Using Vibrio fluvialis Ⅱ as host bacteria, 19 bacteriophages have been isolated from the 76 samples which were collected from Haliotis discus hannai ～growing seawater in Dalian marine culture company Dalian, liaoning province from May in 1996 to August I 1997. Ultrastructure of 19 bacteriophages were observed with electron to Bradley the results showed that of these bacteriophages belonged to Bradley A type, they have hexagonal heads of bacteriophages were identified with VP1,VP2,VP4,VP8 as representatives respectively. The phages remain stable at pH6. 0～10. 0, moreover VP2,VP4 and VP8 are rather stable at basic pHs. Although the characterization of heat inactivation course of VP4 is different from others, four phages are sensitive to heat and inactivated at 80℃ in 5 minutes. One step growth experiment showed that the eclipse period of VP1,VP2,VP4,VP8 are sensitive to heat and the eclipse period of VP1, VP2, VP4, VP8 are 42, 30, 46, 28 minutes. In this experiment we have isolated at least four different types phages, it suggest that in fact there is a population of phages in the seawater environment. The result of this study provided a way to find the potential value of phages as an indicator of pathogenic microorganisms Vibrio fluvilis Ⅱ in marine environment.

Objective To study the temporal changes of alveolar epithelial type Ⅱ cells and surfactant pro-tein A in young rats with acute lung injury induced by lipopolysaecharide. Method Totally 110 SD young rats (male:53, female : 57) were randomly divided into ALI and normal control groups (six subgroups in each group).LPS(4 mg/kg) was given intraperitoneally in ALI group. The same amount of normal saline was given in the con-trol groups. Eight rats in each subgroup were sacrificed at 6, 12, 24, 36, 48 and 72 hours after the injection.Lung samples were taken for transmission electron microscope examination. RT-PCR was epmloyed for the mea-surement of SP-A mRNA. Western blot was used for the detection of SP-A in the lung tissue. ANOVA and homo-geneity of variance test were performed by SPSS 12.0. Results The microvilli disappeared at 24 hours after the injection of LPS. The number of lamellar body (LBs) was provisionality increased at 24 hours and 48 hours. The ring-like an'angement of LBs around nucleus and the giant LB with vacuole-like deformity were found as the main characteristics of AEC- Ⅱ in ALI at 48 hours. The number of LBs reduced and broken and residual LB remained at 72 hours. SP-A elevated greatly from 24 to 48 hours (P < 0.01), reached peak at 36 hours (6.94 ± 0.80, P <0.01),reached the lowest level(3.87 ±0.50, P <0.01)at 72 hours. Conclusions The pathological changes of AEC-Ⅱ and SP-A in lung tissue wiht ALI are time-dependent. The typical alterations of AEC- Ⅱ occurs at 48 hours accompanied by the compensatory increase of SP-A. AEC- Ⅱ is seriously injuried with the typical changes of LBs and the diminishing of SP-A in lung tissue.

