Activation of focal adhesion kinase enhances the adhesion of
Fusarium solani to human corneal epithelial cells via the tyrosine-specific protein kinase signaling pathway
Xiaojing Pan,1,2 Ye Wang,1 Qingjun Zhou,1 Peng Chen,1 Yuanyuan Xu,1 Hao Chen,1 Lixin Xie 1(The first two authors contributed equally to the work)
1State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Qingdao,
China; 2Department of Ophthalmology, Affiliated Hospital of Qingdao University, Qingdao, China
Purpose: To determine the role of the integrin-FAK signaling pathway triggered by the adherence of F. solani to human corneal epithelial cells (HCECs).
Methods: After pretreatment with/without genistein, HCECs were incubated with F. solani spores at different times (0–24 h). Cell adhesion assays were performed by optical microscopy. Changes of the ultrastructure were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of F-actin and Paxillin (PAX) were detected by immunofluorescence and western blotting to detect the expression of these key proteins with/without genistein treatment.
Results: Cell adhesion assays showed that the number of adhered spores began to rise at 6 h after incubation and peaked at 8 h. SEM and TEM showed that the HCECs exhibited a marked morphological alteration induced by the attachment and entry of the spores. The expression of PAX increased, while the expression of F-actin decreased by stimulation with F. solani . The interaction of F. solani with HCECs causes actin rearrangement in HCECs. Genistein strongly inhibited FAK phosphorylation and the activation of the downstream protein (PAX). F. solani-induced enhancement of cell adhesion ability was inhibited along with the inhibition of FAK phosphorylation.
Conclusions: Our results suggest that the integrin-FAK signaling pathway is involved in the control of F. solani adhesion to HCECs and that the activation of focal adhesion kinase enhances the adhesion of human corneal epithelial cells to F.solani via the tyrosine-specific protein kinase signaling pathway.
Fungal keratitis is a common blinding disease, which over the past decades, has had an increased incidence in many agricultural countries [1]. The dominant filamentous fungal pathogens are Fusarium species, with Fusarium solani (F.solani ) being the most frequent isolate among the Fusarium species of keratomycosis in north China [1,2]. Poor knowledge of the pathogenesis of this disease makes effective treatment difficult [2]. Previous studies suggest that the interaction between host cells and fungus may play a critical role in the pathogenesis of fungal diseases [3-5]. Our previous work [6] demonstrated the roles of adherence and matrix metalloproteinases (MMPs) in growth patterns of major fungal pathogens (including F. solani ) in the cornea.However, the precise molecular mechanism in keratomycosis remains unknown. Furthermore, several phosphate-containing proteins have been shown in many cancer cells [7] and gastric epithelial cells [8], the stimulation of tyrosine phosphorylation by several substrates correlates with
Correspondence to: Lixin Xie, M.D., Shandong Eye Institute, 5Yanerdao Road, Qingdao 266071, China; Phone:+86-532-85885195; FAX: + 86-532-85891110; email:lixin_xie@https://www.doczj.com/doc/0b12621817.html, increased adhesion, motility, invasion and alteration in the cytoskeleton, and overexpression and phosphorylation of focal adhesion kinase (FAK) in epithelial cells promotes adherence to Candida yeast cells [9]; however, the role of FAK and tyrosine phosphorylation in the regulation of the interaction of human corneal epithelial cells (HCECs) with F.solani has been poorly elucidated. Therefore, the study of signal transduction pathways in HCECs stimulated by F.solani is especially important in view of their putative implications in the regulation of interaction.
Adherence to host cells, such as endothelial and epithelial cells, is the first step in colonization by fungus and the subsequent establishment of infection [10-12]. Similarly, the adherence of fungus to epithelial cells and to extracellular matrix (ECM) components is considered a crucial event in pathophysiology [9,13-15]. In other issues, multiple adhesions, such as mannoproteins, lectin-like receptors,carbohydrates and integrin-like molecules, can mediate fungus-host cell adhesion [13,16]. Integrins are a large family of highly conserved heterodimers composed of noncovalently linked α and β subunits that mediate cell-matrix and cell-cell interactions in embryogenesis, hemostasis, wound healing,tumor and microorganism invasion, immune response,
and Molecular Vision 2011; 17:638-646
Received 24 December 2010 | Accepted 25 February 2011 | Published 5 March 2011
? 2011 Molecular Vision
inflammation [8]. These receptors mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions. Within focal adhesions, the cytoplasmic domains of the integrin heterodimers provide a site to which cytoskeletal proteins are tethered.
