Adult ; Drowning ; Humans ; Hydrogen Sulfide ; poisoning ; Male ; Radiography, Thoracic ; X-Ray Film ; Young Adult

China ; Education, Medical ; organization & administration ; standards ; Periodontics ; education ; Research Design ; Surveys and Questionnaires

OBJECTIVESince glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary hemolytic erythrocyte enzyme deficiency, most cases have single nucleotide mutations in the coding region, and current test methods for gene mutation have some missed detections, this study aimed to investigate the feasibility of RT-PCR sequencing in the detection of gene mutation in G6PD deficiency.

METHODSAccording to the G6PD/6GPD ratio, 195 children with anemia of unknown cause or who underwent physical examination between August 2013 and July 2014 were classified into G6PD-deficiency group with 130 children (G6PD/6GPD ratio <1.00) and control group with 65 children (G6PD/6GPD ratio≥1.00). The primer design and PCR amplification conditions were optimized, and RT-PCR sequencing was used to analyze the complete coding sequence and verify the genomic DNA sequence in the two groups.

RESULTSIn the G6PD-deficiency group, the detection rate of gene mutation was 100% and 13 missense mutations were detected, including one new mutation. In the control group, no missense mutation was detected in 28 boys; 13 heterozygous missense mutations, 1 homozygous same-sense mutation (C1191T) which had not been reported in China and abroad, and 14 single nucleotide polymorphisms of C1311T were detected in 37 girls. The control group showed a high rate of missed detection of G6PD deficiency (carriers) in the specimens from girls (35%, 13/37).

CONCLUSIONSRT-PCR sequencing has a high detection rate of G6PD gene mutation and a certain value in clinical diagnosis of G6PD deficiency.

Adolescent ; Child ; Child, Preschool ; Female ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; genetics ; Humans ; Infant ; Male ; Mutation ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

Adolescent ; Anemia, Aplastic ; complications ; Humans ; Leukemia, Myeloid, Acute ; etiology ; Male

Adenocarcinoma ; blood supply ; metabolism ; mortality ; secondary ; Adult ; Aged ; Female ; Humans ; Lymphatic Metastasis ; Male ; Microcirculation ; Middle Aged ; Neovascularization, Pathologic ; metabolism ; pathology ; Prognosis ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Receptors, Urokinase Plasminogen Activator ; Stomach Neoplasms ; blood supply ; metabolism ; mortality ; pathology ; Survival Rate ; Vascular Endothelial Growth Factor A ; metabolism

The L-cysteine desulfhydrase gene (cd) of Pseudomonas sp.TS1138 was amplified by PCR,and the amplified gene was recombined in the cloning vector pBluescript SKII.The 1.2kb DNA fragment containing cd was sequenced,and its homology with other desulfhydrases was blast; then the cd was cloned into the expression vector pET-21a(+), and afterward expressed by IPTG inducement.The expression protein was purified by Ni-NTA His-Bind Resin.Then the expression protein was identified by the method of activity staining of desulfhydrase, and the characterization of L-cysteine desulfhydrase and the critical role it played in the L-cysteine biosynthetic pathway were discussed.

The L-ATC hydrolase and L-SCC amidohydrolase which convert L-ATC to L-cysteine in Pseudomonas sp.TS-1138 are purified about 83.9 and 90.3 fold by salting-out method, Sephadex G-75 gel chromatography, DEAE-cellulose 52 ion exchange and Sephadex G-100 gel chromatography, etc. The purified enzyemes are both demonstrated by SDS-PAGE to be a homogeneous protein. Their molecular weight are about 37.5kD and42.8kDa respectively. The optimum reaction temperature are both 35℃, and the optimum pH are 7.0 and 8.0 respectively. The Km of the two enzymes are 0.67 mmol/L and 0.15 mmol/L, and the Vmax are 0.39?10 -3mmol/L?min and 0.42?10 -3mmol/L?min respectively.

