Objective To study the production of ?-lactamase in multidrug-resistant Pseudomonas aeruginosa and to guide the proper use of antibiotics in clinical practice. Methods The modified three-dimensional extract test was employed to detect ?-lactamase in 30 multidrug-resistant Pseudomonas aeruginosa strains screened from antimicrobial susceptibility test in our hospital,and isoelectric focusing electrophoresis was performed on the enzyme-producing strains. Results No metalloenzyme was detected in all the 30 strains.Twenty-six strains produced ?-lactamase,among which 25 continuously yielded large amount of AmpC enzyme and the other one both AmpC enzyme and extended-spectrum ?-lactamases(ESBLs).86.7% of multidrug-resistant Pseudomonas aeruginosa produced enzyme. ConclusionThe majority of the multidrug-resistant Pseudomonas aeruginosa in our hospital yielded large amount of AmpC enzyme in a continuous way.The modified three-dimensional extract test can eliminate some interference such as the decrease of outer membrane permeability and overexpression of efflux pump,facilitating the effective and accurate detection of ESBLs in Pseudomonas aeruginosa.

In order to investigate the mechanism of stat3 nuclear import. positioned a characterized NLS of the SV40 large T antigen into Stat3-GFP, Dstat3-GFP respectively between the C-terminus of Stat3 and the N-terminus of GFP to create Stat3-NLS-GFP and Dstat3-NLS-GFP. With NLS-GFP as the positive control, Expression of the Stat3-NLS-GFP without IL-6 stimulation and Stat3-GFP with IL-6 stimulation resulted in a predominantly nuclear localization in 293T cell. Expression of Stat3-GFP and Dstat3-NLS-GFP without IL-6 stimulation resulted in predominantly cytoplasm localization in 293T cell. The results suggest that latent Stat3 is not anchored in the cytoplasm, and that nuclear localization in response to IL-6 is facilitated by gain of an NLS function.
Active Transport, Cell Nucleus ; Cell Nucleus ; metabolism ; Nuclear Localization Signals ; Plasmids ; metabolism ; STAT3 Transcription Factor ; metabolism

To examine the self-association of CD4 molecules and preliminary studies on its biological function by several indirect methods. A series of CD4 chimeras were generated including truncated CD4 lacking the short cytoplasmic tail, deleted mutantsD1/D2 devoid of D3 and D4 and D3/D4 devoid of D1 and D2 by PCR techniques, as well as another three CD4 chimeric genes by fused human Fas cytoplasmic death domain to the downstream of the above chimeras respectively. All these molecules were subcloned into pEGFP-N1, forming the corresponding expression vectors. After introducing into HEK293 cells, gene-modified cell morphological changes and target protein subcellular localization were observed and analyzed by a confocal microscopy. Moreover, stable 293/CD4 clones were obtained by transfecting the truncated CD4 recombinant plasmid into the HEK293 cell line and selected by G418. The fluorescene intensity and rosette formation of different clones was each analyzed by a confocal microscopy and cell adhesive assays. It's seen that CD4-Fas fusion gene could induce approximately 80% cell apoptosis of transfected HEK293 cells, compared to FKBP12-Fas is about 30% and CD4 gene only is 7%. Furthermore, both D1/D2-Fas and D3/D4 Fas chimeras could trigger nearly all transfected HEK293 cells to death. Cell adhesion assays showed that neither the D1/D2 nor D3/D4 chimeras when expression in HEK293 cells binds to MHC class II + Raji B cells. Interestedly, there were two type stable clones among 293/CD4. Fluorescence intensity analysis displayed that one' mean fluorescence intensity value is about twice of the other while cell-cell binding examination showed that the former is capable of forming rosette with Raji cells but the latter. All these results suggest that CD4 molecules most likely could exist as a dimer or even an oligomer on transfected HEK293 cell surface, which constitute a functional form for stable binding to MHC class II molecules.
Antigen-Presenting Cells ; immunology ; metabolism ; CD4 Antigens ; chemistry ; genetics ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; metabolism ; Cell Line ; Dimerization ; Fas Ligand Protein ; metabolism ; Histocompatibility Antigens Class II ; genetics ; immunology ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Protein Binding ; genetics ; Protein Multimerization