BACKGROUND: Edaravone can alleviate brain injury and improve neurological functions and symptoms. This study aimed to investigate the effect of edaravone on the p38Mitogen-activated protein kinases/Caspase-3 (p38MAPK /Caspase-3) pathway after diffuse brain injury (DBI) in rats. METHODS: DBI models were established according to the description of Marmarou's method. A total of 250 rats were divided (random number) into four groups: control group (CG, n=45), model group (MG, n=77), low-dose edaravone group (n=67, dosage 5 mg/kg) and high-dose edaravone group (n=61, dosage 10 mg/kg). After 1, 6, 24, 48, and 72 hours after injury, brain tissues were collected. The changes of neuron morphous in the hippocampal region were observed through Nissl staining. The expression levels of phosphorylated p38MAPK and caspase-3 were detected by immunohistochemistry and Western blotting respectively. Learning and memory function were tested with Morris water maze from the 3rd to 7th day after injury. RESULTS: Some neurons had histopathologic changes of necrosis and apoptosis in the model group compared with the control group. The phosphorylated p38MAPK expressions increased at 1, 6, 4, and 48 hours (P<0.05), but no significant difference was observed at 72 hours (0.54±0.19 vs. 0.40±0.14, P>0.05). Caspase-3 expressions increased at 6, 24, 48, and 72 hours respectively (P<0.05), but there was no significant difference at 1 hour (0.59±0.29 vs. 0.40±0.17, P>0.05). From the 3rd to 6th day during the Morris water maze test, the latency to find the platform was significantly prolonged (P<0.05) and times of rats crossing the platform was decreased on the 7th day (2.28±1.18 vs. 8.20±1.52, P<0.05). The phosphorylated p38MAPK expressions decreased at 6, 24 and 48 hours respectively in the low dose edaravone group compared with the model group (P<0.05), whereas no significant difference was seen at 1 hour (1.66±0.80 vs. 1.85±0.86, P>0.05). Caspase-3 expression decreased at 6, 24, 48, and 72 hours (P<0.05). The latency to find the platform was significantly shortened (P<0.05), and times of rats crossing the platform increased (4.17±1.15 vs. 2.28±1.18, P<0.05). The above mentioned parameters changed more significantly in the high-dose edaravone group than in the low-dose edaravone group. CONCLUSION: Edaravone can alleviate brain tissue damage after DBI, inhibit p38MAP signal activation after early injury, reduce the expression of caspase-3, and promote the recovery of neurological function in the late period.

BACKGROUNDPoly(ADP-ribose) polymerase (PARP) plays an important role in the death of retinal capillary cells in diabetic retinopathy (DR) partly via its regulation of nuclear factor kappa B (NF-κB). The current study investigated the effect of the regimen of Gaoshan Hongjingtian (RG) on the mechanism of PARP regulation of NF-κB, and demonstrated the possible impact of the RG and Gaoshan Hongjingtian (Rhodiola sachalinensis, RS) on diabetic retinopathy.

METHODSWistar rats were made diabetic by administering streptozotocin. They were then assigned to three groups at random. After 2 months, the three groups of these diabetic rats were treated with RS or RG, or untreated. Analyses of expression levels of PARP, NF-κB, and intercellular adhesion molecule-1 (ICAM-1) in the retinas of rats in different groups were performed by Western blotting and immunohistochemical assays, and mRNA levels of NF-κB and ICAM-1 were determined by real-time polymerase chain reaction (PCR). In addition, the basement membranes of capillaries in the rats' retinas were observed using electron microscopy, and diabetes-induced capillary degeneration (ghost pericytes and acellular capillaries) were quantitated.

RESULTSFrom the third month after the injection of streptozotocin, the diabetic rats were given daily RG, RS or tap water separately. The diabetic rats failed to gain weight compared with normal age-matched rats, whereas their glycated hemoglobin levels were significantly increased. After 5 months, the mRNA levels of NF-κB and ICAM-1 and the protein expression of PARP, NF-κB, and ICAM-1 were significantly increased in the retinas of diabetic rats in the untreated group compared with the nondiabetic controls. After 8 months, the number of degenerated retinal capillaries (ghost pericytes and acellular capillaries) was significantly increased in the diabetic rats in the untreated group compared with normal age-matched rats. RG and RS inhibited diabetes-induced over-expression of PARP, NF-κB, and ICAM-1 in the retinas of diabetic rats at the end of 5-month diabetic duration. Treatment using RG and RS significantly inhibited increases in the number of acellular capillaries and pericyte ghosts and suppressed the basement membrane thickening in the retinas of rats with diabetes for 8 months compared with the control diabetic rats.

CONCLUSIONSThese results indicate that PARP plays an important role in the pathogenesis of diabetic retinopathy. RS and RG may have acted on the mechanism of PARP regulation of NF-κB, which suppressed the expression of NF-κB and ICAM-1, and led to the inhibition of retinal capillary degeneration.

