BACKGROUND:Nitrogen monoxide (NO) plays dual effects in the occurrence and development of tumor. High concentration of NO exerts anticancer effect through cellular cytotoxicity and inducing the apoptosis of tumor; under the changed microenvironment in tumor tissue, its effect on tumor tissue is mainly promoting the growth of tumor.OBJECTIVE: To study the effect of the nitric oxide synthase(NOS)inhibitor NG-nitro-L-arginine methyl ester(L-NAME) on invasion and metastasis of human colorectal cancer cell line LS174T.DESIGN: Single sample observationSETTING: Department of Surgery, Tumor Hospital affiliated to Harbin Medical University MATERIALS: The study was conducted in Cancer Research Institute of Harbin Medical University between January 2004 and October 2004. The human colorectal cancer cell line LS174T was purchased from Cancer Research Institute of Harbin Medical University. L-NAME was purchased from Sigma Company.METHODS: LS174T cells were treated with L-NAME of 0.2,0.4,0.8 and 1.0 mmol/L. Effect of drugs on invasion and migration of LS174T cells was observed in reconstructed basement membrane invading experiment.MAIN OUTCOME MEASRES: Inhibitory rate of L-NAME on LS174T line invading reconstructed basement membrane and the inhibitory rate of cellular migration.RESULTS: ①0.2, 0.4, 0.8, 1.0 mmoL/L L-NAME were used to treate LS174T cells for 72 hours, and the inhibitory rate in cell invading reconstructed basement membrane was 10.29%, 19.62%, 34.08%,42.23% respectively (P ＜ 0.05 or 0.01 ). ② 0.2, 0.4, 0.8, 1.0 mmol/L L-NAME were used to treate LS174T cell for 72 hours , its inhibitory rate in inhibiting cells migration was 20.76%,24.95%,39.43%,46.85% respectively(P ＜ 0.01 ).CONCLUSION: L-NAME has anti-invasive and anti-metastasic effects on LS174T cells.

Objective To investigate the effect of N~G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the growth of colorectal cancer xenografts in vivo and on tumor-associated neovascularization. Methods Twenty BALB/c nude mice were randomly divided into control group and study group equally. Human colorectal cancer cell line SL174T was inoculated subcutaneously into nude mice to form transplantation tumors. Saline 0.2 ml was intragastric-administrated to mice in control group and L-NAME (4 mg toties guoties) was administrated orally to mice in study group three times per week for four weeks. The changes of tumors in both groups were recorded and the microvessel density (MVD) was also measured by immunohistochemistry assay. Results L-NAME significantly inhibited the growth of colorectal cancer xenografts in nude mice. The weight of transplantation tumor reduced with the inhibitory rate of 41.36%, and the inhibitory rate of tumor volume was 43.48 % in study group. MVD in the study group and control group were 14.83?2.10 and 21.04?3.11, respectively, which showed that the former was significantly lower than that of the control group (P

Objective: To analyze the etiology and epidemiological characteristics of influenza in Wenzhou from 2009 to 2014, so as to provide the scientific basis for control and prevention of influenza. Methods: Throat swab specimens of influenza like illness (ILI) were collected from national influenza surveillance sentinel hospitals for nucleic acid detection with real-time PCR and virus isolation, culture and sequencing, and the results were analyzed with statistical methods. Results: During the 8 years, a total of 10 577 089 cases from outpatient and emergency department were monitored in sentinel hospitals. There were 337 896 ILI cases with an average ILI treatment rate of 3.19%. A total of 4 046 ILI samples were detected in children, 511 were positive for influenza, the positive rate was 12.63%. Among the detected influenza types, type B had the highest proportion, followed by H3N2. Among the 6 age groups, the number of flu patients was the highest in 0-3 years old group, the positive rate in 10-12 years old group was the highest (35.03%). There were 28 and 45 amino acid sequence mutations of HA fragment in influenza A and B, respectively, which included multiple mutation of 391 and 145 amino acids. The phylogenetic analysis showed that the strains of type B were different in different years, and Yamagata evolved into Y1 and Y2 two branches. Conclusions The prevalence peaks of influenza in children occurred in winter and spring in Wenzhou city, accompanied by small peaks in summer. Three subtypes of serotypes B, H3N2 and A(H1N1) dominated alternatively in Wenzhou during the 8 years. We should focus on strengthening the prevention and control of influenza in preschool children and primary and secondary school students.

