Humans ; Lipodystrophy ; congenital

Mechanical ventilation (MV) with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses,resulting in ventilator-induced lung injury (VILI).The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear.VILI-related genes such as cysteine-rich angiogenic inducer 61 (Cyr61)have been demonstrated to play a detrimental role in the aggressive ventilation strategies.In the present study,we investigated the involvement of Cyr61 in the VIM and the underlying mechanism.A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting,respectively.Additionally,after exposure ofA549 cells to cyclic stretch for 5 min to 1 h,the expression levels of nuclear factor kappaB (NF-κB) and IL-8 were detected by ELISA and Western blotting.Thereafter,Cyr61 expression was depressed in A549 cells with the siRNA pGenesill.1-Cyr61-3 before the cyclic stretch,and IL-8 secretion and the activation of NF-κB pathways were probed by ELISA and Western blotting,respectively.Moreover,a NF-κB inhibitor (PDTC) and an activator (TNF) were used before mechanical stretch.Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8,respectively.The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells.Additionally,cyclic stretch also mobilized NF-κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells.The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch.Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA.It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells.These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

OBJECTIVE To investigate the develop mental toxicity of muscone to embryos. METHODS With zebrafish embryos as a model,The 3 h post fertilization (hpf)embryos were exposed to muscone at 5,10,20,40,80 and 160 μmol·L -1 culture solutions for 96 h and inspected daily with mi-croscopy for larval morphology.The drug solution was replaced every 24 h.Spontaneous move ments were checked at 24 hpf.Heart rate at 48 hpf,hatching rate,e mbryo deformity rate and mortality rate were evaluated.The expression of sepn1 was determined with real-ti me quantitative PCR technique at 96 hpf.RESULTS The 24 hpf spontaneous move ments showed no significant difference.At 48 hpf, spine curvature,pericardial ede ma,yolk sac ede ma,and abnormal swi mming were observed.In addition, the 48 hpf heart beats(10 s)was decreased fro m 26.5 ±1 .0 to 18.0 ±1 .9(P <0.01 ).At 48 hpf , hatching rate of 5 ~40 μmol·L -1 decreased(P <0.05),while of 160 μmol·L -1 increased (P <0.05) co mpared with muscone 0 μmol·L -1 .Muscone had little effect on hatching rate at other ti me points;Mal-formation rate and mortality rate at higher concentrations were up to 100%.The sepn1 gene expression at 96 hpf in the exposure groups decreased co mpared with that of control group(P <0.01 ).CONCLU-SION Muscone had toxic effects on the develop ment of zebrafish embryos,including spine curvature, abnormal swi mming,and pericardial ede ma.These effects may be related to the inhibition of sepn1 gene expression by muscone.

BACKGROUNDTreponema pallidum (T. pallidum) subsp. pallidum is the causative agent of syphilis. Analysis of recombinant antigens of T. pallidum led to the identification of potential candidate antigens for vaccine development and syphilis serodiagnosis. Tp0965 was predicted to be a membrane fusion protein and was found to be reactive with infected human sera in previous studies, but the results were controversial. In this research, the antigenicity and immunoreactivity of recombinant protein Tp0965 were assessed.

METHODST. pallidum subsp. pallidum (Nichols strain) was propagated and isolated and the genomic DNA was extracted. The Tp0965 gene was amplified by polymerase chain reaction (PCR). Then the recombinant protein Tp0965 was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid (Ni-NTA) purification system. The reactivities of protein Tp0965 were examined by immunoblot analysis and indirect enzyme-linked immunosorbent assay. The antisera against protein Tp0965 were obtained by immune rabbits and the immunogenicity of antisera were detected by indirect enzyme-linked immunosorbent assay.

RESULTSRecombinant protein Tp0965 was expressed successfully in vitro. Immunoblot assay showed that the recombinant protein Tp0965 could be recognized by human syphilitic sera of all stages. Indirect enzyme-linked immunosorbent assay showed there were only 4 of 74 human syphilitic sera that failed to show reactivity to recombinant antigen Tp0965, and lack of reactivity of Tp0965 to all 28 uninfected sera. A low titer of antiserum against Tp0965 in immune rabbits could be detected after the third time of immunization.

CONCLUSIONSThe recombinant antigen Tp0965 shows excellent sensitivity for the reactivity with sera from syphilitic individuals at all stages. The results also demonstrate a potential application for the serodiagnosis of syphilis.

Animals ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Proteins ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Membrane Proteins ; genetics ; immunology ; Polymerase Chain Reaction ; Rabbits ; Syphilis ; immunology ; microbiology ; Treponema pallidum ; immunology ; metabolism

OBJECTIVETo establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.

