Objective To investigate the effects of early nasal intermittent positive pressure ventilation (NIPPV) compared with early continuous positive airway pressure (NCPAP) in low birth weight preterm infants with respiratory distress syndrome (RDS).Methods We performed a prospective,randomized controlled trial involving 364 low birth weight preterm infants with respiratory distress syndrome within 6 hours of birth.The infants were randomly assigned to NIPPV (n=188) or NCPAP (n=176) groups.Non invasive ventilation was initiated in the neonatal intensive care unit (NICU).The rate of mechanical intubation (within 3 days or 7 days),the use of pulmonary surfactant (PS),the rate of complications and mortality were compared between the two groups.Mann Whitney U,t and Chi-square tests were used for statistical analysis.Results The average time of invasive mechanical ventilation in NIPPV group were lower than that in NCPAP group[2.0 (1.0-4.0) d vs 7.0 (3.0-8.5) d,U=-3.457,P=0.001].The need for intubation and mechanical ventilation by day 3 and day 7 in the NIPPV group were less than those in the NCPAP group [day 3:4.8％ (9/188) vs 10.8％ (19/176),x2=4.621,P=0.032; day 7:9.0％ (17/188) vs 16.5％ (29/176),x2=4.551,P=0.033].In the NIPPV group,infants who got PS therapy was less than that in the NCPAP group [3.2％ (6/188) vs 8.5％ (15/176),x2=4.752,P=0.029].There was no significant difference in the fatality rate between the NIPPV and the NCPAP group [12.8％ (24/188) vs 10.8％ (19/176),P ＞ 0.05].There were no significant difference in the incidence of air leak,intracranial hemorrhage,periventricular leukomalacia,retinopathy of prematurity,necrotizing enterocolitis,patent ductus arteriosus,and bronchopulmonary dysplasia between the NIPPV group and the NCPAP group.Conclusion Among low birth weight prcterm infants with RDS,the early use of NIPPV reduces the need for PS,intubation and invasive ventilation compared with NCPAP.

OBJECTIVEMutations of the iduronate-2-sulfatase (IDS) gene is the ultimate cause of Hunter syndrome. Clarification of the nature of mutations will create a necessary premise for prenatal gene diagnosis. A mucopolysaccharidosis (MPS) type II patient and his parents from an ethnic minority in Yunnan province were studied to identify their possible mutation in IDS gene to establish the basis for prenatal gene diagnosis.

METHODSThe patient was a boy, 6 years and 10 months old. Urine glycosaminoglycans (GAGs) assay was used for preliminary diagnosis of the patient and his parents with the disease. The three related persons' DNA was extracted and the concentration and purity of the DNA were measured after the urine test results confirmed the diagnosis. Polymerase chain reaction-denaturing high performance liquid chromatography (PCR-DHPLC) analysis was performed to detect the position of the mutation around the hot spots of mutation in exon 9, 3, 8 of the IDS gene. DNA bidirectional direct sequencing was applied to analyze the mutation detected by PCR-DHPLC.

RESULTSThe results of GAGs test showed that in the child with MPS, dermatan sulfate (DS) was positive (+++), heparan sulfate (HS) (+++), chondroitin sulfate (CS) and keratan sulfate (KS) were negative (-); while in his parents none of DS, HS, CS and KS was positive. Abnormal peaks in exon 9 of IDS gene shown by PCR-DHPLC were found in the patient. His mother had heterozygotic peaks. A new frame-mutation (1343-TT) in exon 9 of IDS gene of this patient was confirmed by DNA sequencing. The position where mutation occurred was inside codon 407 (TTT), that means two "T" deleted at position 1343 base pair (1343-TT) in cDNA of the IDS gene, caused a new frame-mutation. It caused elongation of the amino acid chain to a terminal codon TGA at position 429. Thus the peptide chain was shortened from 550 to 428 amino acids. The patient is a hemizygote of the mutation and his mother is a heterozygote.

CONCLUSIONA new frame-mutation (1343-TT) on the IDS gene was identified in this study. The patient is a hemizygote and his mother is a heterozygote. The mutation (1343-TT) resulted in loss of 122 amino acids, which probably caused seriously decreased enzyme activity of IDS, and the authors speculate that this mutation may be the pathological basis of the disease. So, if the mother becomes pregnant again, a prenatal gene diagnostic test for the same mutation should be performed. Furthermore, PCR-DHPLC followed by DNA sequencing are effective methods for diagnosis, including prenatal diagnosis of MPS II.

Amino Acid Sequence ; Asian Continental Ancestry Group ; Base Sequence ; Child ; China ; Chromatography, High Pressure Liquid ; Genotype ; Humans ; Iduronate Sulfatase ; genetics ; Male ; Molecular Sequence Data ; Mucopolysaccharidosis II ; diagnosis ; genetics ; Mutation ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo identify the mutations of iduronate-2-sulfatase (IDS) gene, and to establish a basis of prenatal gene diagnosis of Hunter syndrome.