The FAK family consists of two evolutionarily conserved protein tyro-kinase (PTKs) localized in the focal adhesions, namely, p125 focal adhesion kinase (p125FAK) and proline-rich tyrosine kinase 2 (Pyk-2) [17,18]. Several studies have shown that FAK functions as part of a cytoskeletal-associated network of signaling proteins, including paxillin (PAX), Src (a proto-oncogenic tyrosine kinase)-homology collagen (Shc), and growth factor receptor-bound protein 2 (Grb-2), which act in combination to transduce integrin-generated signals to mitogen-activated protein kinase (MAPK) cascades [4,5,18].Tyrosine phosphorylation of the FAK family is regulated by different stimuli, which include adhesive events, in that several components of the ECM, such as fibronectin (FN), vitronectin (VN), laminin [19], and collagen IV, or clustering of β1, β3, and β5 integrins, trigger p125FAK tyrosine phosphorylation.
Recently available information suggests that integrin-FAK is one of the best characterized intracellular signaling pathways, which play a critical role in the control of cell adherence, migration, and internalization when activated by a series of stimuli [20]. It seems likely that the activation of the integrin-FAK signaling pathway may be involved in the interaction between fungus and cell surface receptors responsible for transmitting downstream signals. Fungus might associate either directly or indirectly with integrin to modulate FAK and downstream signals leading to cell adherence and migration. The present study sought to determine whether a putative p125FAK that is expressed in HCECs co-culture with F. solani and whether cross-linking of the β1 integrin receptors or adhesion to HCECs can regulate tyrosine phosphorylation. Furthermore, we investigated the mechanism of activation of FAK and its downstream PAX signaling following adhesion to ECM. Our results suggest that activation of FAK enhances the adhesive and migration capabilities of HCECs through the tyrosine-specific protein kinase signaling pathway.
METHODS
Unless otherwise stated, all chemicals used were of analytical grade or higher. The tyrosine-specific protein kinase signaling pathway inhibitor, genistein, was purchased from Sigma-Aldrich Shanghai Trading Co. Ltd. (Shanghai, China). DMEM/F-12 (1:1) was purchased from Thermo Fisher Scientific (Beijing, China).
Strains and culture conditions: The strain of Fusarium solani (CGMCC 3.1829) was purchased from China General Microbiologic Culture Collection Center, (Beijing, China). The two strains were cultured on potato dextrose agar (PDA;Qingdao Hope Bio-Technology Co. Ltd., China) at 28 °C for 5 days, and spores were harvested into 1 ml sterile saline solution and then diluted with sterile saline to yield 108 U/ml (culturable).
Cell adhesion assay: Simian Virus 40-immortalized HCECs were used in the present study [21]. They were kindly gifted by Dr. Choun-K i Joo (Catholic University of Korea, Seoul, Korea). The cells were maintained in DMEM/F 12, 5% fetal bovine serum (FBS), 100 IU of penicillin/ml, and 100 mg of streptomycin/ml in a humidified 5% CO2incubator at 37 °C. The HCECs were pretreated with/without genistein (200 μΜ) [22] for 1 h and then incubated with fungi at different times (0 to 24 h). Untreated monolayers (controls) were incubated in DMEM/F12. Adhesion was verified microscopically every hour and the spore’s phases were maintained throughout the adhesion assays. Unattached fungi were removed by extensive washing with PBS. The number of fungi spores was counted and the results were analyzed by the measurement of integral optical density (IOD) with an image analyzer (Vidas 21; Kontron Corp., Eching, Germany). Experiments were repeated at least three times.