To screen NFAT antagonistic drugs and research signal transduction pathway related to NFAT. Four recombinant vectors were constructed. Each consists of three tandem copies of the human IL-2 distal NFAT-AP1 binding site in the context of the minimal IL-2 enhancer, either the sequence from -326 - +46 or the sequence from -89 - +46 (containing only the TATA box), driving a luciferase reporter gene or a destabilized enhanced green fluorescence protein (d2EGFP) reporter gene, respectively. Transient transfection of Jurkat cells was achieved by electroporation with 5 - 10 microg of the above plasmid and one pulse at 200V, 65ms. Plasmid pEFBos-mNFAT1 constitutively expressing murine full length NFAT1 protein was used for transient cotransfection. The results showed that neither of non-stimulation nor PMA or ionomycin stimulation alone could activate the reporter gene except PMA plus ionomycin costimulation. Furthermore, overexpressed murine NFAT1 augmented the activation of either IL-2 promoter or NFAT-AP1 enhancer drived reporter gene compared to the endogenous did. However, the reporter gene expression was nearly completely inhibited by pretreatment for 1h with FK506 at 5 microg/mL and then stimulation for 6-12h with PMA plus ionomycin in the presence of FK506. These findings indicated that such a transient Jurkat cell model offered a potential platform for preliminary screening of FK506 or CsA-like immunosuppressive agents.
Animals ; Drug Evaluation, Preclinical ; Enhancer Elements, Genetic ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Immunosuppressive Agents ; pharmacology ; Interleukin-2 ; genetics ; Jurkat Cells ; Luciferases ; genetics ; metabolism ; Mice ; Models, Biological ; NFATC Transcription Factors ; genetics ; metabolism ; Promoter Regions, Genetic ; Signal Transduction ; Tacrolimus ; pharmacology

This study was aimed to investigate the biological characteristics of B hematological tumor cells such as proliferation, immunological phenotype and apoptosis by silencing pax5. The specific pax5 small hairpin RNA (shRNA) was synthesized by in vitro transcription. For evaluating the inhibition efficiency, the expression change at mRNA and protein levels were assessed by real-time RT-PCR and Western blot respectively. To detect the biological characteristics of pax5-silenced hematological tumor cells, the immunological phenotype, apoptosis and cell proliferation were measured by using real-time RT-PCR, MTT assay and flow cytometry respectively. The results showed that two shRNA were synthesized, both of which were effective to block pax5 expression. After being blocked by RNAi the immunological phenotype of pax5-silenced lymphoma cells was changed, the expressions of CD19 mRNA and protein were reduced, but the expression of IgM was not changed. As compared with control group, the effect on proliferation and apoptosis of lymphoma cells not could be detected after pax5 silencing. It is concluded that the pax5 plays important role in late differentiation of B cells, and may participate in signal transduction of lymphoma cells, but the effect on proliferation and apoptosis of lymphoma cells were not detected after RNAi, which need to be elucidated further.
Apoptosis ; genetics ; Gene Silencing ; Humans ; Lymphoma, B-Cell ; genetics ; metabolism ; pathology ; PAX5 Transcription Factor ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Tumor Cells, Cultured

Objective To establish a novel convenient loop?mediated isothermal amplification(LAMP)method with the unique genes coding Plasmodium helical interspersed sub?telomeric superfamily(PHIST)for the rapid molecular diagnosis of P. falciparum. Methods The unique genes coding PHIST with high expression mRNA profile during the ring form or schizont period of P. falciparum were screened and selected from the PlasmoDB database. The LAMP primers of targeted genes were de?signed by the online software(PrimerExplorer V4). The LAMP assay was executed by the color?displaying method with SYBR Green. The dried blood spots of P. falciparum from clinical isolates were collected and the genomic DNA(gDNA)was extracted. For evaluation of sensitivity,the gDNA was diluted to four gradients(10?1,10?2,10?3,and 10?4). For assessment of specificity, the gDNA(s)of P. vivax,P. yoelii,Taenia saginata,and Schistosoma japonicum were also extracted. Results Totally,61 P. falciparum unique genes coding PHIST were found. The PF3D7_1372300 with high expression value during the ring form and PF3D7_1401600 with high expression value during the schizont period were selected for LAMP assay. The lowest detectable lim?its of PF3D7_1372300 and PF3D7_1401600 were 130.5 parasite/μl and 1 305.3 parasite/μl,respectively. Specific tests showed the amplified products of P. falciparum was positive and all the others including P. vivax,P. yoelii,T. saginata,and S. japoni?cum were negative. Conclusions The established LAMP method with PF3D7_1372300 gene is sensitive,specific,simple and useful. It can be applied to the field investigation and clinical diagnosis for falciparum malaria.

