Aim To study inhibitory effect of 5-fluorouracil encapsulated by galactosylceramide liposomes (5-Fu-GCL)on 5-Fu-resistent HepG_2 cells and its mechanisms. Methods Inhibitory effect of 5-Fu-GCL on established model of 5-Fu-resistant HepG_2 cells was assessed with MTT assay in vitro. The concentration-time course of 5-Fu-GCL in intracellular fluid was detected with high performance liquid chromatography (HPLC). Thymidylic acid synthase (TS) expression was observed with immunohistochemical method,and NO content was determined with chemical method. Results Obvious inhibitory effects of 5-Fu-GCL (75,150,300,600,1200?mol?L-1) on 5-Fu-resistant HepG_2 cells were observed with IC_ 50 of 158.6 ?mol?L-1,far lower than that of free 5-Fu (400.9 ?mol?L-1). 5-Fu-GCL (300 ?mol?L-1) inhibited 5-Fu-resistant HepG_2 cells in a time-dependent manner,and the inhibitory effect of 5-Fu-GCL was stronger than that of free 5-Fu during 12～48 h. Compared with free 5-Fu,5-Fu-GCL (300 ?mol?L-1) increased the content of intracellular fluid in 5-Fu-resistant HepG_2 cells. 5-Fu-GCL(62.5,300,1200 ?mol?L-1) not only inhibited the expression of TS,but also increased the production of NO in 5-Fu-resistant HepG_2 cells,and these effects of 5-Fu-GCL(300,1200 ?mol?L-1) were stronger than those of free 5-Fu. Conclusion 5-Fu-GCL has inhibitory effect on 5-Fu-resistant HepG_2 cells. The effect may be related to the increased concentration of 5-Fu-GCL in intracellular fluid,inhibited expression of TS and increased production of NO.

Objective To evaluate the effects of 131 adrenoceptor anfisense on blood pressure and?1 adrenoceptor mRNA and protein levels in 2 kidney 1 clip(2K1C)rats.Method 2KIC hypertensive rots were produced by clipping renal artery of SD rats.Liposome/AS-ODNs 2.0 were tested intravenously in rats with 2KIC hypertension.Animals were divided into 5 groups(n=18 in each group):?1-AS-ODN group,?1-IN-ODN group,2K1C group,Sham group and SD group.Blood pressure was measured by tail-cuff method,the levels of myocardial?adreneceptor mRNA and protein were tested by RT-PCR and binding assay.Results On the basis of the magnitude and duration of hypotension,?1-AS-ODN decreased blood pressure by 39 mmHg at the most for 4 weeks.Compared with the 2KIC group,?1-AS-ODN did not significantly change the levels of myocardial?1 adrenoceptor mRNA but significantly decreased the levels of myocardial?1 adrenoceptor protein at 2,7,30 days (P

Objective To investigate the changing pattern of antimicrobial resistance among gram-negative bacilli isolated from respiratory intensive care unit (RICU) for rational use of antimicrobial agents.Methods Antimicrobial susceptibility of 1 047 isolates of gram-negative bacilli from 2000 to 2004 was tested by disk diffusion method.WHONET 5.3 software was used to analyze the data.Results The most common pathogens were Pseudomonas aeruginosa (42.9%),Stenotrophomonas malto- philia (17.1%),Acinetobacter baurnannii (10.0%) and Klebsiella pneumoniae (6.5%).The susceptibility rate of P.aerug- inosa was relatively higher to ceftazidime (50%-74%),amikacin (33.3%-81.0%),piperacillin-tazobactam (30.4%-64.6%) and cefoperazone-sulbactam (33.5%-47.5%),while the susceptibility to imipenem decreased.The susceptibility rate of S. maltophilia was relatively higher to cefoperazone-sulbactam (47.2%-78.6%) and ticarcillin-clavulanic acid(28.3%-86.6%). More than 90% of Acinetobacter baumannii isolates were susceptible to imipenem.The susceptibility rates of K.pneumoniae to imipenem and cefepime were 92.9%-100% and 55.6%-80.0%,respectively.The susceptibility rate to piperacillin-tazobac- tam decreased from 58.3% to 21.7%.The prevalence of extended-spectrum?-lactamases (ESBLs) in K.pneumoniae increased from 11.1% in 2002 to 47.8% in 2004.Conclusions Most pathogens show significant resistance to the most commonly used an- tibiotics.It is very important to select antibiotics for the treatment of infections in ICU based on the results of susceptibility.

To improve the efficiency of halophilic actinobacteria screening and carry out the rapid selection of targeted strains, we tested 34 strains of Streptomonospora by PCR-single strand conformation polymorphism analysis (PCR-SSCP) based on genus-specific primers for the PCR identification. This approach employs PCR with two pairs of primers located in the 16S rRNA sequence flanking two variable region, then build clustering tree according as SSCP data. Synchronously, we sequenced all the 16S rRNA partial sequences for these strains to verify them. The results showed that the PCR-SSCP analysis was an efficient, easy-to-handle and economic method for rapid selection of halophilic actinobacteria resources.

