We report a long-term follow-up of unstable hemoglobin (Hb) patient. He was diagnosed as Hb Madrid [beta115(G17)Ala-->Pro] by direct DNA sequencing and restriction enzyme analysis. Hydroxyurea had been given for beta-chain hemoglobinopathies through activation of gamma(gamma)-chain synthesis. Nowadays he still needs transfusion three or four times per year, but he had been free of hemolytic crisis after hydroxyurea. Although he has been treated for hemochromatosis with parenteral and oral iron-chelating agents, liver cirrhosis complicated by esophageal varix was developed and treated with endoscopic ligation. In addition, he is on warfarin maintenance for anticoagulation therapy for extensive portal vein and superior mesenteric vein thrombosis which presented with abdominal pain and diagnosed by CT scan. In management of unstable Hb patients, physician should monitor and control the serum ferritin level with iron-chelating agents, and be aware of possible long-term complication including hemochromatosis, cirrhosis or thromboembolism.
Abdominal Pain ; Esophageal and Gastric Varices ; Ferritins ; Fibrosis ; Follow-Up Studies ; Hemochromatosis ; Hemoglobinopathies ; Hemoglobins ; Hemoglobins, Abnormal ; Humans ; Hydroxyurea ; Ligation ; Liver Cirrhosis ; Mesenteric Veins ; Organothiophosphorus Compounds ; Portal Vein ; Restriction Mapping ; Sequence Analysis, DNA ; Thromboembolism ; Thrombosis ; Warfarin

PURPOSE: We applied a simplified method using polymerase chain reaction (PCR)-based enzymatic digestion for the detection of epidermal growth factor receptor (EGFR) mutation. MATERIALS AND METHODS: We selected 74 samples of adenocarcinoma of the lung with EGFR exons 19 and 21 that had been previously sequenced. We designed PCR primers and chose a DNA restriction enzyme. Seventy four additional lung cancer samples were tested as a test set. For test sets, the PCR-based method was performed first, followed by validation of the result by DNA sequencing. RESULTS: In the first sample group, we found 15 (20.3%) mutations in exon 19, and 9 (12.2%) mutations in exon 21 using the sequencing method. By using the PCR-based method, we were able to identify all of the mutated samples detected by the sequencing method. The PCR-based method also detected mutations in exon 19 in three additional samples and in exon 21 in one additional sample. In the second sample group, by performing the PCR-based method, we found 10 (13.5%) and 7 (9.5%) mutations in exons 19 and 21, respectively. Additional mutations in exon 19 were identified in 2 samples by the sequencing method. However, the sequencing method failed to identify a mutation in exon 21 in one sample. CONCLUSION: The sensitivity of the PCR-based enzymatic digestion method seems to be comparable to that of the traditional sequencing method for detecting EGFR mutations. Our method can be widely used as a screening test to select patients who may benefit from EGFR targeted therapy.
Adenocarcinoma ; Carcinoma, Non-Small-Cell Lung ; Digestion ; DNA ; DNA Restriction Enzymes ; Epidermal Growth Factor ; Exons ; Genes, erbB-1 ; Humans ; Lung ; Lung Neoplasms ; Mass Screening ; Polymerase Chain Reaction ; Receptor, Epidermal Growth Factor ; Restriction Mapping

Serratia marcescens jn01 was employed as the host for the isolation of phages from environmental sewage. One strain of phage named SmPjn was purified by picking transparent plaque with 2mm diameter and clear edge on the double-layer agar repeatedly. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of (58 +/- 2.16) x (55 +/- 0.47) nm and (7 +/- 1.25) nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage was capable of infecting the other two isolates of S. marcescens, P25 and CMCC41002. The optimal multiplicity of infection was 1. A one-step growth curve of SmPjn indicated that the latent period and burst size were estimated at 50 min and 1,125 pfu/cell, respectively . Genomic DNA of SmPjn was above 27kb in size and could be digested by Hind Ill and EcoR I into 11 and 9 visible fragments after electrophoresis, respectively. A novel Podoviridae-phage infecting S. marcescens was firstly reported in China.
China ; DNA, Viral ; genetics ; isolation & purification ; metabolism ; Host Specificity ; Podoviridae ; genetics ; growth & development ; isolation & purification ; Restriction Mapping ; Serratia marcescens ; physiology

OBJECTIVETo identify the RUNX2 gene mutation in two unrelated Chinese families with cleidocranial dysplasia (CCD), and to assess the feasibility of gene diagnosis for patients with CCD.

METHODSGenomic DNA was isolated from peripheral blood samples of 4 patients and 4 healthy members in the two pedigrees as well as 102 unrelated healthy controls. All 7 coding exons and their flanking intronic sequences of the RUNX2 gene were amplified by PCR, then the PCR products were sequenced bi-directionally. The sequencing results were compared with normal sequences in GenBank to identify the mutation. The mutation was confirmed by RFLP with restriction endonuclease.