Objective To investigate the anti-tumor effect of ZM-66 on multidrug-resistant leukemic cell line K562/ADM.
Methods The K562/ADM cells were treated with varying concentrations (0, 1, 2, 4×10-3 mmol/L) of ZM-66 or etoposide for 24 hours. The proliferation was detected by Sulforhodamine B Sodium Salt (SRB) assay and apoptosis was detected by flow cytometry analysis and fluorescent staining. In addition, the expression levels of p53 and bax genes in K562/ADM cells were detected by RT-PCR analysis. The level of P-glycoprotein (P-gp), P53 and Bax protein in K562/ADM cells were detected by Western blot assay.
Results SRB assay demonstrated that etoposide had little inhibitory effect on K562/ADM cells, whereas ZM-66 (1, 2, 4×10-3 mmol/L) had significantly inhibitory effect on K562/ADM cells (all P<0.01). The acridine orange/propidium iodide dual staining showed that there were typical condensation of chromatin and nuclear fragmentation nuclei with red color in ZM-66 treated cells. Flow cytometric analysis showed that there was a significantly increase of apoptotic cells in K562/ADM cells after treated with ZM-66. RT-PCR showed that the p53 and bax mRNA expression levels in K562/ADM cells treated with ZM-66 at 1, 2, 4×10-3 mmol/L were higher than those in the cell without treatment. Western blot showed that the P53 and Bax protein expression levels in K562/ADM cells treated with ZM-66 at 2, 4×10-3 mmol/L were higher than those in the cell without treatment. But the P-gp protein expression level in K562/ADM cells treated with ZM-66 at 2, 4×10-3 mmol/L was gradually lower than those in the cell without treatment.
Conclusion ZM-66 is able to induce cell death by apoptosis in vitro, as a result of the reverse of the apoptosis resistance in drug-resistant K562/ADM cells by modulating expression of key factors associated with apoptosis induction.

Animals ; Disease Progression ; Kidney Diseases ; physiopathology ; Kidney Glomerulus ; drug effects ; pathology ; Rats ; Renal Agents ; pharmacology ; Renin-Angiotensin System ; drug effects ; Sclerosis

Objective To screen mutations in genes including ASXL1, TET2, IDH1, IDH2, SETBP1, MPL515, JAK2 exon 12 and JAK2V617 in 135 polycythemia vera (PV) patients. To assess progreasson and genomics characteristics post polycythemic myelofibrosis. Methods DNA sequencing of ASXL1(Exon12),TET2 (Exons 3-11),IDH1(Exon4),IDH2(Ex-on4),SEPBP1(Exon4),JAK2 exon 12 and MPL515 (Exon 10) genes were carried out using Sanger method. JAK2V617 muta-tion was detected by allele-specific PCR in patients with PV. In the mean time, the mutation load of JAK2V617F allele (V617F%) was evaluated by real-time PCR using Tagman MGB probe. Then, the significant of gene mutations and clinical outcomes of post-PV Myelofibrosis(PPMF)was analyzed. To study risk factors of PPMF, logistic regression were employed. Results ASXL1, TET2, IDH1, IDH2 were mutated in 7.69%(8/104), 5.26%(1/19) , 0.08%(1/120) and 0.08%(1/121) of all PV patient respectively. JAK2 was mutated in 82.22%(111/135) of PV patients with mutation rate of exon12 of 2.96%(4/135) and there were no mutation of MPL515 and SETBP1 in PV patients. ASXL1 mutation was found in 31.82%(7/22) PPMF patients. Spearman analysis showed that ASXL1 is correlated with JAK2V617F (V617F%) (rs=0.298,P=0.002). The hemo-globin was lower in patients with ASXL1 mutation than patient without mutation (wild type). Leukocyte count, V617F%>50%rate, thrombosis and PPMF were higher in patients with ASXL1 mutation than that of ASXL1 wild type(P＜0.05). ASXL1 mu-tation, V617F%>50% rate and splenomegaly were all risk factors of PPMF. Conclusion ASXL1 mutation is the risk-fac-tor of PPMF and may promote V617F%by some mechanism.