Bardet-Biedl Syndrome ; diagnosis ; therapy ; Child ; Female ; Humans

AIM: To clone the full-length of 75A EST. METHODS: After the extraction of total RNA from primary cultured rat cerebellar granule cell of 7DIV in the medium containing 25 mmol/L KCl, T_4 DNA ligase-mediated 5' RACE was used to retrieve 5' unknown sequence of 75A EST, and the first round 5' RACE PCR product was subcloned into pGEM-T easy vector for sequence and homogeneous analysis. RESULTS: The first round of 5' RACE produce a 2.5 kb band, and 75A EST was identified to be partial sequence of Neuron-derived orphan receptor (Nor1) gene. After two more rounds RACE, we firstly cloned the full-length of Nor-1 cDNA. CONCLUSION: T_4 DNA ligase mediated 5' RACE is an efficient method to retrieve information about the 5' termini of mRNAs, and lay a foundation for further study which role Nor1 play in the cerebellar granule cell differentiation or survive.

AIM: To analyze the subcellular localization of Arnt2 in rat cerebellar granule neurons (CGNs). METHODS: Based on the amino acids sequence of Arnt2 (LOCUS:NP_036913), the subcellular localization of Arnt2 in eukaryotic cells and the nuclear export signals (NES) of Arnt2 were predicted in CBS bioinformatics database. The subcellular localization of Arnt2 in rat cerebellar granule neurons was detected by the method of laser scanning confocal microscopy (LSM) analysis. RESULTS: It was predicted that Arnt2 located in nuclei of eukaryotic cells with the most probability, while located in cytoplasmic mitochondria with a slight possibility. A nuclear export signal was found in Arnt2 amino acids sequence, it was identified to be the leucine of No.143 that located in N-terminal of Arnt2 amino acids sequence. Finally, the result of LSM analysis shows nuclear localization of Arnt2 in rat CGNs. CONCLUSION: Arnt2 is located in nuclei of normal rat CGNs, it suggests that Arnt2 has the tendency to translocate into mitochondria after induced by some of inducible factors, for both the possibility of mitochondria localization and NES exist in Arnt2 amino acids sequence.

AIM: To Screen and identify differentially expressed genes that involved in apoptosis model in rat cerebellar granule neurons (CGNs).METHODS: The rat cerebellar granule neurons were isolated and primarily cultured. Fluorescent differential display RT-PCR (FDD RT-PCR) was performed to screen differentially expressed ESTs in the apoptosis model of primarily cultured rat CGNs. ESTs were subcloned into pGEM-T EasyTM vector and then sequenced. Alignment assay in non-redunant database was applied for encoding information. Reverse Northern blotting was used to appraise the results from DDRT-PCR.RESULTS: 164 pieces of differentially expressed ESTs were obtained by FDDRT-PCR. 17 of them were subcloned and sequenced. 5 ESTs of 17 were confirmed to be positive results by reverse Northern blotting. CONCLUSION: DD-PCR is a rapid, simple-operation and sensitive method for screening differentially expressed genes, which would contribute to the molecular mechanisms of apoptosis/survive of CGNs.

Background Normal adult optic disc rim width follows inferior ≥ superior ≥ nasal ≥ temporal (ISNT) rule.Analysis of the optic disc structural parameters with Heidelberg retina tomograph (HRT) can understand whether ISNT principle is suitable to other optic disc structure parameters.Objective This study was to investigate the characteristics of optic nerve head structure parameters in normal Chinese adults.Methods The parents of twins who participated Guangzhou Twin Eye Study were enrolled in Zhongshan Ophthalmic Center from 2006 June to 2011 August.All subjects received the routine eye examinations,including visual acuity,slit lamp microscope,direct ophthalmoscope examination,auto-refraction examination and HRT Ⅲ examination.All data from the right eyes were collected and analyzed.HRT values from different quadrants of optical nerve head were compared with one-way analysis of variance,and the multiple comparison between quadrants was carried out by Bonferroni test.The differences of optical nerve head parameters in different gender were analyzed using independent sample t test and Bland-Altman test.Results In this study,1672 eyes of 1672 subjects were included,with the 759 male and 913 female persons and average age of (41.8±5.3)years.No significant differences were seen in the disc area (DA)between the superior and inferior,as well as the nasal and temporal quadrants among the subjects (P＞0.05).In addition,there were not significantly different in the rim area (RA) between the superior and nasal disc as well as the rim volume (RV) among the inferior,superior and nasal disc (P＞O.05).However,significant differences were found in the cup area (CA),cup/disk area ratio (C/DAR),cup volume (CV),mean cupdepth (MCD),maximum cup depth (MxCD),height variation contour (HVC),cup shape measure (CSM),mean retinal nerve fiber layer thickness (mRNFLT) and retinal nerve fiber layer cross section area (RNFLcsa) among the different quadrants (P＜0.001).The local differences from only DA and RA complied with an ordination of the ISNT rule.The values of DA,CA,C/DAR,CV,MCD,MxCD from male individuals were higher,but those of the RV,HVC,mRNFLT RNFLcsa were lower in comparison with female individuals.Nevertheless,all the differences were ＜ 0.1 with a maximal mean difference in DA and CA (both 0.07 mm2).Conclusions The optical nerve head configurations measured by HRT have different distributing characteristics in four quadrants in normal adults.Most parameters show the higher values in the inferior and superior than those in nasal and temporal of disc,but DA and RA meet ISNT rule.The differences of most parameters between male and female subjects can be ignored.