Animals ; Basement Membrane ; drug effects ; pathology ; Diabetes Mellitus, Experimental ; drug therapy ; pathology ; Diabetic Retinopathy ; drug therapy ; pathology ; Intercellular Adhesion Molecule-1 ; genetics ; Male ; Medicine, Chinese Traditional ; NF-kappa B ; genetics ; physiology ; Poly(ADP-ribose) Polymerases ; physiology ; Rats ; Rats, Wistar ; Rhodiola ; Streptozocin

OBJECTIVETo investigate the heparitinase (HPA) expression and cell invasiveness in human ovarian cancer cell line SKOV3 during hypoxia, and explore their relationship with hypoxia inducible factor-1alpha (HIF-1alpha).

METHODSSKOV3 cells were incubated with normoxia, hypoxia, and hypoxia plus rapamycin, respectively. SKOV3 cells of hypoxia group were incubated in 5% CO2 + 1% O2. Cells in hypoxia plus rapamycin group were incubated with 10 ng/ml of rapamycin before cultured under hypoxic condition. Cells in each group were collected for analysis after incubated with hypoxia for 12, 24, and 36 hours, respectively. Western blotting and RT-PCR were performed to detect the expressions of HIF-1alpha and HPA. Cell invasiveness was measured by matrigel invasion assay.

RESULTSWestern blotting showed that the expression of HIF-1alpha significantly increased compared with normoxic group (P < 0.05). However, hypoxia had no obvious impact on HIF-1alpha mRNA expression. The expressions of HPA protein and mRNA of SKOV3 cells of hypoxia group were significantly higher than normoxic group (P < 0.05). The up-regulation of HPA expression in hypoxic group decreased after the utilization of rapamycin. The cell invasion of hypoxic group was significantly higher than that of normoxic group (P < 0.05); meanwhile, the expression of HPA was positively correlated with the invasiveness of SKOV3 cells (r = 0.9863, P < 0.05).

CONCLUSIONHypoxia may promote the expression of HPA and the invasiveness of SKOV3 cells through the HIF-1alpha pathway, which plays an important role in the pathogenesis of ovarian cancer.

Cell Hypoxia ; Cell Line, Tumor ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Neoplasm Invasiveness ; Ovarian Neoplasms ; enzymology ; genetics ; metabolism ; pathology ; Polysaccharide-Lyases ; genetics ; metabolism ; Signal Transduction ; Up-Regulation

BACKGROUNDHypoxia-inducible factor-1alpha (HIF-1alpha) is a transcriptional factor that could improve the stimulation of angiogenesis and the metabolic adaptation of tumor cells to hypoxia. A recent study showed that HIF-1alpha could induce colon cancer cells epithelial-mesenchymal transition (EMT). However, no evidence indicates a similar correlation in human prostate cancer cells. This study was designed to evaluate the effect of HIF-1alpha over-expression on the EMT in human prostate cancer cells.

METHODSWe selected the appropriate cell line for HIF-1alpha induction from those EMT negative prostate cell lines through vimentin gene detection by RT-PCR. As the result, LNCaP cell line is the best one for further experiment. LNCaP cells were transfected with recombinant plasmid pcDNA3.1 (-)/HIF-1alpha and pcDNA3.1 (-) control vector by Lipofectamine 2000 system. The positive cell colonies were confirmed by indirect immunofluorescence labeling. Then Transwell polycarbonate filter was used to analyze the invasive potency. The expression of EMT associated proteins, E-cadherin and vimentin, was detected by Western blotting.