The authors explained the process of organizing and implementing of the designed experiments.The designed experi- ments can improve students' creative thinking ability and activate students'initiative and meanwhile,it can improve teachers' level and contribute to making progress in teaching and studying although there are still some problems to solve.

Objective To investigate the effect of hydrogen sulfide(H2S)on gene expression of adenosine triphosphate(ATP)-sensitive K+ channel(KATP)of aortic smooth muscle cells(ASMC)in rat.Methods Male Sprague-Dawley(SD)rats were used in the study.The original generation cells were obtained by modified tissue piece inoculation.The passaged cells were used.The ASMC were divided into 2 groups:H2S group of different dosages and control group.The contents of sodium hydrosulfide in the culture media of H2S group were 10-5,10-4 and 10-3 mol/L respectively,and the same volume of normal saline was added to control group.Each group was treated for 24 hours.Morphology of the cells was observed by inverted microscope and identified by immunohistochemical method employing ?-smooth muscle-actin.The expressions of SUR2B and Kir6.1 were identified by immunohistochemical SP technology.The SUR2B mRNA and Kir6.1 mRNA levels were assayed by real time fluorescence relative quantitative polymerase chain reaction.Results The passaged cells developed typical growth pattern peak and valley and the 97th percent of the cells expressed the smooth muscle specific differentiation marker ?-smooth muscle-actin.SUR2B and Kir6.1 could be detected in ASMC by immunohistochemical technology.They were both located at cytoplasmic and cytomembrane but not in at nucleus.Compared with control group,SUR2B mRNA and Kir6.1 mRNA levels in H2S groups were higher than those in control group in a dosage-dependent mode.Conclusions H2S can increased the expression KATP channel SUR2B mRNA and Kir6.1 mRNA levels of ASMC.J Appl Clin Pediatr,2009,24(1):21-23

OBJECTIVETo explore the effect and mechanism of hirudin on atherosclerotic plaques in apolipoprotein E knockout (ApoE(-/-)) mice.

METHODSTotally 24 ApoE(-/-) mice, 7-8 weeks old were fed with high fat diets. They were randomly divided into the recombinant hirudin treatment group (drug group) and the model group according to body weight and different dens, 12 in each group. Twelve C57BL/6J mice, 7-8 weeks old fed with high fat diet were recruited as the normal control group. Recombinant hirudin (0.25 mg/kg) was intraperitoneally injected to mice in the drug group from the 10th week old once every other day for five successive weeks. Equal volume of normal saline was injected to mice in the model group. Mice in the normal control group received no treatment. All mice were sacrificed after fed with high fat diet until they were 20 weeks old. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), high-sensitive C-reactive protein (hs-CRP), E-selectin, interleukin-6 (IL-6), and stromal metalloproteinase-2 (MMP-2) were detected. The plaque/lumen area and extracellular lipid composition/ plaque area were analyzed by HE staining and morphometry. Changes of signaling molecules in store-operated calcium channels, including stromal interacting molecule 1 (STIM1), Orail protein, and transient receptor potential channel 1 (TRPC1) were determined by Western blot. Results Lipid plaque formed in the aorta vessel wall of 20-week old mice in the model group. Compared with the normal control group, serum levels of TC, TG and LDL increased (P<0.01), hs-CRP, E-selction, IL-6, and MMP-2 obviously increased (P<0.01, P<0.05) in the model group; expression levels of STIM1, TRPC1, and Orail significantly increased (P<0.01). Compared with the model group, the plaque/lumen area and the extracellular lipid composition/plaque area significantly decreased in the drug group (P<0.05, P<0.01); serum levels of TC and LDL, hs-CRP, E-selction, IL-6, and MMP-2 obviously decreased (P<0.05, P<0.01); expression levels of STIM1, TRPC1, and Orail were significantly down-regulated (P<0.05, P<0.01).

CONCLUSIONHirudin could significantly improve lipids and endothelial functions of ApoE(-/-) mice, down-regulate expression levels of STIM1, Orai1, and TRPC1, and thus delaying the occurrence and development of atherosclerosis.

Animals ; Aorta ; Apolipoproteins E ; metabolism ; Atherosclerosis ; C-Reactive Protein ; Cholesterol ; Diet, High-Fat ; Drugs, Chinese Herbal ; E-Selectin ; Hirudins ; metabolism ; Interleukin-6 ; Lipids ; Lipoproteins, HDL ; Lipoproteins, LDL ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Plaque, Atherosclerotic ; metabolism ; Recombinant Proteins ; metabolism ; Triglycerides

