April 2001 to October 2004, 49 knees with PCL tears in 49 patients were verified with arthroscope in this department. Of them, 13 were combined with anterior cruciate ligament tears, 14 with disruptions of the posterolateral corner, 6 with ruptures of the posteromedial corner and medial collateral ligament, 9 with lateral meniscus tears, 5 with medial meniscus tears and 2 with popliteal vascular tears. All the damaged PCLs were reconstructed with single-bundle of autogenous quadrupled hamstring tendons under arthroscope. Biodegradable interference screws or blunt titanium interference screws were used for direct anatomic fixation of the reconstructed ligament.

RESULTSAfter operation, no severe complications occurred at early stage in the 49 patients. All of them were followed up for 10-52 months with an average of 22.0 months+/-10.7 months. Lysholm score was remarkably improved from 30-60 (mean: 47.96+/-8.16) preoperatively to 70-95 (mean: 89.08+/-6.10) at the last postoperative follow-up (P less than 0.01). Furthermore, there was a significant improvement in International Knee Documentation Committee (IKDC) score from abnormal (Grade C) in 10 knees and severely abnormal (Grade D) in 39 preoperatively to normal (Grade A) in 20, nearly normal (Grade B) in 24 and abnormal in 5 at the last follow-up. Of the 49 patients, 40 returned to the same activity level as before and 9 were under the level.

CONCLUSIONSSingle-bundle reconstruction of PCL with quadrupled hamstring tendons has the advantage of minimal trauma in surgery and satisfactory outcome.

Adolescent ; Adult ; Arthroscopy ; Female ; Follow-Up Studies ; Humans ; Knee Injuries ; diagnosis ; rehabilitation ; surgery ; Knee Joint ; Male ; Middle Aged ; Posterior Cruciate Ligament ; injuries ; surgery ; Reconstructive Surgical Procedures ; Tendons ; transplantation

OBJECTIVETo investigate the clinical characteristics and GCH I gene mutations in patients with dopa-responsive dystonia (DRD).

METHODSThe clinical features of 3 families with 6 affected members and 8 sporadic cases were analyzed to determine the clinical characteristics, and 2 families with 4 affected members and 2 sporadic cases were screened for mutations of the GCH I gene.

RESULTSAge at onset was (10 +/- 3) years. Onset occurred earlier in female (9 +/- 4) years than in male (12 +/- 1) years. The initial symptom was a gait disorder, dystonia or tremor in most patients and nine patients (64%) presented with diurnal fluctuation. Thirteen patients (93%) were cured and one was improved after administration of low doses of levodopa for 3 months and no long-term side effects of levodopa had occurred. Two independent mutations were found in three patients. Gln161Pro, a new missense mutation, was found in a sporadic case, leading to a relatively severe phenotype. The two patients with mild phenotype in one family were found to have Lys224Arg mutation, as previously described.

CONCLUSIONSDRD patients have diverse phenotypes and diurnal fluctuation is an important feature. They have dramatic and sustained response to levodopa. There may be a correlation between genotype and phenotype. The detection of GCH I mutations is helpful in early diagnosis of non-typical cases.

Age of Onset ; Child ; China ; DNA Mutational Analysis ; Dopamine Agents ; therapeutic use ; Dystonia ; diagnosis ; drug therapy ; genetics ; physiopathology ; Early Diagnosis ; Female ; GTP Cyclohydrolase ; genetics ; Genotype ; Humans ; Levodopa ; therapeutic use ; Male ; Molecular Sequence Data ; Mutation ; Mutation, Missense ; Pedigree ; Phenotype ; Polymerase Chain Reaction ; Sex Factors ; Treatment Outcome