Background Heidelberg Retinal Tomograph (HRT) has been widely used in clinical diagnosis of glaucomatous optic neuropathy,importantly,the measurement results of HRT may be affected by myopia.Therefore,studying the effect of different refractive status and axial length on structural parameters of the optic disc is important.Objective This study was to evaluate the influence of refractive error and axial length to optic nerve head parameter measured by HRT.Methods The parents who participate in Guangzhou Twin Study were enrolled in this investigation from July 2006 to August 2011.The regular eye examinations,including visual acuity,slit lamp,fundus,auto-refraction,HRT-Ⅲ and intraocular lens (IOL) Master,were performed on all the subjects.Thc data from right eyes of subjects were collected and analyzed.The linear correlation analysis and multiple linear regression analysis were applied to analyze the correlation between HRT optic papilla configuration parameter and equivalent sphere and ocular axis length.Results A pilot prospective series cases observational study was designed.1748subjects were enrolled in this study.The disc area (DA) (P=0.021),rim area (RA) (P＜0.001),rim volume (RV) (P＜0.001),mean retinal nerve fiber layer thickness (mRNFLt) (P＜0.001) and retinal nerve fiber layer cross section area (RNFLcsa) in the high myopia were greater than those of the emmetropic group,with significant difference between the two groups (P＜0.001).However,the cup area (CA),cup/disk area ratio (C/DAR) and cup volume (CV) were less in the high myopic group than those of the emmetropic group (P=0.004,P＜0.001 and P＜O.001),respectively.In the mild-moderatc myopic group,the RV,mRNFLt and RNFLcsa were greater than those of the emmetropic group (all of the P value less than 0.001),but the numbers of DA,CA,C/DAR,and CV were less than those of the emmetropic group,the differences were significant and their P valuables were 0.038,＜ 0.001,＜0.001 and 0.001 respectively.The differences of structure parameters between the hyperopia group and the emmetropic group were not statistically significant (P＞0.05).The DA,CA,RA,C/DAR,CV,RV,mean cup depth (MCD),maximum cup depth (MxCD),cup shape measure (CSM),mRNFLt and RNTLcsa were (2.04±0.43) mm2,(0.49±0.34) mm2,(1.55 ± 0.34) mm2,0.23-0.13,(0.11 ± 0.12) mm3,(0.45 ± 0.19) mm3,(0.21 ± 0.09) mm,(0.60±0.21) mm,-0.21 ± 0.07,(0.29 ± 0.07) mm and (1.46 ± 0.36)mm2,respectively,in the 1748 eyes of 1748 subjects.No significant correlation was found between the DA value with SE (r=-0.032,P =0.186).However,weaker positive correlations were seen between the CA,C/DAR,CV,MCD,MxCD with SE (r=0.139,0.163,0.130,0.004,0.054,P＜ 0.05) and negative correlation between RA,RV,mRNFLt and RNFLcsa with SE (r =-0.178,-0.331,0.241,-0.239,P＜0.001).With the increase of the ocular axial length,the DA,RA,RV,mRNFLt and RNFLcsa values were enlarged,and the CV value was smaller.With the increase of CA,the RA,C/DAR,CV,RV,MCD,MxCD,CSM and RNFLcsa values elevated,but mRNFLt value was decreased.Conclusions Refractive error is not associated with DA,but the measuring results are different among various refractive groups.Refractive error,ocular axial length and DA affect the measuring result of HRT.