RESULTSIn one family, a novel heterozygous missense mutation c.346T to A (W116R) in exon 1 of the RUNX2 gene was detected in the two affected individuals, and the mutation was further confirmed with Bsr I restriction endonuclease digestion. In the other family, a novel nonsense mutation c.610A TO T (K204X) was identified in the two patients. No above sequence change was found in the 102 healthy controls.

CONCLUSIONTwo novel RUNX2 mutations were found in two unrelated Chinese families with cleidocranial dysplasia. The identification of these mutations further extended the mutation spectrum of RUNX2 gene and will facilitate prenatal diagnosis and gene diagnosis of CCD.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Case-Control Studies ; Cleidocranial Dysplasia ; genetics ; physiopathology ; Core Binding Factor Alpha 1 Subunit ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Infant ; Male ; Mutation ; Pedigree ; Restriction Mapping

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.
Animals ; Feces/*microbiology ; Helicobacter/*isolation & purification ; Polymerase Chain Reaction/veterinary ; Restriction Mapping/veterinary ; Saliva/*microbiology ; Stomach/*microbiology ; Stomach Ulcer/microbiology/pathology/*veterinary ; Swine ; Swine Diseases/*microbiology/pathology

BACKGROUND: Extended-spectrum cephalosporins (ESCs)-resistant Enterobacter cloacae is one of the pathogens of nosocomial infection the incidence of which is on the rise. Moreover, plasmid-mediated quinolone resistance genes (PMQR genes) that reduce quinolone sensitivity have been shown to be widely distributed among clinical isolates of Enterobacteriaceae. This study was carried out to observe the distribution of PMQR genes in clinical isolates of E. cloacae resistant to ESCs. MATERIALS AND METHODS: Fourty-three ESCs-resistant E. cloacae strains from the blood collected during the span of 7 years, from 1994 to 2001, at Seoul National University Children's Hospital (SNUCH) were included in this study. Isoelectric focusing and enzyme specific PCR were performed to characterize beta-lactamase. The presence of qnrA, qnrB, qnrC, qnrS, qepA, and aac(6')-Ib-cr was determined by PCR, restriction enzyme analyses of PCR products, and DNA sequencing. Minimal inhibitory concentration (MIC) of several quinolones was measured by agar dilution method. RESULTS: The PMQR genes were detected in 9 (21%) of 43 ESCs-resistant E. cloacae isolates. Among them, five isolates were positive for qnrB2, and each two isolates harbored qnrB4 or qnrB5, respectively. qnrA, qnrC, qnrS or qepA was not identified. aac(6')-Ib was detected in 27 isolates, but aac(6')-Ib-cr was not found. Among the 9 qnrB-positive isolates, 5 produced SHV-12, 3 were derepressed mutants, and 1 produced pI 7.5 beta-lactamase. MIC ranges and percent resistances of nalidixic acid, ofloxacin, levofloxacin, and moxifloxacin for the PMQR genes-positive isolates were higher than PMQR genes-negative isolates. CONCLUSION: In this study, ESCs-resistant E. cloacae showed a high prevalence of PMQR genes, and qnrB was the only PMQR gene identified.
Agar ; Aza Compounds ; beta-Lactamases ; Cephalosporins ; Cloaca ; Cross Infection ; Enterobacter ; Enterobacter cloacae ; Enterobacteriaceae ; Incidence ; Isoelectric Focusing ; Nalidixic Acid ; Ofloxacin ; Polymerase Chain Reaction ; Prevalence ; Quinolines ; Quinolones ; Restriction Mapping ; Sequence Analysis, DNA