To investigate transcription factor PAX5 expression characteristics in childhood acute leukemic cells, expression levels of PAX5 and CD19 mRNA in 6 hematological tumor cell lines and bone marrow cells of 6 normal children, 58 de novo patients and 4 relapse acute leukemic children, including 39 cases of B-ALL, 10 cases of T-ALL and 13 cases of AML, were detected by a real-time RT-PCR. The results showed that PAX5 and CD19 mRNA expression levels were 2.35% and 2.52% in Namalwa (B-cell lines) respectively, but almost not detectable in other T- and myeloid cell lines. Among clinical samples, expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML (P = 0.029 and P = 0.013 respectively). PAX5 expression was significantly lower in T-ALL and AML than that in normal controls. The difference of PAX5 mRNA expression levels between T-ALL and AML was not significant. Individual difference of PAX5 mRNA expression levels in children with B-ALL was great. Moreover, PAX5 mRNA expressions in de novo and relapse patients with B-ALL were significantly higher than those in remission (P = 0.011 and P = 0.006 respectively). As binding sites for B-cell specific activator protein have been identified in the promoter regions of CD19, the study found that in B-ALL, there was clear correlation between the expression levels of PAX5 and CD19, which was also studied by real-time RT-PCR. It is concluded that PAX5 transcripts are readily detectable and quantifiable in clinical materials with B-ALL by real-time RT-PCR. The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.
Adolescent ; Antigens, CD19 ; biosynthesis ; genetics ; Cell Line, Tumor ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; PAX5 Transcription Factor ; biosynthesis ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics

OBJECTIVETo study the clinical and genetic characteristics of a Chinese family with benign familial convulsions (BFNC).

METHODSThe clinical data of this family was analyzed. The blood samples were collected from 13 members of this family. By four microsatellite markers which are located in the gene loci of both K+ channel KCNQ2 and KCNQ3, the linkage analysis was performed in the family. With DNA direct sequencing and restriction endonuclease cutting analysis, the mutation analysis of KCNQ3 gene was made for the proband, other 12 family members and 76 unrelated normal individuals.

RESULTSThere were 7 patients with BFNC observed in the three generation of family. The BFNC seizures of all patients disappeared during one month and no recurrence of seizures was found. The linkage analysis suggested the disease gene linked to KCNQ3 gene locus in the family. The mutation 988(C to T) of KCNQ3 gene was found in the proband by DNA-direct sequencing. Cosegregation of this mutation with BFNC was confirmed by restriction endonuclease cutting analysis.

CONCLUSIONChinese patients with BFNC can be caused by KCNQ3 gene mutation.

Base Sequence ; Child ; China ; DNA Mutational Analysis ; Epilepsy, Benign Neonatal ; genetics ; pathology ; Family Health ; Female ; Genetic Linkage ; genetics ; Genotype ; Humans ; KCNQ3 Potassium Channel ; genetics ; Male ; Mutation ; Pedigree ; Sequence Analysis, DNA

OBJECTIVETo analyze and compare the epidemiological features of rotavirus diarrhea among infants in the different areas so as to provide data for rotavirus vaccine research.

METHODSFrom Sep. 2001 through Sep. 2003, sentinel sites were set up in Suzhou Children's Hospital and Maanshan Hospital. Fecal samples from children (< 5 years) with acute diarrheal were collected and enzyme linked immunosorbent assay was used to detect rotavirus antigen. Reverse transcription-polymerase chain reaction was used to determine the G serotypes and P genotypes of rotavirus strains. The features of strains in the two places and other areas of China were analyzed and compared.

RESULTS(1) Rotavirus infection appeared in autumn and winter, but the peaks varied. In Suzhou the peaks were from December to next February in 2001, and November to next January in 2002. But in Maanshan, it was November to next January for both two years. (2) Rate of rotavirus infection in Suzhou was much higher than that in Maanshan, infective rates of Inpatient Department and Outpatient Department are 47.28%, 28.39% and 30.38%, 14.77% respectively in the two hospitals. (3) Rates of infection in two hospitals showed age difference but the highest group was in 6 - 35 month-olds. No gender difference was found. (4) Secular distribution of G-typing and P-typing of rotavirus strain was different in Suzhou and Maanshan. G3 was mainly found in Suzhou and G1 in Maanshan. From 2002-2003 on, G3 became dominant in Maanshan.

CONCLUSIONRotavirus caused diarrhea among infant and children were different in terms of areas, period and types, suggesting that the introduction of rotavirus vaccine should be adjusted according to different strains with specific types and optimal timeline.

Child, Preschool ; China ; epidemiology ; Diarrhea, Infantile ; epidemiology ; virology ; Female ; Humans ; Incidence ; Infant ; Male ; Rotavirus Infections ; epidemiology ; Seasons ; Sentinel Surveillance