Background The pathogenesis and development of cataract is associated with oxidative stress-induced apoptosis of human lens epithelial cells(LECs).BH3-only protein is a factor that can initiate apoptosis,and thus the apoptotic process is probably related to the activation of the c-Jun N-terminal kinase(JNK).However,the relationship between oxidative stress-induced apoptosis of human LECs and the JNK pathway remains to be illuminated.Objective This study was to investigate the effects of the JNK/c-Jun pathway and its target gene,Bim (Bcl-2 interacting mediator of cell death)and PU M A(p53 up-regulated modulator of apoptosis),on oxidative stressinduced apoptosis of human LECs.Methods The human LECs cell line(HLEC-B3)was cultured and passaged in DMEM with 10％ fetal bovine serum in vitro.Confluent cells were incubated in 24 well plates and divided into 4 groups.Hydrogen peroxide(H2O2)(50 μmol/L)was used to treat the cells for 4,8 or 12 hours,and cells without H2O2 treatment served as the control group.Apoptosis was detected using Hoechst 33258 staining and quantified by counting the number of cells with pyknotic nuclei.In addition,confluent cells were seeded in 6 well plates,and Western blot and RT-PCR were used to detect the expression of the caspase-3,c-Jun,Bim and PUMA proteins and their mRNA in HLEC-B3,respectively.The JNK/c-Jun pathway inhibitors,CEP11004 or SP600125,were added into cultured media with H2O2,and cells treated with DMSO or H2O2 only served as negative and positive control groups.The expression of the p-JNK,JNK,p-c-Jun,c-Jun,Bim,PUMA proteins was detected by Western blot and apoptosis was assayed using Hoechst 33258 staining.200 pmoL/L of Bim or PUMA small interference RNA(siBim or siPUMA)fragments were transfected into the cells for 24 hours,respectively,and H2O2 was then used to treat the cells for 8 hours.The expression of the Bim and PUMA protein and their mRNA in the cells was detected by Western blot and RT-PCR,respectively.Results After H2O2 treatment in HLEC-B3 cells for 4,8,or 12 hours,the rates of apoptosis were 4.30％±1.15％,27.08％±0.74％ and 46.59％±0.91％,showing a significant difference among them (F=1909.433,P=0.000),and those of the 4,8,12 hour groups were significantly increased in comparison to the control group(P =0.049,0.000,0.000).Compared to untreated cells,the levels of expression of the JNK,Bim,PUMA proteins and their mRNA in HLEC-B3 cells were significantly elevated.After the addition of CEP11004 or SP600125,the expression of these protein and mRNA in HLEC-B3 cells in the presence of H2O2 was significantly weaker than that in the DMSO control group(P =0.000,0.000).After the tranfection of siBim or siPUMA,the apoptosis rates of the H2O2 treated groups were significantly higher than those in the Bim-/-or PIMA-/-group (P＜0.05).Conclusions H2O2 can activate the JNK/c-Jun pathway and up-regulate the expression of its target genes Bim and PUMA in human LECs in a time-dependent manner.Inhibiting the JNK/c-Jun pathway and interfering with the expression of Bim and PUMA can protect human LECs against oxidative stress-induced apoptosis.

OBJECTIVETo establish a simple but effective method of laser scanning confocal microscopic imaging for Ca2+ oscillations of pancreatic acinar cells in adult mice.

METHODSPancreatic acinar cells from adult Kunming mice were isolated acutely with collagenase, and then loaded with fluo-4-AM, a Ca2+ indicator. A laser scanning confocal microscope armed with 488 nm laser was employed to record the dynamic fluorescent signals in-time and synchronously while acetylcholine (ACh) was added in the pancreatic acinar cells.

RESULTS(1) The classic pancreatic acinar cell Ca2+ oscillations were induced by a certain concentration of ACh (100 nmol/L) successfully and steadily, which could be blocked by atropine completely. (2) Plasmic Ca2+ oscillations from different parts of one acinar cell were usually with different amplitudes and almost the same frequencies. But both of amplitudes and frequencies were different among different cells. (3) The acinar cell Ca2+ oscillations were induced by ACh in a concentration-dependent manner.

CONCLUSIONThe laser scanning confocal microscopic imaging for adult mouse pancreatic acinar cell Ca2+ oscillations was established successfully. The features of being easy to use, direct to see lively, high efficiency and good flexibility make it a popular tool for researchers to choose.

Acinar Cells ; chemistry ; Animals ; Calcium ; analysis ; Calcium Signaling ; Cells, Cultured ; Mice ; Microscopy, Confocal ; methods ; Pancreas ; cytology

INTRODUCTIONHereditary spastic paraplegia (HSP) belongs to a large, heterogeneous group of progressive neurodegenerative diseases characterised by progressive lower extremity weakness and spasticity, which is caused by developmental failure or degeneration of motor axons in the corticospinal tract. Classical genetic studies have identified at least 46 genetic loci responsible for HSP.

