The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.

Objective To determine the diagnostic value of anti-mutated citrullinated vimentin(anti- MCV)antibodies for rheumatoid arthritis(RA).Methods The anti-MCV were determined in 136 patients with RA,80 non-RA patients and 19 normal peoples.The diagnostic value of anti-MCV was assessed and compared with anti-CCP,AKA and RF.Results The sensitivity and specificity of anti-MCV in the 136 RA patients was 95.6% and 80.8% respectively,there was significanl difference between the test group and the control group(P

Objective To explore the impact factors for post-thaw embryo survival rate and clinical pregnancy rate in frozen-thawed embryo transfer program. Methods The clinical data of 573 cycles of frozen-thawed embryo transfers were retrospectively analysed. Groups were divided according to the pre-freeze embryo quality, pre-freeze embryonic developmental stage, frozen-thawed embryo quality and cryopreservation technique, respectively, and post-thaw embryo survival rates and/or clinical pregnancy rates were compared among groups. Results The clinical pregnancy rate of high quality pre-freeze embryo was significantly higher than that of low quality pre-freeze embryo (31.8% vs 20.0%) (P< 0.05). There was no significant difference in the post-thaw survival rates and clinical pregnancy rates between embryos frozen at day 2 of ferrtilization and those frozen at day 3 of ferrtilization(79. 1% vs 82.9% and 25.5% vs 31.2%, respectively) (P>0.05). The clinical pregnancy rates of the transfer cycles only with fully intact embryos and with mixed embryos were significantly higher than that only with partially damaged embryos(36.7% vs 24.1% and 29.2% vs 24.1%, respectively)(P<0.05). The post-thaw survival rate and post-thaw high-quality embryo rate were significantly higher in those processed with modified cryopreservation technique than in those processed with original cryopreservation technique (82.0% vs 66.3% and 50.0% vs 27.5%, respectively)(P<0.05). Conclusion Pre-freeze embryo quality, post-thaw embryo survival rate and post-thaw embryo quality have a positive correlation to subsequent clinical pregnancy rate. Favorable cryopreservation technique may ensure the success of post-thaw embryo recovery and transfer.

OBJECTIVETo compare the thermal conductivity of the related tissues along meridian line and non-meridian area.

METHODSForty healthy volunteers were observed with a infrared thermal imaging system.

RESULTSDuring heating the acupoint or the non-acupoint along the meridian line, the velocity of spreading of the temperature-increasing response along the meridian line was more easy and rapid, with a definite direction. However, during heating the non-meridian spots, the change of temperature was confined to the local area, with no definite direction. The spreading of skin temperature response along the meridian line was more rapid than the non-meridian area during both the two were heated simultaneously, and finally, 3 infrared radiant tracks along the meridian courses conformed basically with that of the classical three yin- meridians of the hand appeared.

CONCLUSIONThe thermal conductivity of the related tissues along the meridian line is better than that of non-meridian area and has definite direction, with differences in physical characteristics between them.

Acupuncture Points ; Hand ; Humans ; Meridians ; Skin Temperature ; Thermal Conductivity

OBJECTIVETo study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1.

METHODSHeLa epithelial cells were treated with either histone deacetylase inhibitor (HDACi) trichostatin A (TSA) or DNA methylation inhibitor 5-Aza-C before detecting SATB1 expression. Luciferase reporter system was applied to measure effects of EZH2 on SATB1 promoter activity. Over-expression or knockdown of EZH2 and subsequent quantitative reverse transcription-polymerase chain reaction were performed to determine the effect of this Polycomb group protein on SATB1 transcription. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of EZH2 and trimethylated H3K27 (H3K27me3) at SATB1 promoter in HeLa cells. K562 cells and Jurkat cells, both having high-level expression of SATB1, were used in the ChIP experiment as controls.

RESULTSBoth TSA and 5-Aza-C increased SATB1 expression in HeLa cells. Over-expression of EZH2 reduced promoter activity as well as the mRNA level of SATB1, while knockdown of EZH2 apparently enhanced SATB1 expression in HeLa cells but not in K562 cells and Jurkat cells. ChIP assay Results suggested that epigenetic silencing of SATB1 by EZH2 in HeLa cells was mediated by trimethylation modification of H3K27. In contrast, enrichment of EZH2 and H3K27me3 was not detected within proximal promoter region of SATB1 in either K562 or Jurkat cells.

CONCLUSIONSATB1 is a bona fide EZH2 target gene in HeLa cells and the repression of SATB1 by EZH2 may be mediated by trimethylation modification on H3K27.