BACKGROUND: Extended-spectrum cephalosporins (ESCs)-resistant Enterobacter cloacae is one of the pathogens of nosocomial infection the incidence of which is on the rise. Moreover, plasmid-mediated quinolone resistance genes (PMQR genes) that reduce quinolone sensitivity have been shown to be widely distributed among clinical isolates of Enterobacteriaceae. This study was carried out to observe the distribution of PMQR genes in clinical isolates of E. cloacae resistant to ESCs. MATERIALS AND METHODS: Fourty-three ESCs-resistant E. cloacae strains from the blood collected during the span of 7 years, from 1994 to 2001, at Seoul National University Children's Hospital (SNUCH) were included in this study. Isoelectric focusing and enzyme specific PCR were performed to characterize beta-lactamase. The presence of qnrA, qnrB, qnrC, qnrS, qepA, and aac(6')-Ib-cr was determined by PCR, restriction enzyme analyses of PCR products, and DNA sequencing. Minimal inhibitory concentration (MIC) of several quinolones was measured by agar dilution method. RESULTS: The PMQR genes were detected in 9 (21%) of 43 ESCs-resistant E. cloacae isolates. Among them, five isolates were positive for qnrB2, and each two isolates harbored qnrB4 or qnrB5, respectively. qnrA, qnrC, qnrS or qepA was not identified. aac(6')-Ib was detected in 27 isolates, but aac(6')-Ib-cr was not found. Among the 9 qnrB-positive isolates, 5 produced SHV-12, 3 were derepressed mutants, and 1 produced pI 7.5 beta-lactamase. MIC ranges and percent resistances of nalidixic acid, ofloxacin, levofloxacin, and moxifloxacin for the PMQR genes-positive isolates were higher than PMQR genes-negative isolates. CONCLUSION: In this study, ESCs-resistant E. cloacae showed a high prevalence of PMQR genes, and qnrB was the only PMQR gene identified.
Agar ; Aza Compounds ; beta-Lactamases ; Cephalosporins ; Cloaca ; Cross Infection ; Enterobacter ; Enterobacter cloacae ; Enterobacteriaceae ; Incidence ; Isoelectric Focusing ; Nalidixic Acid ; Ofloxacin ; Polymerase Chain Reaction ; Prevalence ; Quinolines ; Quinolones ; Restriction Mapping ; Sequence Analysis, DNA

OBJECTIVETo study the bacterial community structure of the microbiota in the vaginal fluid from patients with bacterial vaginosis.

METHODSThe composition of bacteria in the samples of vaginal fluid from 3 patients with bacterial vaginosis and 1 normal premenopausal control was investigated by amplified ribosomal DNA restriction analysis(ARDRA).

RESULTSLactobacillus species were the predominant bacteria in the woman without bacterial vaginosis. Bacterial vaginosis was associated with higher concentrations of a variety of bacterial groups. Women with bacterial vaginosis had greater bacterial diversity, with 31 to 37 OTUs operational taxonomic units detected per sample. The species associated with bacterial vaginosis were Leptotrichia, Prevotella sp. and Megasphaera including several species with no close cultivated relatives.

CONCLUSIONSWomen with bacterial vaginosis have complex vaginal infections with many newly recognized species. ARDRA allows rapid analysis of the diversity of microorganisms in the vagina, and is capable of identifying potentially pathogenic bacteria that can not be identified by general culture.

Adult ; Bacteria ; classification ; genetics ; isolation & purification ; Bacterial Typing Techniques ; methods ; DNA, Ribosomal ; analysis ; genetics ; Female ; Humans ; Leptotrichia ; genetics ; isolation & purification ; Megasphaera ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods ; Phylogeny ; Prevotella ; genetics ; isolation & purification ; Restriction Mapping ; Vaginosis, Bacterial ; microbiology

OBJECTIVETo investigate the possible association of a single nucleotide polymorphism (SNP) at position 9250 in exon 7 of osteopontin gene (OPN gene 9250) with lupus nephritis (LN) in southern Chinese patients.

METHODSAltogether 109 patients with LN (12 males and 97 females) diagnosed according to the American College of Rheumatology criteria (revised in 1982) for systemic lupus erythematosus (SLE) and 180 healthy ethnically matched controls (34 males and 146 females), all Han people living in South China, were enrolled in this study with informed consent. Blood samples from all subjects were collected in EDTA tube for genomic DNA extraction according to the standard isolation procedures. OPN gene 9250 polymorphism was typed by PCR-restriction fragment length polymorphism (PCR-RFLP).

RESULTSAll the genotype frequencies of OPN gene 9250 were in Hardy-Weinberg equilibrium. Compared with the controls, LN patients showed significantly lower frequency of TT genotype of OPN gene 9250 (70% vs 51%, P<0.05) and significantly higher frequency of TC genotype (29.4% vs 45%, P<0.05). There were significant differences in OPN gene 9250 allele and phenotype frequencies between LN patients and the controls (P<0.05). Compared with the female controls, the frequency of TT genotype of OPN gene 9250 was significantly lower in female LN patients (68.5% vs 54%, P<0.05), and there was a significant difference in OPN gene 9250 allele frequency between them (P<0.05).

CONCLUSIONOPN gene 9250 polymorphism appears to be associated with the susceptibility to LN in southern Chinese Han population.

Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; ethnology ; genetics ; Case-Control Studies ; China ; ethnology ; Exons ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Lupus Nephritis ; genetics ; Male ; Middle Aged ; Osteopontin ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Restriction Mapping ; Young Adult

OBJECTIVETo develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.

METHODSThe double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.

RESULT AND CONCLUSIONDNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.

Base Sequence ; Gene Expression ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Inverted Repeat Sequences ; Molecular Sequence Data ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Restriction Mapping ; methods ; Sequence Analysis, DNA ; Time Factors