OBJECTIVE: To observe the changes of apolipoprotein E (apoE) protein expression of pulmonary tissue in mice with pulmonary hypertension induced by hypoxia. METHODS: The animal model of hypoxic pulmonary hypertension was established by exposing the mice to isobaric hypoxic chamber for 3 weeks (23 h/d, regular chow feed).Twenty male wild type (WT) C57BL/6 mice and twenty apoE gene knockout (apoE-KO) mice were randomly divided into normoxia group and hypoxia group. The plasma concentrations of low density lipoprotein (LDL), high density lipoprotein (HDL) and total cholesterol were detected by ELISA method. The protein expression of apoE in lung and liver, and peroxisome proliferators-activated receptor gamma (PPARγ) in lung were measured by Western blot. RESULTS: ①In WT mice, the right ventricular systolic pressure (RVSP) and the weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) of hypoxia group were significantly higher than those of normoxia group by 68% and 59% (<0.05), respectively. The plasma concentration of HDL and HDL/LDL of hypoxia group were significantly lower than those of normoxia group by 17% and 40% (<0.05), respectively.The protein expression of apoE in lung and in liver of hypoxia group were significantly down-regulated than those of normoxia group by 48% and 52% (<0.05), respectively.The protein expression of PPARγ in lung was significantly down-regulated than that of normoxia group by 37%(<0.05).RVSP were significantly negative correlated with the protein levels of apoE and PPARγ in lung (<0.01).② In apoE-KO mice, RVSP and the weight ratio of RV to LV+S of hypoxia group were significantly higher than those of normoxia group by 96% and 86% (<0.05), respectively.RVSP and RV to (LV+S) of hypoxia group in apoE-KO mice were significantly higher than those of hypoxia group in WT mice by 29% and 24% (<0.05), respectively. CONCLUSIONS Down-regulated expression of apoE in lung tissue participates in the pathological proceeding of pulmonary hypertension induced by hypoxia.
Animals ; Apolipoproteins E ; Hypertension, Pulmonary ; Hypoxia ; Lung ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVETo investigate the influence of neonatal maturity on active oxygen metabolism in neutrophils and possible causes of a high susceptibility to bacterial infection in preterm infants.

METHODSThirty-five preterm infants born at a gestation age of 26-32 weeks (< or =32 weeks group, n=15) and at 33-36 weeks (> 32 weeks group, n=20) and 23 full-term infants (control group) were enrolled in this study. The samples of whole cord blood from the two preterm groups and the control group were stimulated in vitro with live bacteria,Staphylococcus aureus ( S. aureus) and Escherichia coli (E. coli) and stained with hydroethedine, an indicator of superoxide. The percentage of neutrophils which produced superoxide and the mean fluorescence intensity for superoxide production were measured by flow cytometry. The incidence of bacterial infection during hospital stay was compared between the two preterm groups.

RESULTSUnder S. aureus or E. coli stimulation, the percentage of neutrophils which produced superoxide in the < or =32 weeks group was significantly lower than that of the > 32 weeks group and the control group (P < 0.01). The percentage of neutrophils which produced superoxide was closely related to gestational age in preterm infants ( y=2.66 x, P < 0.01).There were no significant differences in the blood level of superoxide production in neutrophils among the three groups. The incidence of bacterial infection during hospital stay in the < or =32 weeks group (40%) was significantly higher than that the > 32 weeks group (10%) (P < 0.05).

CONCLUSIONSThe capability of active oxygen metabolism in neutrophils was significantly related to the gestational age in preterm infants. The decreased capability of active oxygen metabolism might be contributed to a higher susceptibility to bacterial infection in preterm infants.