Azacitidine ; pharmacology ; Base Sequence ; Cell Line ; Chromatin Immunoprecipitation ; DNA Methylation ; DNA Primers ; DNA-Binding Proteins ; physiology ; Enhancer of Zeste Homolog 2 Protein ; Epigenesis, Genetic ; physiology ; Epithelium ; metabolism ; Gene Silencing ; Humans ; Hydroxamic Acids ; pharmacology ; Matrix Attachment Region Binding Proteins ; genetics ; Polycomb Repressive Complex 2 ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; physiology

OBJECTIVETo investigate the role of PERK/eIF2alpha signaling pathway in hepatocyte apoptosis of alcoholic liver injury rats.

METHODSRat models with ethanol-induced liver injury were successfully developed by gastric gavage with ethanol-corn oil mixtures for 12 weeks. At different time points (4, 6, 10, 12 week), liver pathology was dynamically observed. The hepatocyte apoptosis was quantitatively analyzed by Annexin V-FITC/PI double-labeled flow cytometry, the serum total homocysteine (tHCY) level was detected by ELISA and the expressions of eIF2a, p-eIF2a, GRP78/Bip, GRP94, caspase-3 and caspase-12 in liver were examined using Real-time PCR and Western blot.

RESULTSTypical acute liver injury and chronic liver injury were observed at week 4 and week 12 respectively. The hepatocyte apoptosis rates in 6-week model rats significantly increased compared with normal rats (P value less than 0.05), and the degree of hepatocyte apoptosis continued to increase with the modeling time, and the percentages of early and total apoptosis reached 26% and 29% at week 12. From week 6 to week 12, the serum tHCY levels in model rats were obviously higher than in normal rats (P value less than 0.01). Since week 4, eIF2a protein phosphorylation in model rat livers remarkably elevated compared with that in normal rat livers (P value less than 0.01), and at week 12 the peIF2a protein expression in model rat livers increased by 2.81-fold. Since week 4 the expressions of GRP78/Bip, GRP94, caspase-12 and caspase-3 mRNA and protein in model rat livers showed a significant increase as compared to normal rat livers, and at week 12, these gene and protein levels increased 4.70, 12.95, 3.83, 4.05 fold and 3.93, 6.93, 9.88, 3.31 fold, respectively (P value less than 0.01).

CONCLUSIONActivation of PERK/eIF2a signaling pathway contributes to the occurrence and development of hepatocyte apoptosis in alcoholic liver injury rats and it might be as a potential target for therapeutic applications in alcoholic liver diseases.

Animals ; Apoptosis ; Eukaryotic Initiation Factor-2 ; metabolism ; Hepatocytes ; cytology ; pathology ; Liver Diseases, Alcoholic ; metabolism ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; eIF-2 Kinase ; metabolism

This study was purposed to investigate the therapeutic effects of early transfusion of immunized donor lymphocytes after haploidentical transplantation by means of mouse model of nonmyeloablative haploidentical bone marrow transplantation. CB6F1 female mouse was served as recipient and C57BL/6 male mouse was served as donor. Each CB6F1 female mouse was subjected to intravenous transfusion with 1×10(6) erythroleukemia (EL9611) cells at day 4 before transplantation, followed with intraperitoneal injection of Ara-C (0.015 g) respectively at day 2 and day 1, then conditioned for BMT with TBI (450 cGy) at day 1 before transplantation. After conditioning (day 0), each of recipients was transplanted with 6×10(7) mixture of bone marrow and spleen cells from the C57BL/6 mice, and was infused with 6 × 10(7) immunized donor lymphocytes at day 15 after transplantation. All treated animals were evaluated for survival, development of leukemia and aGVHD. The donor CD3(+) cell chimerism and sex determining region Y gene (SRY)in recipients were monitored periodically after transplantation. The results showed tht all mice with only inoculation of 10(6) EL9611 cells survived for 15 ± 1 days (n = 4); all mice of other groups obtained the varying degrees of implantation. SRY could be detected at day 30 and 60 after transplantation. The chimerism of donor CD3(+) cells in mixed bone marrow transplantation (MT) group at day 14, 30 and 60 respectively reached 17.95% ± 12.03%, 37.34% ± 2.78% and 47.06% ± 6.1%. In donor lymphocyte infusion (DLI) group it reached 69.78% ± 12.62%, 75% ± 15.97%, 83.41% ± 16.07% at day 30, 45 and 60 after transplantation. The mice of MT and DLI group survived for 66.66 ± 1.47 days and 78.2 ± 7.82 days. It is concluded that the high tumor burden before transplantation can affect donor cell engraftment and prognosis.Early post-transplanted infusion of immunized lymphocytes from donor can help to improve the therapeutic efficacy and survival.
Animals ; Bone Marrow Transplantation ; methods ; Female ; Haplotypes ; Leukemia, Erythroblastic, Acute ; therapy ; Lymphocyte Transfusion ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Tissue Donors ; Transplantation Conditioning ; methods ; Transplantation, Homologous

OBJECTIVETo evaluate the clinical manifestations of laryngeal tuberculosis and its laryngoscopic characteristics.