RESULTSAmong four of the EMT negative cell lines, LNCaP was the only one expressed the vimentin gene but not the associated protein. The expression level of HIF-1alpha in LNCaP/HIF-1alpha was distinctly higher than that in LNCaP/pcDNA3.1 and LNCaP. The cell numbers of LNCaP/HIF-1alpha that penetrated through the Transwell filter were higher than that of LNCaP/pcDNA3.1 and LNCaP. Compared with the LNCaP/pcDNA3.1 and LNCaP cells, the expression of vimentin was up-regulated in LNCaP/HIF-1alpha, whereas the expression of E-cadherin was down-regulated.

CONCLUSIONSOver-expression of HIF-1alpha stimulates the invasion potency of human prostate carcinoma cells through EMT pathway. The expression of E-cadherin and vimentin, playing established roles in EMT, could be regulated by HIF-1alpha in human prostate cancer cell line.

Cadherins ; genetics ; Cell Line, Tumor ; Epithelial Cells ; chemistry ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; physiology ; Male ; Mesoderm ; pathology ; Prostatic Neoplasms ; metabolism ; pathology ; RNA, Messenger ; analysis ; Vimentin ; genetics

To analyze the antigenic and genetic characteristics of the influenza A (H3N2) virus in mainland China during the surveillance year of 2013-2014, the antigenic characteristics of H3N2 virus were analyzed using reference ferret anti-sera. The nucleotide sequences of the viruses were determined by Sanger dideoxy sequencing, phylogenetic trees were constructed with the neighbor-joining method, and the genetic characteristics of the viruses were determined in comparison to current vaccine strains. The results showed that most of the H3N2 viruses were antigenically closely related to the A/Victoria/361/2011 vaccine strain cell-propagated prototype virus (99.6%). Using the A/Texas/50/2012 egg isolate as the reference antigen, 15.1% of the viruses were found to be closely antigenically related to it, while 11.9% of strains were closely antigenically related to the egg-propagated epidemic strain, A/Shanghai-Changning/1507/2012. Phylogenetic analysis of HA genes indicated that the A(H3N2) viruses in this surveillance year were in the same clade, but no drug resistant mutation was identified in the NA genes. During the 2013-2014 influenza surveillance year, no significant genetic change was detected in either the HA or NA genes of the A(H3N2) viruses, while significant mutations were found in egg isolates resulting from their adaptation during propagation in eggs. The antigenic and genetic changes should be investigated in a timely manner to enable the selection of an appropriate vaccine strain in China.
Animals ; Antigenic Variation ; Base Sequence ; Chick Embryo ; China ; Genetic Variation ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; immunology ; isolation & purification ; Influenza, Human ; virology ; Molecular Sequence Data ; Mutation ; Phylogeny

AIMTo assess the antioxidative properties and the mechanism of action of dihydromyricetin (DMY) from Ampelopsis grossedentata.

METHODSThe antioxidative properties of DMY were measured by scavenging 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and inhibiting lipid peroxidation induced by FeSO4-edetic acid in linoleic acid. The mechanism of antioxidative properties of DMY was tested by measuring the chelating activities of DMY for Fe2+ with ultraviolet spectrum (UV) method.

RESULTSThe specific absorption of DPPH radical solution at 517 nm was reduced 73.3%-91.5% when DMY was added into the reaction solution in the concentration range from 0.01% to 0.04%. DMY was shown to greatly inhibit the increase of lipid peroxidation (LPO) values in linolei acid system catalyzed by FeSO4-edetic acid. The reaction rates (A532.min-1) of lipid peroxidation were 0.0021-0.0004 in the concentration range from 0.01% to 0.04% and the inhibition activities of DMY was found to be in a concentration-dependent manner. The mechanism of antioxidative properties of DMY was chelating Fe2+ in the Fe(2+)-dependent lipid peroxidation system.

CONCLUSIONDMY showed great antioxidative effect and would be a good natural antioxidant.