METHODSA pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis.

RESULTSThe 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR.

CONCLUSIONSThe method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.

Bacterial Infections ; diagnosis ; microbiology ; DNA, Bacterial ; chemistry ; genetics ; DNA, Ribosomal Spacer ; genetics ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Sequence Analysis, DNA

OBJECTIVETo identify 6 major human herpesviruses with consensus primers and to explore its clinical application.

METHODSBased on the highly-homogeneous regions of DNA polymerase gene in human herpesviruses,Two pairs of primer were synthesized. One pair was designed to amplify herpes simplex virus type 1, type 2, Epstein-Barr virus and cytomegalovirus; and another was used to amplify varicella-zoster virus or human herpesvirus 6. Virus species identification was performed by restriction enzyme digestion with BamH I and BstU I. Thirty-eight CSF specimens of clinically diagnosed viral encephalitis,and 49 blood specimens from 27 confirmed cases and 22 clinically diagnosed ones were tested for herpes virus DNA using the PCR-RFLP assay with these primers.

RESULTSThirteen out of 38 CSF specimens (34.2%) were herpes virus positive. All blood specimens from 27 confirmed cases showed positive results, while for 22 clinically diagnosed cases 16 (72.7%) were positive. The types of herpes virus were determined using restriction enzyme digestion with BamH I and BstU I. Two CSF specimens from the patients, who were treated with aciclovir for 2 - 3 days, were still positive for herpes virus DNA by this method. None of the control blood or CSF controls were positive for herpesvirus by PCR.

CONCLUSIONThe PCR-RFLP method used in this study is a specific, sensitive and practicable one for diagnosis of herpes virus infection.

Child ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; virology ; DNA Primers ; DNA, Viral ; blood ; cerebrospinal fluid ; Epstein-Barr Virus Infections ; virology ; Female ; Herpesviridae ; isolation & purification ; Herpesviridae Infections ; virology ; Herpesvirus 4, Human ; isolation & purification ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Simplexvirus ; isolation & purification

OBJECTIVETo investigate the genotype of the single nucleotide polymorphisms(SNP) of CYP1A2 IVS4 + 43A/G in patients with prostate cancer by TaqMan allelic discrimination.

METHODSTaqMan SNP allelic discrimination was performed with the ABI 7300 system to detect the genotype of CYP1A2 IVS4 + 43A/G in 85 patients with prostate cancer.

RESULTSThe genotypes of CYP1A2 IVS4 + 43A/G in the patients were as follows: AA subtype in 5 cases (5.9%), AG in 33 (38.8%), and GG in 47 (55.3%). Each subtype of CYP1A2 IVS4 + 43A/G was not correlated with the Gleason score of prostate cancer.

CONCLUSIONThere was no significant correlation between SNP of CYP1A2 and the differentiation of prostate cancer.

Cytochrome P-450 CYP1A2 ; genetics ; Gene Frequency ; Genotype ; Humans ; Male ; Polymorphism, Single Nucleotide ; Prostatic Neoplasms ; genetics ; pathology

OBJECTIVETo establish a restriction endonuclease pattern which could detect and differentiate four major human herpesviruses by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequence analysis.

METHODSA pair of primer, which was designed according to sequences in well-conserved regions of the DNA polymerase gene in human herpesviruses, was designed to amplify herpes simplex virus type 1 and 2 (HSVI/II), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Sequences of the primers are as follows: P(1) (5'-CGACTTTGCCAGCCTGACC-3') and P(2) (5'-AGTCCGTGTCCCCGTAGATG-3'). DNA of four strains of standard herpesviruses were amplified by PCR, and further studied by DNA cloning, sequence analysis and RFLP. At last, the authors established the PCR-RFLP technique to differentiate the four different herpesviruses. Meanwhile, 75 clinical blood specimens from infants with suspected viral infection and 38 blood specimens from healthy children were evaluated for herpesviruses DNA or virus-specific IgM antibody by PCR-RFLP or by ELISA.

RESULTSThe PCR amplified products of four human herpesviruses were from 510 bp to 592 bp in length and were analyzed for herpesvirus types with restriction endonuclease technique. The specificity and sensitivity of this PCR-RFLP were examined. There was no cross-reaction with Escherichia coli, Staphylococcus aureus, hepatitis B virus (HBV), Clostridium neoformans and human-genomic DNA and the lowest detection level was 0.1 fg DNA. Among 75 specimens, 23 were positive by PCR and the positive rate was 30.7%, including 13 for CMV, four for EBV, five for HSVII and one for HSVI after restriction enzyme digestion with BamHI and BstUI, while 10 were positive by ELISA and positive rate was 13.3%. All ELISA-positive specimens were likewise positive by PCR. Thirteen of 65 specimens that were ELISA-negative were tested positive by PCR. An infant with CMV infection was determined with viral DNA and virus-specific IgM antibody in blood at 3, 4 and 6 months after birth, respectively. The result showed that she was still CMV DNA-positive in blood whereas IgM antibody was positive only at month 3 after birth. None of the 38 control blood specimens was positive for herpesvirus by this PCR-RFLP or by ELISA.

