The desired DNA product of KGPcd and KGP-hag was obtained from the total DNA of Porphyromonas gingivalis by PCR with two pairs of gene specific primers. The segment of KGPcd and KGP-hag (about 1.5kb and 1.6kb) was inserted into pGEM-T easy Vector. The double-stranded DNA of the postitive clone was analyzed by restriction endonuclease mapping and DNA sequenceing. The sequences of KGPcd and KGP-hag were consistent with those of the references appeared. The proteins of KGPcd and KGP-hag will be obtained for further study.

OBJECTIVETo observe the protection against periodontal bone loss in the Sprague-Dawley (SD) rats periodontitis model, with the recombined plasmid pcDNA3.1+/kgpcd as DNA gene vaccine.

METHODSPcDNA3.1+/kgpcd was delivered into rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. Immunohistochemistry staining was used to assess the protection in the animal model.

RESULTSThe level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model.

CONCLUSIONSThe results indicate that pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model.

Animals ; Bacterial Vaccines ; immunology ; therapeutic use ; Immunoglobulin A, Secretory ; analysis ; Periodontitis ; immunology ; microbiology ; prevention & control ; Porphyromonas gingivalis ; genetics ; immunology ; Rats ; Rats, Sprague-Dawley ; Vaccines, DNA ; immunology ; therapeutic use

OBJECTIVEThis study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.

METHODSEukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.

RESULTSIt proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.

CONCLUSIONThe eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.

Animals ; Bacterial Proteins ; genetics ; COS Cells ; Cercopithecus aethiops ; Cysteine Endopeptidases ; genetics ; Genetic Vectors ; Plasmids ; Porphyromonas gingivalis ; genetics ; Transfection

Objective To investigate standardized self-management education for diabetics outside the hospital treatment by continuous subcutaneous insulin pump insulin (CSⅡ) effect.Methods The 51 patients who received hospital stay treatment by continuous subcutaneous insulin pump insulin and post-discharge treatment by continuous subcutaneous insulin pump insulin were selected as research subjects,and randomly divided into two groups:observation group of 26 patients and the control group of 25 patients,both groups received education in the hospital and after discharge follow-up of education; observation group was to take standardized self-management education,control group to take the traditional education method.Knowledge master CSII therapy in the two groups of patients before discharge was compared.Biochemical indicators,body mass index and the abnormal situation 3 months post-discharge was compared in the two groups.Results Glycosylated hemoglobin,total cholesterol,triglycerides,body mass index were (5.81 ±0.36)％,(3.62 ±0.48)mmol/L,(1.61 ±0.46)mmol/L and (22.91 ±2.33) kg/m2,respectively,those in the control group were ( 6.57 ± 1.34 ) ％,( 4.50 ± 0.99 ) mmol/L,(1.94 ±0.74)mmol/L,and (24.20 ±2.95) kg/m2,respectively,and the differences were significant (t=2.78,4.06,1.92,1.74,respectively; P ＜ 0.05).7.6％ hypoglycemia,11.5％ skin inflammation in the infusion site,15.4％ plugging,and 15.4％ tape loose occurred in the observation group during the pump period,and those in the control group were 32.0％,64.0％,48.0％,72.0％,respectively; the incident rates in the observation group were lower than the control group,and the difference was significant (x2=4.78,15.01,6.29,16.65,respectively; P ＜0.05).Conclusions The hospital CSII-treated diabetes,standardized self-management education is better than traditional education methods; it is worth in clinical practice.

OBJECTIVETo study the incidence of various enzyme deficiency in tetrahydrobiopterin (BH4) metabolism and the related gene mutation among the patients with motor disturbance and mental retardation.

METHODSOne hundred patients with unknown motor disturbance and mental retardation were referred to this study. All patients were performed by phenylalanine (Phe) and BH4 loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity. Some patients received the dopa treatment for diagnosis of dopa-responsive dystonia (DRD). The analysis of GTP cyclohydrolase 1 gene (GCH1) mutation for DRD patients and the analysis of 6-pyruvoyl tetrahydropterin synthase (PTS) gene mutations for PTS deficient patients were done under the consent from their parents.