Electron microscopy: The protocol for scanning electron microscopy (SEM) and the protocol for transmission electron microscopy (TEM) are at specific websites. After incubation with fungi spores for different times, the cells were washed three times with PBS. Then, the cells were fixed in 4% buffered glutaraldehyde, washed in a buffered solution of 0.2% sucrose-kakodyl for 4–10 h, and dehydrated in graded alcohol concentrations. For SEM (JEOL JSM-840; JEOL, Tokyo, Japan), the specimens were replaced with isoamyl acetate, air-dried, and sputter-coated with gold before examination under the microscope. For TEM, semi-thin sections (1 μm in thickness) of the specimens were embedded in an epoxy resin for orientation purposes and were subsequently stained with toluidine blue. In addition, ultrathin sections were stained with uranyl acetate-lead citrate and were examined on a JEOL JEM-1200 transmission electron microscope (JEOL). The central and paracentral regions were also observed.
Flow cytometry: The effect of fungi spores on β1 integrin expression in HCECs was determined by flow cytometry (FCM) analysis. Briefly, cells treated as described above were harvested from the 6-well plates following treatment with trypsin. The cell suspension, at a concentration of 1.0×106 cells/ml, was fixed for 20 min in 40 mg/l paraformaldehyde, blocked for 20 min at room temperature in 1% BSA, washed twice in cold PBS, and stained overnight at 4 °C with a rabbit monoclonal anti-β1 integrin antibody. The bound antibody was visualized with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody at room temperature for 1 h and washed three times with PBS. The labeled cells were determined over 10,000 events by flow cytometry (BD FACSCalibur; Becton Dickinson, San Jose, CA) and analyzed using CellQuest Pro Software (Becton Dickinson).
Immunofluorescence: The expression of PAX and F-actin were shown by immunofluorescence. The cells were fixed in 40 mg/l paraformaldehyde for 20 min, blocked for 10 min at room temperature in 1% BSA, and then incubated overnight at 4 °C with the appropriately diluted primary antibody. In addition, normal rabbit IgG or mouse IgG was used as a negative control. The bound antibody was visualized with a fluorescent secondary antibody at room temperature for 1 h, following standard protocols. Finally, the cells were covered with mounting media (Ultra Cruz TM Mounting Medium; DAPI; sc-24941; Santa Cruz Biotechnology, Santa Cruz, CA) and examined by fluorescence microscopy (Eclipse TE2000-U; Nikon, Tokyo, Japan). Phalloidin-FITC (ALX-350–268-MC01; Alexis Biochemicals, Lausanne, Switzerland) was also used to observe the changes in the cellular cytoskeleton.
Western blot analysis: Protein was extracted from the HCECs using RIPA lysis buffer (50 mM Tris PH 7.4, 150 mM NaCl, 1%Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, and sodium fluoride; Galen, Beijing, China) according to the manufacturer’s instructions. Each of the prepared samples, in a final volume of 15 μl (containing a total of 50 μg of protein), were run on a 10% SDS–PAGE and then transferred into a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). The blots were blocked in 5% non-fat dry milk dissolved in TBST (20 mM Tris PH 7.5, 0.5 mM NaCl, 0.05%Tween-20) for at least 1 h and incubated with the primary antibody in TBST for 1 h at room temperature. Subsequently, the blots were incubated for 1 h at RT with a horseradish peroxidase-conjugated secondary antibody in TBST. The membranes were then developed with a SuperSignal West Femto Maximum Sensitivity substrate (Pierce Biotechnology, Rockford, IL) and exposed to X-ray film (Kodak, Rochester, NY). Immunoreactive bands were visualized via chemiluminescence and quantified using NIH Image 1.62 software (National Institutes of Health, Bethesda, MD). The primary antibodies were rabbit monoclonal anti-p-FAK antibody (ab4803; Abcam, Cambridge Science Park, Cambridge, UK), rabbit polyclonal anti-FAK antibody (cst-3285; Cell Signaling Technology, Beverly, MA), rabbit monoclonal anti-β1 integrin antibody (ab52971; Abcam), and goat polyclonal anti-p-PAX antibody (sc-14036; Santa Cruz Biotechnology).
Statistical analyses: The statistical differences of each sample comparing the treated and experimental groups were analyzed using the one-way ANOVA (ANOVA) and Student-Newman-Keul's (SNK) test. All p values less than 0.05 were considered statistically significant.