METHODSUrine glycosaminoglycan (GAG) assay was used to preliminary diagnosis of mucopolysaccharidosis. PCR-denaturing high-performance liquidchromatograptly (PCR-DHPLC) analysis was performed to detect the mutation in exons 9, 3, 8 of the IDS gene. DNA sequencing was applied to analyze the mutation detected by PCR-DHPLC.

RESULTSAbnormal peaks were found by PCR-DHPLC. A new frame-mutation (1569+TT) in exon 9 of IDS gene was identified by DNA sequencing. Two "T"q inserted in position 1569 base pair (1569+TT) caused a substitution of codon 482 (TTA, leucine) to 482 (TTT, phenylalanine). The "TT" insertion results in the decrease of amino acids from 550 to 482. The patient is a hemizygote and his mother is a heterozygote.

CONCLUSIONA new frame-shift mutation of IDS gene is found to report. The mutation (1569+TT) results in 68 amino acids lost. Probably it causes the enzyme activity seriously dropped down and being pathologically the basis of disease.

Child, Preschool ; Chromatography, High Pressure Liquid ; DNA Mutational Analysis ; Humans ; Iduronate Sulfatase ; genetics ; Male ; Molecular Sequence Data ; Mucopolysaccharidoses ; genetics ; Mucopolysaccharidosis II ; enzymology ; genetics ; Mutation

OBJECTIVETo investigate the relationship between complex 1311 mutation of C-->T in exon 11 and 93 T-->C in intron 11 of G6PD gene and the G6PD deficiency.

METHODSUsing NBT paper strip method to screen and quantitative NBT method to confirm G6PD deficiency. PCR-SSCP technique was used to find the abnormal exon 11 and the amplification refractory mutation system (ARMS) to identify 1311 mutation, and DNA sequencing to identify the complex mutation at 1311 in exon 11 and 93 in intron 11.

RESULTSAbnormal band in exon 11 was found in 12 cases. DNA sequencing showed that they were 1311 mutation together with 93 mutation.

CONCLUSIONThis complex mutation may be the cause of reduced activity of G6PD enzyme.

Base Sequence ; DNA Mutational Analysis ; Exons ; genetics ; Genetic Testing ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; enzymology ; genetics ; Humans ; Introns ; genetics ; Molecular Sequence Data ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Polymorphism, Single-Stranded Conformational

OBJECTIVEStudying on G6PD polymorphism from Hakka population in Guangdong province.

METHODSIdentifying the variants of G6PD gene and determining the frequencies respectively with the use of amplified refractory mutation system(ARMS), polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and ABI 3100 DNA sequencing technologies.

RESULTSMutations of G6PD gene in cDNA 1388 (G-->A), 1376 (G-->T), 95 (A-->G), 392 (G-->T), 1024 (C-->T), 1311 (C-->T) have been found.

CONCLUSIONG6PD cDNA 1388 (G-->A), 1376 (G-->T), 95(A--> G), 392 (G-->T), 1024 (C-->T) and 1311 (C-->T) accompanied with intron 11 (93 T-->C) are the common mutations in Chinese population. cDNA 1388 (G-->A), cDNA 1376 (G-->T) are the most popular G6PD gene variants in Hakka population. In this study, no new type of G6PD gene mutation was found in the Hakkas of Guangdong.

Asian Continental Ancestry Group ; genetics ; China ; DNA Mutational Analysis ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; ethnology ; genetics ; Humans ; Introns ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

OBJECTIVETo study the gene expressions in the stromal cells of the human prostate peripheral zone (PZ) in men of different ages.

METHODSWe primarily cultured stromal cells from the normal prostate PZ of men aged 23 -32 (young group) and 56 -75 years (old group), profiled the gene signature of the PZ cells by cDNA microarray, and defined the differential gene expression patterns by hierarchical cluster analysis. Among the differential genes, we selected and confirmed up-regulated genes by quantitative real time PCR (Q-PCR), and identified their protein coding by Western blotting.

RESULTSThere were significant differences in the gene expressions of the PZ cells between the old and young groups. Based on the fold change ratio of > or = 2 or < or = 0.5, 509 up-regulated and 188 down-regulated genes were selected in the PZ cells. A subset of significantly differential genes influencing the growth of adjacent epithelial cells were identified, including HGF, IGF2, IGFBP5 and MMP1 in the old males.

CONCLUSIONStromal cells in the prostate PZ were more active in older males in promoting the malignant progression of adjacent prostate epithelial cells, which might be due to the increased expression of extracellular paracrining mediators.

Adult ; Age Factors ; Aged ; Cell Proliferation ; Cells, Cultured ; Gene Expression ; Gene Expression Profiling ; Humans ; Male ; Middle Aged ; Prostate ; metabolism ; Stromal Cells ; metabolism ; Young Adult

OBJECTIVETo study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions.

METHODSThe phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h.

RESULTSThe phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment.

CONCLUSIONERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; embryology ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVEOf denaturing high performance liquid chromatography (DHPLC), a technique platform was developed for screening G6PD deficient variants.

