Objective To prepare a high‐titer rabbit specific serum antibody against Kaposi′s sarcoma associated herpesvirus (KSHV) ORF65 capsid protein and identify the specificity of serum antibody .Methods Artificial synthetic peptide of ORF65 pro‐tein was emulsified with Freund adjuvant .4 rabbits were immunized with the prepared antigen by subcutaneous injection at various sites of skin of back and jaw once every two weeks .Immunization was carried out in total 4 times .The serum of the immunized rab‐bits was collected at a week after the last immunization .The titer of rabbit anti‐serum was assayed by ELISA .Specificity of the rab‐bit anti‐serum was analyzed by immunofluorescence assay and Western blot .Results The immunized rabbits produced high‐titer se‐rum antibody after total immunization .The highest titer of anti‐serum against ORF65 protein peptide was 1∶12 800 .The results of Immunofluorescence assay showed that antibody was binded in plasma of BCBL‐1 cell mostly ,which was consistent with the expres‐sion location of ORF65 in BCBL‐1 cell .Subsequently ,the data of Western blot revealed a specific band about 21 kD which accorded with the size of ORF65 protein .Meanwhile ,the expression of ORF65 in TPA treated BCBL‐1 cells was higher than the control cells ,which was consistent with the expression characteristics of lytic protein .Conclusion High‐titer specific rabbit serum antibody against KSHV capsid ORF65 antigen could be successfully prepared by rabbits immunization with ORF65 protein peptide .The pre‐pared antibody could be revealed immune reaction specificity with KSHV ORF65 protein .

Objective To explore the role of Kv channel interacting protein 1(KChIP1)in the process of epileptic seizure and the relationship between KChIP1 and gamma-aminobutyric acid-(GABA)ergic neurons.Methods Normal female Sprague-Dawley rats were treated with pentylenetetrazole to make acute pentylenetetrazole models of epilepsy.Laser Scanning Confocal Microscope(LSCM)combined with double-labeled immunohistochemical technique was applied to observe the expression of the KChIP1 and the GABAergic neurons in the hippocampus of rats.Results The number of KChIP1-postive neurons in the hippocampus was significantly increased in the acute pentylenetetrazole model rats(P<0.05).There was no significant difference in the number of double-labled neurons(P>0.05),nor of the GABA-postive neurons between the model rats and the controls.The ratio of double-labeled neurons/total positive neurons was 63.9% in the hippocampus.Conclusion The KChIP1 might be involved in epileptogenesis of pentylenetetrazole induced seizure.The KChIP1 was associated with GABAergic neurons,whereas it may be functionally different from GABA.

Objective To evaluate the clinical effects of memory alloy embracing fixators in fixation of the rib fractures and investigate the related surgical indications and surgical techniques.Methods Retrospectively review was conducted on the clinical data of 30 patients with rib fractures treated with memory alloy embracing fixators from October 2010 to April 2011 at General Hospital of Guangzhou Military Command.The number of memory alloy embracing fixators used in operation,the number of fixed positions,and operation time were recorded.The pain scores before and after operation were comparatively studied.Operation efficacy and complications were analyzed.Results Of the 30 patients,the total operation time,number of fixators,and number of fixed ribs were (111.9±48.0) minutes,4.3±2.1 and 3.5±1.3,respectively.Meanwhile,the difference between pre-operative and post-operative pain scores was significant (6.93±0.88) points vs (4.04±0.62) points,P＜0.05).The ambulation perlod was (4.6±1.9) days and length of hospital stay was (27.2±10.8) days.Incisional and thoracic wall hematoma was detected in three patients and pulmonary infection in six post-operatively but none presented intractable chest pain,foreign body rejection or wound infection.Conclusion Memory alloy embracing fixators for rib fractures is reliable,easy,and effective in alleviating pain,improving lung function,reducing the frequency of ventilator use and preventing complications like lung infection.

The gamma-secretase complex mediates the final cleavage of APP to generate the principal component of amyloid plaques in the brains of Alzheimer's disease patients.Four integral membrane proteins (PS,NCT,PEN-2 and APH-1) are essential and sufficient for gamma-secretase activity.To identify the promoter of human nicastrin gene (NCT),its 5' -flanking region has been characterized and a 270 bp fragment containing the TSS (transcription start site) for the promoter activity has been identified.EMSA assays confirmed that all four AP-1 binding sites and two NFAT sites in the NCT promoter region were able to bind relative transcription factors in vitro.Mutations,as well as treatment with PDTC,which adjust the regulatory effect of AP-1 and NFAT,altered NCT promoter activity in both HeLa cells and rat cortical neurons.The results demonstrated that AP-1 and NFAT are involved in the regulation of hNCT transcription and suggest that balanced activation of AP-1 and NFAT ensures a strict temporal and tissue-specific control of NCT transcription.

Many somatic cell typos were obtained by in vitro differentiation or teratoma formation of human embryonic stem ceLls (hESCs). However, it is unclear whether specific cell types can be obtained from hESCs-derived teratoma. It was reported that many kinds of cells, including neural progetfitor/precursor cells (NPCs) and mesenchymal stem cells (MSCs) were isolated efficiently from the teratoma of hESCs through in vitro selection. The teratoma-derived NPCs and MSCs showed specific characteristics of molecular markers similar to the primary NPCs and MSCs. Moreover, these teratoma-induced NPCs and MSCs can be further induced to differentiate into neurons, astrocytes, or adipose and bone cells, reflecting their inherent multi-potencies. Given that teratoma normally contains a mixture of ectoderm, mesodenn, and endoderm lineage cells at different differentiation stage, it was suggested that hESCs-derived teratoma could be an alternative source to generate a variety of uncommitted progenitor cells or terminally differentiated somatic cells, which may be otherwise difficult to obtain through direct in vitro differentiation.