METHODSA genetic study was conducted on a four-generation Chinese family with autosomal dominant HSP. The SPAST gene was investigated using linkage analysis and direct sequencing. Findings were compared with unaffected family members and 50 normal, unaffected individuals who were matched for geographical ancestry.

RESULTSWe identified a novel 14-bp heterozygous deletion that induced a frameshift mutation in exon 15 of SPAST (SPG4). This mutation is predicted to have functional impact and found to cosegregate with the disease phenotype.

CONCLUSIONOur results have expanded the mutation spectrum of the SPAST gene. These findings could help clinicians provide prenatal diagnosis of affected foetuses in families with a known history of such neurodegenerative diseases.

Adenosine Triphosphatases ; genetics ; Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; China ; Exons ; Family Health ; Female ; Frameshift Mutation ; Gene Deletion ; Genetic Linkage ; Genetic Predisposition to Disease ; Heterozygote ; Humans ; Infant ; Male ; Middle Aged ; Pedigree ; Phenotype ; Sequence Analysis, DNA ; Spastic Paraplegia, Hereditary ; diagnosis ; genetics ; Spastin ; Young Adult

Cholestatic liver injury,which is mainly caused by the disruption of bile acids,is common in the clinic.The pathogenesis of cholestatic liver injury is directly related to the changes of bile acid-related transporters,synthetic and metabolic enzymes.Nuclear receptors play a crucial part in cholestatic liver injury by regulating the expression of transporters and metabolic enzymes that maintaining the homeostasis of bile acids.In this review,we focus on the role of hepatic transporters and metabolic enzymes in cholestatic liver injury and the mechanism of nuclear receptors on the regulation of transporters and metabolic enzymes.

Liver is an important metabolic and detoxification organ in the body.Hepatic transporters are a series of functional membrane proteins that are extensively expressed in the liver.They are responsible for the uptake of endogenous and exogenous substances such as medicines into hepatocytes and excretion of their metabolic products into bile.Recent studies have provided that transporters and metabolic enzymes play important roles in the chemical substances-induced liver injury,and its various regulatory mechanisms have become hot topics of research.In this paper,we summarize the classification of hepatic transporters and metabolic enzymes and the changes of transporters and metabolic enzymes in the chemical substances-induced liver injury and its regulatory mechanism.

Objective To explore the therapeutic effect of lipid emul-sion combined with hemoperfusion and hemofiltration on acute paraquat poisoning and its consequence of acute lung injury.Methods A total of 24 dogs were randomly divided into A, B, C, D group.After paraquat ( PQ) 30 mg? kg -1 was given by intramuscular injection, if there was no loss during the injection process, the model of poisoning was considered to be made successfully.Then the dog models in four groups were respec-tively treated with 4 mL? kg -1 0.9%NaCl intravenous injection in group A, 4 mL? kg -1 lipid emulsion intravenous injection in group B, 4 mL? kg -1 0.9%NaCl intravenous injection combined with hemoperfu-sion and hemofiltration in group C, and 4 mL? kg -1 lipid emulsion com-bined with hemoperfusion and hemofiltration in group D.The standards of tumor necrosis factor -α( TNF -α) , interleukin -1β( IL -1β) , IL-10 , human insulin like growth factor -1 ( IGF-1 ) , transforming growth factor-1 (TGF-β1) were determined at 0, 8, 12, 24, 36 and 48 h after starting the hemoperfusion and hemofiltration.Results Every index TNF-α, IL-1β, IL-10, IGF-1, TGF-β1 of the group A was significantly higher than that in the group B, C, D at the same section (P<0.05).IngroupB,thecytokinelevelsTNF-α,IL-1β,IL-10,IGF-1,TGF-β1weresignificantlyhigher compared with group D ( P<0.05).Conclusion Lipid emulsion combined with hemoperfusion and hemofiltration can be beneficial to relieve the lung injury induced by acute paraquat poisoning.