RESULTSSeventy of 100 patients had normal basic blood Phe levels, six (6%) patients were diagnosed as DRD. Thirty patients had hyperphenylalaninemia (HPA), eight (8%) were diagnosed as PTS deficiency and 22(22%) were diagnosed as phenylalanine hydroxylase (PAH) deficiency. All patients had normal DHPR activity. The mutation IVS5+3insT of GCH1 was found in 2 patients with DRD. Seven kinds of PTS mutations were found in 8 patients with PTS deficiency, and 75% of the mutations were 259C-->T,286G-->A and 155A-->G.

CONCLUSIONSome patients with unknown motor disturbance and mental retardation may suffer from BH4 metabolism related diseases. Theses patients are necessary to be screened for such kind of diseases in order to confirm the diagnosis.

Adolescent ; Biopterin ; analogs & derivatives ; metabolism ; Child ; Child, Preschool ; Dihydropteridine Reductase ; genetics ; metabolism ; Dystonia ; genetics ; metabolism ; Female ; GTP Cyclohydrolase ; genetics ; metabolism ; Humans ; Infant ; Intellectual Disability ; genetics ; metabolism ; Male ; Mutation ; Phenylalanine Hydroxylase ; genetics ; metabolism ; Phosphorus-Oxygen Lyases ; genetics ; metabolism

OBJECTIVETo obtain the mutation spectrum of exon 12 of the phenylalanine hydroxylase(PAH) gene.

METHODSThe samples from 127 patients with phenylketonuria(PKU) were studied by polymerase chain reaction(PCR)-single strand conformation polymorphism(SSCP) PCR-denaturing gradient gel electrophoresis(DGGE) and direct DNA sequencing.

RESULTSFour heterozygous mutations including R413P S411X R408W R408Q in 10 of 127 patients were identified by DNA sequencing. The frequencies of R413P S411X R408W R408Q mutation were 2.76%, 0.39%, 0.39% and 0.39%, respectively. The S411X mutation in Chinese was first reported. Only 2 cases with R413P mutation were detected by SSCP. DGGE analysis showed that 10 cases displayed 3 kinds of abnormal electrophoretic bands. There were no obvious differences in the frequency of R413P mutation between northern and southern Chinese with PKU, and between classical PKU and hyperphenylalaninemia.

CONCLUSIONDGGE is more sensitive than SSCP in screening the mutations of exon 12 of the PAH gene. DGGE combined with DNA sequencing can be used to define all the mutations of exon 12 of the PAH gene. These results indicate the similarity in the frequency of R413P mutation between northern and southern patients.

Electrophoresis, Polyacrylamide Gel ; Exons ; Humans ; Mutation ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; genetics ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA

OBJECTIVETo confirm the diagnosis of multiple carboxylase deficiency (MCD) on the gene level and explore the mutations in Chinese children with MCD.

METHODSBiotinidase (BT) and holocarboxylase synthetase (HLCS) genes were analyzed by PCR and direct sequencing for the 4 BT deficiency patients and 8 HLCS deficiency patients, respectively. The identified mutations in the parents of the patients and 50 normal controls were screened by PCR-restriction fragment length polymorphism and direct DNA sequencing.

RESULTSTotal detection rate of gene mutation is 100% in the 12 children with MCD. Six mutations were detected in the 4 children with BT deficiency, they were c. 98-104del7ins3, c. 1369G>A (V457M), c. 1157G>A(W386X), c. 1284C>A(Y428X), c. 1384delA and c. 1493_1494insT. The last four were novel mutations. Four mutations were found in the 8 children with HLCS deficiency. They were c. 126G>T (E42D), c. 1994G>C (R665P), c. 1088T>A (V363D) and c. 1522C>T (R508W). The last two were hot-spot mutations [75%(12/16)], and c. 1994G>C (R665P) was a novel mutation.