AIM: To establish an experimental model of high intraocular pressure in mice by laser photocoagulation and to prepare for future research.
METHODS: Experimental model of high intraocular pressure was induced unilaterally in 44 C57BL/6 mice. The fellow eye served as a control. TONO-PEN AVIA Tonomter was used to measure intraocular pressure (IOP) to guarantee IOP value at 1, 2, 4, 8wk. Slit-lamp biomicroscopy was performed throughout the period and the structural changes were assessed histologically. And then, their eyes were enucleated, postfixed, cryoprotected, and embedded in optimal cutting temperature medium. After hematoxylin and eosin stain ( HE stain ) , cryosections of the retina were observed under light microscope. TdT-mediated biotin-dUTP nick end labeling ( TUNEL ) was performed on the retinal sections to determine apoptosis rate.
RESULTS: IOP of laser-treated eyes was significantly higher than that of control eyes from 1-8wk (P<0. 05). The highest IOP was 31mmHg, but only one eye. The IOP was mainly around 20mmHg. In laser-treated eyes, the angle of anterior chamber were narrow. Number of cells in the inner nuclear layer and retial gangllion cell layer was slightly lower than that in control eyes at 2wk, but by 4 and 8wk the number of cells was significantly lower than that in the control contralateral eyes.
CONCLUSION: The laser photocoagulation of limbus causes chronic elevation of IOP and this method may be a promising experimental model for the investigation of biological mechanisms of glaucomatous retinal ganglion cell damage.

OBJECTIVETo study the clinicopathologic features, diagnosis and differential diagnosis of splenic marginal zone B-cell lymphoma (SMZL).

METHODSThe clinical data, histologic findings and immunophenotype of 8 SMZL cases were studied. IgH gene rearrangement was performed in 1 case. Follow-up information was available in 4 patients.

RESULTSThe median age of the patients was 61.5 years (range: 36 to 75 years). The male-to-female ratio was 1.7:1. All cases presented with massive splenomegaly. Five of six cases had abnormal blood counts: neutropenia and thrombocytopenia with two of them showing anemia. After splenectomy, the blood counts in 3/3 cases returned to normal levels. Post-operative fludarabine-based chemotherapy was given to 3 patients, two of them achieved complete remission and 1 case died during the course of chemotherapy. The average survival time was 21.5 months (range: 6 to 60 months). Histologically, all of the 8 cases showed micronodular white pulp lesions. Six of them exhibited the classic biphasic appearance with central aggregates of small B cells rimmed by a peripheral zone of atypical monocytoid B cells. The remaining 2 cases had a monomorphous appearance, consisting mainly of atypical monocytoid B cells. There was infiltration of tumor cells in the red pulp, sheets in appearance in all 8 cases. Immuno-histochemical staining showed CD20-positive (8/8), IgD-positive in 2 of the 4 cases (2/4), CD5-positive in 1 of the 4 cases (1/4), 6 of the 6 cases were bcl-2-positive, cyclin D1-negative and bcl-6/CD10-negative, CD43-negative in 5 of the 6 cases (5/6). The proliferation index, as highlighted by Ki-67 immunostaining, was low (< 15%).

CONCLUSIONSSMZL is an indolent B-cell non-Hodgkin lymphoma. The main clinical manifestations are splenomegaly and abnormalities in blood counts. The main modality of treatment is splenectomy. Adjuvant fludarabine-based chemotherapy helps to achieve complete remission. In general, the prognosis of this lymphoma type is good. The lymphoma cells predominantly grow in micronodular pattern, with atypical monocytoid B cells rimming around the small B cells, which aggregates in the center. The differential diagnosis includes other small B-cell lymphomas and lymphoid hyperplasia of spleen.

Adult ; Aged ; Antigens, CD20 ; metabolism ; Chemotherapy, Adjuvant ; Female ; Follow-Up Studies ; Humans ; Immunophenotyping ; Ki-67 Antigen ; metabolism ; Lymphoma, B-Cell, Marginal Zone ; drug therapy ; metabolism ; pathology ; surgery ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Spleen ; pathology ; Splenectomy ; Splenic Neoplasms ; drug therapy ; metabolism ; pathology ; surgery ; Survival Rate

The gene coding for beta-glycosidase (EC3.2.1.21) from Thermus nonproteolyticus HG102 has been cloned and expressed in E. coli. The gene open reading frame was 1311 bp and it codes for 436 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of glycosyl hydrolase family I. The enzyme had high content of hydrophobic amino acid (Ala 12.8%, Leu 10.9%), Arg(9.6%), Glu(9.4%) and Pro(8.0%), but low content Cys(0.45%) and Met (0.9%). From the alignment of enzyme amino acid sequence with other glycosyl hydrolase family I members, Glu164 and Glu338 were predicated as the proton donor and nucleophile group. The DNASTAR program was used to predict the secondary structure. According to the Chou-Fasman model, the enzyme has 41.4% of alpha-helics, 16.2%, beta-strands, 14.4%, beta-turns. 14 of the 35 Pro were located at the second sites of beta-turns. Hydrophobic interaction, ion bond, alpha-helics and Pro had important contribution to Tn-gly thermostability.
Amino Acid Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; Glycoside Hydrolases ; biosynthesis ; classification ; genetics ; Hot Temperature ; Molecular Sequence Data ; Open Reading Frames ; genetics ; Phylogeny ; Protein Structure, Secondary ; physiology ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods ; Sequence Homology ; Thermus ; enzymology ; beta-Glucosidase