METHODClinical analysis of 36 laryngeal tuberculosis cases confirmed pathologically was carried out retrospectively.

RESULTSThe patients' ages ranged from 19 to 78 years old (median 39.5 years old). The male to female ratio was 1.8 to 1. The most frequent chief complaint was hoarseness (83.3%). The true vocal cord and the false vocal cord were usually found involved by fibrolaryngoscope observations. The lesions appearances were mainly the proliferation such as nodules or granules. The laryngostroboscopy demonstrated that the wave of the mucosa and vibration of the involved vocal cords were markedly reduced or disappeared. Among 24 patients with multiple lesions in the larynx, 19 (79.2%) had active pulmonary tuberculosis,while 9 (75.0%) had normal lung status among 12 patients with single lesions in the throat. All the patients received a full course of antituberculosis treatment. The results of the antituberculous medication was satisfactory in all 26 cases patients who can be followed up, while 10 cases were lost for the follow-up.

CONCLUSIONSThe local clinical manifestations of laryngeal tuberculosis is nonspecific, while the whole manifestation is not apparent. The lesions commonly involve the true vocal cord and the false vocal cord. It can even occur without pulmonary tuberculosis.

Adult ; Aged ; Female ; Humans ; Laryngoscopy ; Larynx ; pathology ; Male ; Middle Aged ; Retrospective Studies ; Tuberculosis, Laryngeal ; diagnosis ; pathology ; therapy ; Young Adult

AIM:To explore the expression level of long non-coding RNA(lncRNA)myocardial infarction-as-sociated transcript(MIAT)in the tissues and cells of non-small-cell lung carcinoma(NSCLC), and to investigate the effect of MIAT on the function of NSCLC cell line.METHODS:Bioinformatic data in microarray dataset GSE19804 from Gene Expression Omnibus(GEO)were collected for analyzing the difference expression of MIAT between NSCLC tissues and normal lung tissues.Clinical and prognostic data in microarray dataset GSE 30219 from GEO were also collected for an-alyzing the correlation between the expression level of MIAT and the survival time of NSCLC patients.qPCR was applied to detect the expression of MIAT in 25 paired tumor tissues and corresponding adjacent normal tissues,normal lung epithelial HBE cell line and NSCLC A549,NCI-H266 and NCI-H1299 cell lines.The specific small interfering RNA for MIAT(si-MIAT group)or negative control sequence(si-NC group)was transfected into A549 cells,and flow cytometry,colony for-mation experiment and CCK-8 assay were employed to detect the proliferation of the cells in the 2 groups.The expression levels of cyclin D1 and cyclin-dependent kinase inhibitor 1A(CDKN1A)in the 2 groups were determined by qPCR and Western blot.RESULTS:In the GEO dataset GSE19804,the expression of MIAT in NSCLC tissues was significantly ele-vated compared with normal lung tissues(P<0.05).In the GEO dataset GSE30219,the overall survival time was signifi-cantly shorter in the patients with high expression of MIAT than the patients with low expression of MIAT(P<0.05).Fur-thermore,the levels of MIAT in both NSCLC tissues and cells were higher than those in adjacent normal tissues and normal cells(P<0.05).Compared with si-NC group,lower MIAT level,cell viability and cell colony number in si-MIAT group with statistical significance were observed(P<0.05).Meanwhile, compared with si-NC group, the expression of cyclin D1 in si-MIAT group was significantly decreased(P<0.05),and inversely,the expression of CDKN1A in si-MIAT group was significantly increased(P<0.05).CONCLUSION:There is high expression of MIAT in NSCLC tissues and NSCLC cells,and knockdown of MIAT expression inhibits NSCLC cell proliferation, which provides a potential target of targeted therapy for NSCLC.

The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.
Amino Acid Sequence ; Chromatography, High Pressure Liquid ; Molecular Sequence Data ; Peptide Mapping ; Recombinant Fusion Proteins ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Tandem Mass Spectrometry ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; chemistry