Objective:To study protective effects of ranolazine preconditioning on myocardial ischemia reperfusion in-jury(MIRI)in rats.Methods:A total of 32 SD rats were randomly and equally divided into sham operation group, ischemia/reperfusion(I/R)group,low dose ranolazine group(low dose group)and high dose ranolazine group (high dose group).HR,SBP,DBP,left ventricular systolic pressure(LVSP),left ventricular diastolic pressure (LVDP),left ventricular pressure maximum rate of rise(+dp/dtmax),left ventricular pressure maximum rate of de-cline(-dp/dtmax),levels of CK-MB,LDH and cTnI,severity of myocardial infarction and ATP concentration were measured and compared among all groups.Results:Compared with sham operation group,there were significant re-ductions in LVSP[(119.35 ± 5.00)mmHg vs.(92.68 ± 2.95)mmHg vs.(100.60 ± 3.12)mmHg vs.(112.22 ± 3.69)mmHg],LVDP[(24.78 ± 1.71)mmHg vs.(17.26 ± 1.69)mmHg vs.(19.25 ± 1.05)mmHg vs.(22.18 ± 1.55)mmHg],+dp/dtmax[(3736 ± 102.37)mmHg/s vs.(3115 ± 112.72)mmHg/s vs.(3338 ± 51.88)mmHg/s vs.(3446 ± 37.99)mmHg/s],-dp/dtmax[(3634 ± 102.51)mmHg/s vs.(3015 ± 127.00)mmHg/s vs.(3239 ±37.36)mmHg/s vs.(3349 ± 45.49)mmHg/s]and ATP concentration[(22.54 ± 1.52)nmol/mg vs.(14.08 ± 1.80) nmol/mg vs.(16.88 ± 0.74)nmol/mg vs.(19.34 ± 0.88)nmol/mg],and significant rise in levels of CK-MB [(490.88 ± 168.04)U/L vs.(1259.0 ± 78.02)U/L vs.(1127.9 ± 127.23)U/L vs.(956.62 ± 105.22)U/L], LDH[(1494.9 ± 174.84)U/L vs.(2657.6 ± 104.33)U/L vs.(2293.9 ± 99.58)U/L vs.(1932.6 ± 134.25)U/L]and cTnI[(1.03 ± 0.14)ng/ml vs.(10.62 ± 1.34)ng/ml vs.(6.97 ± 1.32)ng/ml vs.(4.87 ± 0.79)ng/ml] in I/R group,low dose group and high dose group,P<0.01 all.Compared with I/R group,there were significant rise in LVSP,LVDP,+ dp/dtmax,-dp/dtmaxand ATP concentration,and significant reductions in levels of CK-MB,LDH and cTnI and MI severity[(0.5289 ± 0.0223)vs.(0.4887 ± 0.0089)vs.(0.4438 ± 0.0154)]in low dose group and high dose group(P<0.05 or <0.01),and those of high dose group were significantly better than those of low dose group(P< 0.05 or < 0.01).Conclusion:Ranolazine preconditioning possesses significant protective effect on MIRI,and it's dose-dependent.

OBJECTIVEThis study investigated the association between a missense SNP in the codon of ADD1 phosphorylation site and the susceptibility of non-cardia gastric cancer in a Chinese population.

METHODSPhosphoSitePlus and dbSNP database were combined to discover missense SNPs in the codon of phosphorylation site. Then, we genotyped the missense SNP in 1, 998 cases with non-cardia gastric cancer and 2, 008 cancer-free controls of Chinese descent. Analysis was conducted by using Logistic model adjusted by gender and age.

RESULTSThe rs4963 in the codon of ADD1 phosphorylation site was found. The frequencies of the 3 rs4963 genotypes, CC, CG, GG, among controls were 25.2%, 50.4%, and 24.4%, respectively, among patients were 20.1%, 50.6%, and 29.3%, respectively. Compared with CC genotype, the rs4963 CG genotype and GG genotype significantly increased the risk of non-cardia gastric cancer with the odds ratios being 1.24 (95%CI: 1.06 ∼ 1.46, P = 0.008) and 1.49 (95%CI: 1.25 ∼ 1.78, P < 0.001), respectively.

CONCLUSIONSFnnctional polymorphism in the phosphorylation site of ADD1 (rs4963) may influence the susceptibility of non-cardia gastric cancer.

Aged ; Asian Continental Ancestry Group ; genetics ; Calmodulin-Binding Proteins ; genetics ; Codon ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Logistic Models ; Male ; Middle Aged ; Mutation, Missense ; Odds Ratio ; Phosphorylation ; Polymorphism, Single Nucleotide ; Stomach Neoplasms ; ethnology ; genetics

OBJECTIVETo observe the performance of liver metastases with contrast-enhanced ultrasound (CEUS) and assess its clinical application.

METHODSTwenty-one patients with 21 untreated liver metastases underwent CEUS with low mechanical index imaging. The characteristic appearances of CEUS in different vascular phases were observed.

RESULTSOf 21 metastases, 19 (90.5%) were identified as fast-in and fast-out enhancement pattern. In the arterial phase, all the 21 lesions showed enhancement but with varied appearances: 12 (57.1%) showed early diffuse enhancement, 8 (38.1%) showed ring-like enhancement, and the remaining one lesion of large size showed slowly enhomogenous minor enhancement. In the late phase, sharp defects were found in 20 lesions (95.2%), and more lesions were detected in 3 patients (14.3%).

CONCLUSIONCEUS can show the characteristic appearance of liver metastases, and can be used for the screening and diagnosis of liver cancers.

Contrast Media ; Humans ; Liver Neoplasms ; diagnostic imaging ; secondary ; Ultrasonography

OBJECTIVETo develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.

METHODSThe heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.

RESULTSThe baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.

CONCLUSIONThe recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Antibodies, Monoclonal ; biosynthesis ; immunology ; Baculoviridae ; genetics ; Hepatitis A virus ; immunology ; Hepatitis Antibodies ; biosynthesis ; immunology ; Humans ; Immunoglobulin Fab Fragments ; biosynthesis ; immunology ; Immunoglobulin G ; biosynthesis ; immunology ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Recombinant Proteins ; biosynthesis ; immunology