Disease Susceptibility ; Gestational Age ; Humans ; Infant, Newborn ; Infant, Premature ; immunology ; Neutrophils ; metabolism ; Superoxides ; metabolism

Acute Lung Injury/chemically induced* ; Animals ; Bronchoalveolar Lavage Fluid ; Lipopolysaccharides ; Lung ; Mice ; Mice, Inbred C57BL

Objective To study the effects of L-NAME on invasion and metastasis of human colorectal (carcinoma) cell line(LS-174T) and explore its possible mechanism.Methods The LS-174T cells were co-incubated with L-NAME at various concertrations.Griess techniqe was used to examine the effect of(L-NAME) on excretion of nitric oxide(NO) of the cells.The effects of L-NAME on invasion and migration of LS-174T were evaluated using Transwell chambers attached with polycarbonate filters and reconstituted(basement) membrane.Expression of MMP2 mRNA and TIMP2mRNA in LS-174T cells was measured by(RT-PCR).Results(1)L-NAME decreased the excretion of NO of the cells in a dose-dependent manner.(2)The ability of the L-NAME treated LS-174T cells to invade the reconstituted basement membrane(increased) significantly with the concentration and time,at the concentration of 0.2 mmol/L,0.4 mmol/L,0.8mmol/L and 1.0mmol/L,respectively,after 72 hour,the inhibition rates were 10.29%,19.62%,34.08% and 42.23%,respetively.(P

OBJECTIVETo explore effect of curcumin in different concentrations on learning and memory of senescence-accelerated mice (SAM) and their possible mechanisms.

METHODSMice were randomly divided into six groups: the SAMR1 normal control group, the SAMP8 model control group, the SAMP8 + solvent (the peanut oil) control group, SAMP8 + low, middle and high dose curcumin groups. Mice were gastrogavage for 25 successive days. On the next day of ending the experiment, changes of learning and memory in mice of each group were observed by Morris water maze. The hippocampal [Ca2+] was determined. Expressions of hippocampal calmodulin-dependent protein kinase II (CaMK II) and Calmodulin (CaM) mRNA were detected using Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively.

RESULTSThe latency to find the hidden platform was remarkably prolonged, the hippocampal [Ca2+]i was markedly increased, the expression of CaMK II in the hippocampal membrane and the level of hippocampal CaM mRNA were significantly reduced in the SAMP8-model control group (P < 0.05, P < 0.01). The latency to find the hidden platform was remarkably shortened in the SAMP8 + middle dose curcumin and the SAMP8 + high dose curcumin groups (P < 0.01). The hippocampal [Ca2+]i was markedly lowered, the expression of CaMK II in the hippocampal membrane and the level of hippocampal CaM mRNA obviously increased in the SAMP8 + low, middle and high dose curcumin groups (P < 0.05, P < 0.01).

CONCLUSIONCurcumin could improve learning and memory Ca2+/capacities of SAM by lowering hippocampal [Ca2+] overload, increase the hippocampal CaM mRNA level and CaMK II expression in the hippocampal dose-dependently.

Aging ; drug effects ; metabolism ; Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Calmodulin ; metabolism ; Curcumin ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Maze Learning ; drug effects ; Memory ; drug effects ; Mice ; RNA, Messenger ; genetics

To identify human monoclonal HIV-l-neutralizing antibodies from an HIV-1 CRF07BC specific phage display antibody library by cell-based screening. 293T cells were transfected by pCH064. 2-Env plas mid and then used to biopan the phage antibody library. The positive phage clones were screened by cell based ELISA and sequenced for the variable region of heavy (VH) and light (VL) chains. The expressed Fabs were purified by Ni(+2) -NTA column and analyzed by SDS-PAGE. The cell- and gp120 protein-based ELISA as well as flow cytometry were used to measure Fab's binding activity. The neutralizing activity of Fabs was assessed by HIV-1 pseudoviruses. After 4-round biopanning, the binding phages to transfected cells were enriched about 650-folds. A total of 28 positive clones were screened out by cell ELISA and sequence analysis identified 5 different Fabs possessing unique VH and VL (2801, 2837, 2863, 2870 and 2920). Interestingly, these Fabs reacted with the Env-transfected 293T cells but not soluble gp120 proteins, suggesting that they might target conformation-dependent epitopes presenting on viral Env complex. We found that three Fabs (2801, 2863, 2870) exhibited potent neutralizing activity against CRF07_BC isolate CH120. 6 with IC50 of 2.24, 0.89 and 3.09 microg/mL respectively, and that 2801 and 2863 cross-neutral ized the subtype B isolate SF162 at IC50 of 0.69 and 3.52 microg/mL respectively. In conclusion, the HIV-1 Env-transfected 293T cells can be used to efficiently enrich and screen the phage antibody library and isolate human monoclonal HIV-1-neutralizing Fabs that target the Env complex-dependent conformational epitopes. Therefore, our studies provide a powerful platform for exploring the mechanism of HIV-1 neu tralizing response and for designing AIDS vaccines.
Antibodies, Monoclonal ; genetics ; immunology ; Antibodies, Neutralizing ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; HEK293 Cells ; HIV Antibodies ; genetics ; immunology ; HIV Envelope Protein gp120 ; genetics ; immunology ; HIV Infections ; immunology ; virology ; HIV-1 ; genetics ; immunology ; isolation & purification ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Neutralization Tests ; Peptide Library ; Transfection