Objective To isolate and identify the potential binding partners of LRRK2,a gene linked to both dominant familial form and sporadic form of Parkinson's disease,thus to further our knowledge of its function.Methods We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait.The bait amplified by polymerase chain reaction(PCR) was then cloned into a yeast expression plasmid pGBKT7.After being sequenced and analyzed,pGBKT7-bait was transformed into the yeast strain AH109.Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain.Then human fetal brain cDNA library was trarnsformed into that yeast strain.which could express pGBKT7-bait fusion protein.The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade)containing X-a-gal.We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7,respectively.At last,these plasmids isolated from these true positive colonies were analyzed by bioinformatics.Results We obtained 9 true positive colonies,these colonies were sequenced, and we performed sequence Blast in GenBank.Three colonies of the 9 positive colonies were not in open reading-frames.Among other 6 colonies,there were known proteins including spermatid perinuclear RNA-binding protein(STRBP)and BCL2-associated athanogene 5 isoform b(BAG5),as well as unknown proteins including tyrosine phosphatase non-receptor type(PTPN23),1(3)mbt-like 3 isoform b(L3 MBTL3),RALY RNA binding protein-like isoform 1(RALYL),and Homo sapiens mRNA for KIAA1783 protein,partial cds(KIAA 1783).Conclusion True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid.Our screened proteins may provide a new research clue for revealing biological functions of LRRK2,pathogenesis of Parkinson's disease,and other neurodegerations.

Hearing impairment (HI) affects 1/1000 children and over 2% of the aged population. We have previously reported that mutations in the gene encoding gap junction protein connexin-31 (C×31) are associated with HI. The pathological mechanism of the disease mutations remains unknown. Here, we show that expression of C×31 in the mouse inner ear is developmentally regulated with a high level in adult inner hair cells and spiral ganglion neurons that are critical for the hearing process. In transfected cells, wild type C×31 protein (C×31wt) forms functional gap junction at cell-cell-contacts. In contrast, two HI-associated C×31 mutants, C×31R180X and C×31E183K resided primarily in the ER and Golgi-like intracellular punctate structures, respectively, and failed to mediate lucifer yellow transfer. Expression of C×31 mutants but not C×31wt leads to upregulation of and increased association with the ER chaperone BiP indicating ER stress induction. Together, the HI-associated C×31 mutants are impaired in trafficking, promote ER stress, and hence lose the ability to assemble functional gap junctions. The study reveals a potential pathological mechanism of HI-associated C×31 mutations.
Animals ; Connexins ; genetics ; Ear, Inner ; metabolism ; Endoplasmic Reticulum ; physiology ; Gap Junctions ; genetics ; metabolism ; physiology ; Golgi Apparatus ; genetics ; metabolism ; Hearing Loss ; genetics ; metabolism ; pathology ; Mice ; Mutation ; Neurons ; metabolism ; Protein Transport ; genetics ; Stress, Physiological

OBJECTIVETo introduce a new technique for rapid construction of gene site-directed mutagenesis.

METHODSThree primers are synthesized. One is a primer with the needed mutation; the other two containing appropriate enzyme sites for construction of the PCR fragment into a suitable plasmid are located at the flanks of the mutation primer. After the amplification of the PCR fragment using the mutation primer and the reverse flanking primer, another PCR is performed using the previous PCR mutation segment as primer and the other flanking primer. The final PCR segment can be cloned into an appropriate plasmid by using the enzyme sites in the primers.

RESULTSTwo site-directed mutagenesis have been successfully constructed in the Parkin gene by this method.

CONCLUSIONThe method is effective and simple for construction of gene site-directed mutagenesis.

Base Sequence ; Cloning, Molecular ; methods ; DNA Primers ; genetics ; Ligases ; genetics ; Mutagenesis, Site-Directed ; Mutation ; Plasmids ; genetics ; Ubiquitin-Protein Ligases

Ring dermoid of cornea (RDC) is an autosomal dominant disorder of cornea. The previous study identified a G185A mutation of PITX2 gene in a Chinese family with RDC. To investigate the pathological mechanism of PITX2 R62H mutation, a PITX2 prokaryotic expression plasmid were constructed and GST-PITX2 fusion protein were purified. EMSA was further conducted. The DNA-banding ability of PITX2 R62H was similar to that of the wild type PITX2 were found. The cell lines stably expressed PITX2 was also generated, and cell cycle were analyzed by flow cytometry, and the expression of ?-catenin and Cyclin D1 were detected by quantitative Real-time PCR. The results showed that proliferating ability of cells expressing PITX2 R62H was lower than that of cells expressing PITX2 WT, as well as ?-catenin and Cyclin D1 mRNA level. These findings revealed that PITX2 R62H mutation affected the Wnt/?-catenin→PITX2 pathway, promoted the genes expressing abnormally, and led to abnormal cell proliferation and the formation of RDC, which may play an important role in pathogenesis of RDC.

The Chinese Standard Aphasia Examination was made according to Chinese language and cultural habits by referring to Japanese Standard Language Test of Aphasia.151 normal people and patients whose brain damaged without aphasia were tested.Mean and standard deviations were calculated.The significant differences were not found by analysis of variance to age,sex,handedness,profession and education except oral instruction and describing pictures in different educational groups.Therefore,the examination is suitable for Chinese aphasics who live in different parts of China and spoken mandarin.