CONCLUSIONThis study confirmed the diagnosis of 12 patients with MCD on the gene level. Six mutations were found in the BT gene and 4 in the HLCS gene, including 5 novel mutations. Two mutations of the HLCS gene are probably hot-spot mutations in Chinese children with HLCS deficiency.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; Biotinidase ; genetics ; Biotinidase Deficiency ; Carbon-Nitrogen Ligases ; deficiency ; genetics ; Case-Control Studies ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Molecular Sequence Data ; Multiple Carboxylase Deficiency ; genetics ; metabolism ; Mutation

OBJECTIVETo explore etiology, clinical manifestation and immunological changes of infectious pneumonia of neonates in Chengdu area.

METHODSSerum specimens were collected from 111 infants with infectious pneumonia. Eight viral and mycoplasmal specific serum IgM antibodies were detected by enzyme linked immunosorbent assay (ELISA); C reactive protein (CRP), total IgG and its subclasses, IgA and IgM were determined by rate scattered nephelometry; T lymphocyte subpopulations were detected by biotin-streptavidin-peroxidase method, and clinical and other laboratory data were analyzed.

RESULTS(1) Etiological agents: specific serum IgM antibodies were positive in 40 of 111 cases (36.0%) with pneumonias. All the 30 control infants were negative for the specific serum IgM antibodies. Among 111 infants with infectious pneumonia, 20.7% had single viral or mycoplasmal infection, 40.5% had bacterial infection, 15.3% had viral and mycoplasmal infection with bacterial infection; 23.4% had infection with unknown agents. (2) The most common clinical manifestations were tachypnea and cyanosis. The next were cough, milk choking, rales, retractions of the supraclavicular, intercostal and subcostal areas. Roentgenographic examination commonly revealed vague opacities, increased density and patchy infiltration. (3) Immune status: (1) CD(3), CD(4) cell counts of infants with pneumonias were lower than those of the controls while their serum IgA, IgM concentrations were higher than those of the control. (2) The CD(3) and CD(4) cell counts of the group with bacterial infection were lower than those of the control group. (3) The serum IgA concentration of the group with viral and mycoplasmal infection was higher than those of the control group and the group with unknown infection. (4) The serum IgM concentration of the group with bacterial infection was higher than those of the control group. (5) There were no significant differences in CD(8) cell counts, CD(4)/CD(8), concentration of serum IgG and IgG(1 - 4) between pneumonia group and the control group, and among various infectious groups and the control.

CONCLUSIONPathogens of neonatal infectious pneumonia in Chengdu area included single viral or mycoplasmic infection or bacterial infection, viral and mycoplasmal infection with bacterial infection, and unknown infection. Immunological changes of newborn infants suffered from infectious pneumonia included declined CD(3) and CD(4) cell counts, particularly in bacterial infection.

Antibodies, Bacterial ; blood ; Antibodies, Viral ; blood ; Bacterial Infections ; complications ; C-Reactive Protein ; analysis ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin M ; blood ; Infant, Newborn ; Male ; Pneumonia ; diagnosis ; etiology ; immunology ; T-Lymphocyte Subsets ; immunology ; metabolism ; Virus Diseases ; complications

OBJECTIVETo investigate the incidence of hyperphenylalaninemia (HPA) caused by different etiologic factors in China and the relationship between the phenylalanine and mental development of patients with HPAs who were diagnosed by neonatal screening and early treated.

METHODSTwo hundred and twenty-three patients with HPA detected by neonatal screening programs were refered to us at the age of (41 +/- 27) days after birth. The differential diagnosis was performed by BH(4) (20 mg/kg) loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity determination respectively. The control of phenylalanine (Phe) metabolism, growth and mental development were evaluated in all treated patients. Related gene mutation analysis was performed in some patients