OBJECTIVETo investigate changes in adenosine triphosphatase (ATPase) activity and expression of ryanodine receptor 1 (RyR1) mRNA in formation of pressure ulcer at early stage, and to analyze its mechanism.

METHODSThirty-six male Sprague-Dawley rats were divided into three groups according to the random number table as follows, with 12 rats in each group. (1) Ischemia-reperfusion (IR) for 3 times (3IR) group: unilateral gracilis of rats were loaded with 22.47 kPa pressure with a special pressure apparatus for 2.0 h to simulate ischemia, and unloaded for 0.5 h to simulate reperfusion. All rats were treated with above-mentioned manoeuvre for 3 times. (2) IR for 5 times (5IR) group: rats were treated with the same manoeuvre as that in 3IR group except for IR for 5 times. (3) CONTROL GROUP: gracilis of rats were subjected to a load of 0 kPa pressure. Rats in 3IR, 5IR groups were sacrificed, and then central part of pressured tissue was harvested for detection of activity of total ATPase, Ca(2+)-Mg(2+)-ATPase, and Na(+)-K(+)-ATPase with spectrophotometer colorimetry, the level of malondialdehyde (MDA) with enzyme linked immunosorbent assay (ELISA), and the level of RyR1 mRNA with real-time fluorescence quantitative RT-PCR. The same part of gracilis muscle of rats in control group was harvested for determination of indexes as above. Data were processed with one-way analysis of variance. Pearson correlation analysis was respectively performed between total ATPase activity and MDA level, total ATPase activity and RyR1 mRNA expression level, and RyR1 mRNA expression level and MDA level.

RESULTSActivity of total ATPase, Ca(2+)-Mg(2+)-ATPase, Na(+)-K(+)-ATPase in control group was respectively (1.629 ± 0.004), (0.907 ± 0.061), (0.697 ± 0.083) U/mg, all significantly higher than those in 3IR group [(1.365 ± 0.004), (0.784 ± 0.020), (0.581 ± 0.017) U/mg, with F value respectively 1707.0, 29.8, 15.2, P < 0.05 or P < 0.01] and 5IR group [(1.055 ± 0.049), (0.619 ± 0.016), (0.436 ± 0.039) U/mg, with F value respectively 1107.0, 169.9, 65.7, P values all below 0.01], and the values of 3 indexes in 5IR group were obviously lower than those in 3IR group (with F value respectively 322.8, 341.7, 94.0, P values all below 0.01). The level of MDA in control group [(7.5 ± 0.6) nmol/L] was lower than that in 3IR group [(9.9 ± 0.6) nmol/L, F = 53.2, P < 0.01] and 5IR group [(13.7 ± 1.3) nmol/L, F = 76.9, P < 0.01]. There was also statistical difference in MDA level between 3IR group and 5IR group (F = 82.9, P < 0.01). Expression level of RyR1 mRNA in control group (8.5 ± 4.2), which was similar to that in 3IR group (3.3 ± 2.1, F = 0.9, P > 0.05), was significantly higher than that in 5IR group (0.6 ± 0.5, F = 23.6, P < 0.05); while the RyR1 mRNA expression level was lower in 5IR group than in 3IR group (F = 39.3,P < 0.05). Activity of total ATPase was negatively correlated with MDA level (r = -0.918, P < 0.01). Activity of total ATPase was positively correlated with RyR1 mRNA expression level (r = 0.713, P < 0.01). RyR1 mRNA expression level was negatively correlated with MDA level (r = -0.702, P < 0.01).

CONCLUSIONSEnergy dysbolism may be an initial factor in the development of pressure ulcer at early stage. Calcium overload injury in pressure tissue can be identified by determination of RyR1 mRNA expression.

Adenosine Triphosphatases ; metabolism ; Animals ; Male ; Muscle, Skeletal ; metabolism ; Pressure Ulcer ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel ; genetics ; metabolism