Ampelopsis ; chemistry ; Antioxidants ; isolation & purification ; pharmacology ; Chelating Agents ; pharmacology ; Flavonols ; chemistry ; isolation & purification ; pharmacology ; Free Radical Scavengers ; isolation & purification ; pharmacology ; Lipid Peroxidation ; drug effects ; Molecular Structure ; Plants, Medicinal ; chemistry

BACKGROUNDTo investigate variant genotyping of CCR2-64I, SDF1-3'A and CCR5Delta32 in HIV-1 infected Chinese Long-term nonprogressors and to study their association with disease progression.

METHODSThe genotypes of CCR2-64I, SDF1-3'A and CCR5Delta32 were detected by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) assay in seventeen HIV-1 infected Chinese Long-term nonprogressors (LTNPs) and thirty-nine Chinese typical progressors (TPs).

RESULTSThe frequency of CCR2-64I and SDF1-3'A in LTNPs are 50% and 62.5%, higher than those (23.08% and 33.33%) in TPs. Only one heterozygous CCR5 mutant was detected in LTNPs, and no CCR5 mutant in TPs.

CONCLUSIONVariant genotyping of CCR2-64ISDF1-3'A and CCR5Delta32 may be protective factors for delaying disease progression in HIV-1 infected Chinese LTNPs.

Chemokine CXCL12 ; genetics ; China ; Gene Frequency ; Genotype ; HIV Infections ; genetics ; pathology ; virology ; HIV Long-Term Survivors ; HIV-1 ; physiology ; Host-Pathogen Interactions ; Humans ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Receptors, CCR2 ; genetics ; Receptors, CCR5 ; genetics ; Receptors, HIV ; genetics

BACKGROUNDThe prospects of using immature CD8a(+) dendritic cells (DC2) to establish transplant immunologic tolerance and treatments for autoimmune diseases in the future are promising. However, the methods for inducing DC2 are still being explored. The present study was aimed to investigate the optimal in vitro conditions for preparing large numbers of predominant DC2 from murine bone marrow cells.

METHODSThree groups of bone marrow cells cultured under different conditions were examined, namely a cytokine-induced experimental group (cytokine group), a control group with a low concentration of granulocyte-macrophage colony stimulating factor (GM-CSF, low GM-CSF group) and a control group without endogenous cytokines. The cytokine group was cultured with 5 ng/ml GM-CSF, 25 ng/ml Flt3 ligand (Flt3L), 20 ng/ml interleukin 4 (IL-4) and 100 ng/ml stem cell factor (SCF). The low GM-CSF control group was cultured with 0.4 ng/ml GM-CSF, 25 ng/ml Flt3L and 100 ng/ml SCF, without IL-4. The control group without exogenous cytokines was cultured without additional cytokines. All cells were cultured at 37 degrees C under 5% CO2. On days 3, 7 and 16, 4-color flow cytometry was carried out to analyze the cell phenotypes, and the total cell numbers were counted to analyze the cell yields. Phase-contrast microscopy was used to observe the cell morphologies.

RESULTSThe cytokine group exhibited higher proportions of typical immature CD8a(+) DC, especially on day 3, but the total cell number and DC2 proportion decreased during prolonged culture. The low GM-CSF control group showed the same tendencies as the cytokine group on days 16 and 22, but produced higher total cell numbers (P<0.05) with lower DC2 proportions and cell numbers. The control group without exogenous cytokines spontaneously generated a certain proportion of DC2, but with low total cell and DC2 numbers that decreased rapidly, especially during prolonged culture (days 7 and 16, P<0.05).

CONCLUSIONSCulture in the presence of 5 ng/ml GM-CSF, 25 ng/ml Flt3L, 20 ng/ml IL-4 and 100 ng/ml SCF can rapidly induce large quantities of predominant immature CD8a(+) DC from murine bone marrow cells. Therefore, these represent optimal culture conditions for preparing murine immature DC2 in vitro.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; CD8 Antigens ; metabolism ; CD8-Positive T-Lymphocytes ; cytology ; drug effects ; Cell Culture Techniques ; methods ; Cells, Cultured ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Microscopy, Phase-Contrast