RESULTSOne hundred and twenty-nine of 223 patients (57.8%) were diagnosed as phenylalanine hydroxylase deficiency (PAHD), 64 patients (28.7%) as BH(4) responsive PAHD, 30 patients (13.5%) as 6-pyruvoyl tetrahydropterin synthase deficiency (PTSD). One hundred and forty-nine patients were followed at age of 4 m - 2 y in our clinic. The 136 of 149 patients were treated according to different etiology at the age of 1.6 m (0.5 - 3.5 m) after birth. Thirteen patients were followed up without the need for treatment. All patients had normal growth development. One hundred and eight (79.4%) of 136 treated patients had normal mental development. The negative correlation (r = -0.439, P < 0.01) between IQ and average Phe levels were observed in 58 patients. Twenty-eight patients were able to go to primary school or even university. Nine kinds of PTS gene mutations were found in 9 cases with PTSD, among which 286G-->A and 259C-->T were most commonly seen, accounting for 45%. Seven kinds of PAH gene mutations were found in 13 cases with BH(4) responsive PAHD with the R241C (43.8%) mutation being the most frequent one.

CONCLUSIONThe differential diagnosis should be quickly made in all HPA patients detected by neonatal screening. Near 80% patients early treated had normal mental development. The good control of blood Phe level is a key factor for mental development.

China ; epidemiology ; Female ; Follow-Up Studies ; Humans ; Incidence ; Infant ; Infant, Newborn ; Male ; Neonatal Screening ; methods ; Phenylalanine ; blood ; Phenylketonurias ; diagnosis ; epidemiology ; prevention & control ; Time Factors

BACKGROUND AND OBJECTIVEMonitoring the therapeutic effects of radiotherapy for nasopharyngeal carcinoma (NPC) is critical to providing individualized treatment. This in-vivo study was initially designed to evaluate the therapeutic effect of radiotherapy using 18F-fluorodeoxyglucose positron emission tomography with computed tomography (18F-FDG PET-CT) imaging.

METHODS18F-FDG PET-CT imaging was performed on all of the 10 nude mice bearing NPC xenografts before radiotherapy, and early-phase and delayed-phase PET-CT images were performed on 7 of the 10 mice. All mice were randomly divided into either a control group or a radiotherapy group. The 5 mice in the control group were immediately killed after the imaging and pathology were performed. After receiving radiotherapy of 12 Gy, 5 animals in the radiotherapy group were given 18F-FDG PET-CT imaging on days 2, 4, and 6, and then were killed for pathologic evaluation. Regions of interest (ROI) technology was used to measure the tumor target/non-target (T/NT) ratio and the volume of the tumors.

RESULTSThe average T/NT ratios of early- and delayed-phase imaging were 1.806 +/- 0.532 and 1.777 +/- 0.597, respectively, with no significance (P > 0.05). For the radiotherapy group, the average T/NT ratios for 18F-FDG PET-CT before radiotherapy, and on days 2, 4, and 6 after radiotherapy, were 1.735 +/- 0.466, 1.818 +/- 0.396, 1.096 +/- 0.101, and 0.604 +/- 0.108, respectively, The tumor volumes were (1.48 +/- 0.27) cm3, (1.57 +/- 0.31) cm3, (1.59 +/- 0.31) cm3 and (1.60 +/- 0.29) cm3, respectively. The average T/NT ratios of day 6 after radiotherapy and the other time points were significant (P < 0.05). The average death ratio of the tumor cells was (93.00 +/- 7.42)% after 6 days of post-radiotherapy.

CONCLUSIONS18F-FDG PET-CT imaging can be used for the early assessment of radiotherapeutic effect of NPC in vivo. Day 6 after radiotherapy may be an appropriate time point for the imaging. However, the T/NT ratio measurement of delayed-phase imaging might make no sense for the diagnosis of NPC.

Animals ; Carcinoma, Squamous Cell ; diagnostic imaging ; pathology ; radiotherapy ; Cell Line, Tumor ; Fluorodeoxyglucose F18 ; Humans ; Ki-67 Antigen ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Multimodal Imaging ; methods ; Nasopharyngeal Neoplasms ; diagnostic imaging ; pathology ; radiotherapy ; Neoplasm Transplantation ; Positron-Emission Tomography ; Random Allocation ; Tomography, X-Ray Computed ; Tumor Burden ; radiation effects