ObjectiveTo explore the expression of CD3+ CD8+ human leukocyte antigen (HLA)-A2+T lymphocytes with specificity to the different hepatitis B virus (HBV) peptides in the peripheral blood mononuclear cells (PBMC)from the patients with hepatitis B associated hepatocellular carcinoma (HCC).MethodsThe HLA-A2+ PBMC from four patients with hepatitis B associated HCC were incubated with five HBV/HLA-A2 pentamers respectively,which were HBV sAg (FLLTRILTI),HBV sAg (GLSPTVWLSV),HBV sAg (WLSLLVPFV),HBV core (FLPSDFFPSV),and HBV pol (FLLSLGIHL),as well as anti-CD3-pacific blue and anti-CD8-fluorescein isothiocyanate (FITC).Then,HBV/HLA-A2-CD3-CD8 positive cells were detected by flow cytometry. The monoclonal HBV/HLA-A2-CD3-CD8+ cells were acquired by fluorescenceactivated cell sorter,and cultured and identified by flow cytometry.The anti-HBV specific T lymphocytes were then cultured with HepG2 (HLA-A2+ ) cells and the release of interferon γ (IFN-γ)were determined by enzyme-linked immunosorbent assay (ELISA),Res(a)ltsThe percentage of antiHBV T lymphoeytes with specificity to GLSPTVWLSV in total CD8+ T lymphoeytes from four patients with hepatitis B associated HCC was 1.44％±0.04％,which was higher than those to other four HBV antigen peptides (0.68％±0.08％ of FLLTRILTI,1.06％±0.09％ of FLPSDFFPSV,0.56％ ±0.04％ of FLLSLGIHL,and 0.46％ ±0.08％ of WLSLLVPFV) (t=0.001,P＜0.05).The two lines of monoclonal cell with specificity to GLSPTVWLSV both exhibited high level of IFN-γ expression after incubated with hepatic carcinoma cell line HepG2 (HLA-A2+)with HBV GLSPTVWLSV peptide.ConclusionsCD3+ CD8+ HLA-A2+ cells with specificity to the different HBV peptides exist in PBMC of patients with hepatitis B associated HCC.The expression level depends on HBV antigen peptide sequences and genomic sites.

Objective] To amplify and sequence the light chain of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum. [Methods] By comparing the conserved regions at each end of the nucleotide sequences of murine germ line genes enco ding FR1 and FR4 regions of immunoglobulin light chain variable regions, we designed a set of primers for amplification of V L gene. The hybridoma cells secreting anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum were cultured and their genome DNAs were extracted and used as templates for PCR. The PCR product was then cloned into pUC19 vector. The recombinants were sequenced by Sanger′s method. The V L gene was compared with GenBank and published mouse V L genes. [Results] The full length of V L gene was 318 bp. The V L gene was a member of mouse Ig ? light chain subgroup IV and generated from rearrangement of germ line V and J? 4 genes. The V L gene sequence has been registered by GenBank(accession No. AF206720). [Conclusion] The obtained V L gene was a potentially functional gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum .

Firstly discovered in 1980s,human immunodeficiency virus (HIV) continues to affect more and more people.However,there is no effective drug available for the therapy of HIV infection.Betulinic acid existing in various medicinal herbs and fruits exhibits multiple biological effects,especially its outstanding anti-HIV activity,which has drawn the attentions of many pharmacists.Among the derivatives of betulinic acid,some compounds exhibited inhibitory activities at the nanomolar concentration,and have entered phase Ⅱ clinical trials.This paper summarizes the current investigations on the anti-HIV activity of betulinic acid analogues,and provides valuable data for subsequent researches.

This study was aimed to investigate the effect of GATA-2 over-expression on function of mouse fetal liver hematopoietic stem cells. GATA-2 was introduced into mouse fetal liver cells via retrovirus mediated transduction with GFP as a detecting marker. Flow cytometry, colony-forming assay and cell cycle assay were used to detect the biologic changes of these retrovirus infected mouse fetal liver hematopoietic stem cells. The results showed that GATA-2 over-expression increased the Lin(-)Sca1(+)C-Kit(+) (LSK) population dramatically. Cell cycle of LSK cells didn't show abnormal, while colony forming ability decreased significantly. These data indicated that GATA-2 over-expression inhibited definitive differentiation of mouse fetal liver hematopoietic stem cells. It is concluded that over-expression of GATA-2 can significantly raise the LSK cell proportion in mouse fetal liver and inhibit the differentiation capability, the underlying mechanisms may be related to up-regulation of Hes-1, which may lead to the blocking of cell differentiation at the stem/progenitor cell stage.
Animals ; Cell Differentiation ; Cells, Cultured ; Female ; GATA2 Transcription Factor ; genetics ; Hematopoietic Stem Cells ; cytology ; Liver ; cytology ; Male ; Mice ; Mice, Inbred C57BL