METHODSWhen applied to screen and identify the G6PD deficient variants from 124 patients who come from 11 nations in China, the DHPLC was compared with amplification refractory mutation system (ARMS) or DNA sequence technique and assessed carefully in its accuracy, sensitivity, efficiency and the cost of experiment.

RESULTSThe G6PD-deficient variants, such as 1388 G-->A (36/124 cases), 1376 G-->T(35), 95 A-->G (14), 1024 C-->T (3), 392 G-->T (4), 871 G-->A /1311 C-->T /IVS XI +93 t-->c (9), 871 G-->A (1), 1311 C-->T/IVS XI +93 t-->c (4), 1376 G-->T /1388 G-->A (1) and so on, were characterized as sharp peaks by DHPLC and verified by DNA sequence. Further, the standard chromatograms were put into database for 8 kinds of common G6PD deficient variants in Chinese populations. And also DHPLC found 3 G6PD variants (1388 G-->A) from 103 negative controls. With taking 8.8 minutes and costing 1 dollar for each sample, DHPLC successfully detected and identified 34 heterozygous females from patients with G6PD deficiency. However, ARMS checked 83 positive controls but got 12 false G6PD mutants, of which 5 were false positive, 7 false negative. Above results show that DHPLC sounds like to be more convenience, sensitive and accurate than ARMS and DNA sequence techniques for checking G6PD mutants.

CONCLUSIONDHPLC is of great advantage to screen the G6PD deficient variants with accuracy, convenience, automation and less cost, and significantly to identify the female heterozygote and clinical type IV individuals with G6PD deficiency.

Chromatography, High Pressure Liquid ; methods ; DNA Mutational Analysis ; Female ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; genetics ; Humans ; Male ; Mutation ; Reproducibility of Results

OBJECTIVETo study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene (B[a]P)-induced changes of cell cycle in human embryo lung fibroblast (HELF) cells.

METHODSThe stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin D1, CDK4, E2F1, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle.

RESULTSB[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C (10, 100, 500, 1000, and 5000 micromol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from G1 to S phase. Both vitamin C and antisense cyclin D1 suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone.

CONCLUSIONSB[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.

Ascorbic Acid ; pharmacology ; Benzo(a)pyrene ; Blotting, Western ; methods ; Cell Cycle ; drug effects ; physiology ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; G1 Phase ; drug effects ; physiology ; Humans ; Lung ; cytology ; embryology ; RNA, Antisense ; genetics ; S Phase ; drug effects ; physiology ; Transfection ; methods

There is a high prevalence of thalassemia in the South of China. To explore the genotype of alpha-thalassemia as well as the distribution of alpha globin gene mutation in the South of China, 356 patients with heterozygote alpha(+) thalassemia, heterozygote alpha(0) or homozygote alpha(+) thalassemia and 78 patients with HbH were analyzed. The gene diagnosis methods including Gap-PCR, nested-PCR, PCR-RE, PCR-SSCP, 4P-ASPCR and DNA sequence analysis were used. The results showed that among 356 patients, 295 patients with --SEA/alphaalpha (82.87%), 1 patient with alphaalpha/alpha-alpha(3.7) (0.28%), 3 patients with alphaalpha/alpha-alpha(4.2) (0.84%), 3 patients with alphaalpha/alpha(CS)alpha (0.84%), 1 patient with alphaalpha/alphaalpha(QS) (0.28%) and 2 patients with alphaalpha/alpha(Westmead) alpha (0.56%) were found. The homozygote with -alpha(4.2) or -alpha(3.7) was not found. In 78 patients with HbH, 29 patients with --SEA/alphaalpha(-3.7) (37.2%), 20 patients with --SEA/alphaalpha(-4.2) (25.6%), 19 patients with --SEA/alphaalpha(CS) (24.3%), 2 patients with --SEA/alphaalpha(QS) (2.6%) were detected, and other remaiming 8 patients were needed to be defined. Among the non-defined 8 patients, the synonymous mutation with C-->G transversion (GCC-GCG) at codon 65 in the exon 2 of alpha 2-globin gene was detected in 2 unrelated HbH patients came from Guangxi province. Whether it correlated with the phenotype of HbH disease or it is only a single nucleotide polymorphism site (SNPs), should be confirmed in the future. In addition, a set of gene diagnosis methods based on PCR to screen deletion and non-deletion genotypes of alpha-thalassemia in Chinese was improved. A new method, 4P-ASPCR, to detect Hb CS and Hb QS was also developed. The method was verified to be more accurate, time-saving and economic. In conclusion, the genotypes of alpha-thalassemia in Chinese are very complicated, the genotypes of alpha-thalassemia in Chinese need to be further studied, the results of this research probably have practical significance for the gene diagnosis or antenatal diagnosis of alpha-thalassemia in the South of China.
Base Sequence ; China ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Gene Deletion ; Gene Frequency ; Genotype ; Globins ; genetics ; Hemoglobin H ; genetics ; Hemoglobins ; genetics ; Hemoglobins, Abnormal ; genetics ; Humans ; Molecular Sequence Data ; Mutation ; Polymorphism, Single-Stranded Conformational ; alpha-Thalassemia ; genetics ; pathology