CONCLUSIONSThis PCR-RFLP technique was specific, sensitive, rapid and accurate in diagnosing herpesviruses infection in infants, and it could detect herpesviruses DNA in specimens which were negative for IgM antibody by ELISA.

Antibodies, Viral ; blood ; Cytomegalovirus ; genetics ; Enzyme-Linked Immunosorbent Assay ; Herpesviridae ; genetics ; Herpesvirus 4, Human ; genetics ; Humans ; Immunoglobulin M ; blood ; Infant ; Infant, Newborn ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA

OBJECTIVETo assess the effectiveness and safety of the alphala/d blocker naftopidil in the treatment of benign prostatic hyperplasia (BPH) patients with overactive bladder (OAB) symptoms.

METHODSFifty BPH patients with OAB symptoms were treated with naftopidil at the dose of 25 mg/d for 6 weeks. A self-controlled clinical trial was conducted. The effectiveness and safety of the drug were observed by comparing the International Prostate Symptom Scores (IPSS), quality of life indexes (QOL), maximum urinary flow rates (Qmax) , average urinary flow rates (Qave), voiding volumes (VV), blood pressures (BP) and heart rates (HR) obtained before and after the treatment.

RESULTSAfter 6 weeks' medication, the 46 assessable cases showed an average decrease of 9.75 in IPSS (P < 0.01), 3.97 in voiding symptom score (P < 0.01), 5.78 in urinary storage symptom score (P < 0.01) and 1.95 in QOL (P < 0.01), and a mean increase of 4.29 ml/s in Qmax (P < 0.01), 3.75 ml/s in Qave (P < 0.01) and 55.12 ml/s in VV (P < 0.05). But no significant changes were observed in BP and HR. Only 1 patient (4.35%) experienced the adverse event of dizziness.

CONCLUSIONThe alphalA/D blocker naftopidil is both effective and safe in the treatment of BPH patients with OAB symptoms.

Adrenergic alpha-Antagonists ; therapeutic use ; Aged ; Humans ; Male ; Middle Aged ; Naphthalenes ; therapeutic use ; Piperazines ; therapeutic use ; Prostatic Hyperplasia ; complications ; drug therapy ; Urinary Bladder, Overactive ; complications ; drug therapy

OBJECTIVETo review clinical features of four male patients with glutaric academia type I and screen glutaryl-CoA dehydrogenase (GCDH) gene mutations.

METHODSThe 4 patients underwent brain computer tomography (CT) and magnetic resonance imaging (MRI) analyses. Blood acylcarnitine and urine organic acid were analyzed with tandem mass spectrometry and gas chromatographic mass spectrometry. Genomic DNA was extracted from peripheral blood samples. The 11 exons and flanking sequences of GCDH gene were amplified with PCR and subjected to direct DNA sequencing.

RESULTSAll patients have manifested macrocephaly, with head circumference measured 50 cm (14 months), 47 cm (9 months), 46 cm (5 months) and 51 cm (14 months), respectively. Imaging analyses also revealed dilation of Sylvian fissure and lateral ventricles, frontotemporal atrophy, subarachnoid space enlargement and cerebellar vermis abnormalities. All patients had elevated glutarylcarnitine (5.8 umol/L, 7.5 umol/L, 8.3 umol/L and 7.9 umol/L, respectively) and high urinary excretion of glutaric acid. Seven mutations were identified among the patients, among which c.146_149del4, IVS6-4_Ex7+4del8, c.508A>G (p.K170E), c.797T>C (p.M266T) and c.420del10 were first discovered.

CONCLUSIONMacrocephaly and neurological impairment are the most prominent features of glutaric academia type I. Blood tandem mass spectrometry and urine gas chromatographic mass spectrometry analysis can facilitate the diagnosis. The results can be confirmed by analysis of GCDH gene mutations.

Amino Acid Metabolism, Inborn Errors ; diagnosis ; genetics ; metabolism ; Amino Acid Sequence ; Base Sequence ; Brain Diseases, Metabolic ; diagnosis ; genetics ; metabolism ; Glutaryl-CoA Dehydrogenase ; deficiency ; genetics ; metabolism ; Humans ; Infant ; Male ; Molecular Sequence Data ; Mutation ; Sequence Alignment