RESULTS
Involvement of FAK with adhesive capabilities of HCECs to F. solani spores: After incubation with HCECs for different time points (0 to 24 h), the spores were respectively observed by light microscopy. In the F. solani and HCEC co-incubated group, the number of adhered spores began to rise at 6 h after incubation, peaked at 8 h, and maintained over 10 h compared with the HCECs (Figure 1). Then, the effects on the adhesive response in HCECs to F. solani spores were investigated along with the inhibition of FAK tyrosine phosphorylation. Inhibition with genistein showed a reduced adhesiveness to spores. When genistein treated HCECs interacted with the spores, a reduced adherence of the spores was observed in comparison to the untreated cells (Figure 1). The result was determined by measurement of the IOD (Figure 2). These data suggest that FAK regulation may play a critical role in the adhesive capabilities of HCECs to F. solani spores. Ultrastructure: Examination of the SEM images showed that the normal HCECs had uniform epithelial cell morphology with numerous microvilli located on the surface. Adhesion, as determined at different times (6, 8, and 10 h) after cell incubation and observed by the cells with attached spores, is shown in Figure 3A-F. In the F. solani and HCEC co-incubated group, the morphology of the HCECs was characterized as being corrugativus and pantomorphic where the microvilli were fewer in number. Such changes were more significant in the 8 h and 10 h groups (Figure 3B,E,C,F). After 10 h adhesion, the cellular areas were even smaller and the microvilli were less numerous than those observed at 8 h. Most of the HCECs retained the characteristic ultrastructures of the ruptured membranes and shrunken and dead cells (Figure 3F). It is interesting to note the obvious clumping of adherent spores attached to the filament-like projections stretching from the plasma membrane (Figure 3E). These clumps were evident after 8 h and contained numerous spores after 10 h of incubation. Ruptured and extensively destroyed membrane with spores adhering to it and the characteristics of the dead cells were also apparent at 10 h after inoculation.
In addition, TEM was used to examine the ultrastructural features of the HCECs at 6, 8, and 10 h after incubation with the F. solani spores (Figure 3G-L). The TEM images show that the normal cells arrayed with the monolayer and took on a polygon shape. Their organelles, such as mitochondria and rough endoplasmic reticulum, were abundant. The nuclear membranes were full and slick and the nuclei were large (Figure 3I,L). During the adherence process, the HCECs came into interact with spores instead of fusing with them (Figure 3G,J). Damage to the HCECs can be seen at 6 h. After 8 h incubation, most cells exhibited a marked morphological alteration. The normal organelles were significantly less well resolved and the vacuoles were larger and more abundant in the cytoplasm (Figure 3H,K). Curiously, at 10 h, some of the HCECs started to die. F. solani spores could be observed inside cells at 10 h after incubation (Figure 3H,K). Expression of β1 integrin on HCECs evaluated by flow cytometry: We further evaluated the expression of β1 integrin on HCECs by flow cytometry. β1 integrin expression on cell surfaces was significantly increased when the cells were
treated with F. solani spores or genistein. The expression of β1 integrin increased by 97.97% when the cells were incubated with F. solani spores for 8 h (Figure 4). However,when the cells were pretreated with genistein, the expression of β1 integrin decreased by 83.60%. However, no significant differences were found in the two groups (p>0.05).Expression of PAX and F-actin: Immunofluorescence and confocal microscopy were used to detect the expression of PAX and F-actin. F-actin was stained with FITC (Figure 5A,D,G) and PAX was stained with Texas red (Figure 5B,E,H). The areas of co-localization appear yellow in the merged sections (Figure 5C,F,I). When the cells were pretreated with genistein, the expression of PAX decreased
(Figure 5B,E,H). Incubation with F. solani spores induced alterations in the F-actin microfilaments of the HCECs (Figure 5A,D,G). These results show that the untreated HCECs exhibited normal morphology, while an actin rearrangement was noted in cells incubated with the F.solani spores. A combined treatment with genistein and the spores decreased the polymerization of actin, and the HCECs became more spreading than in the group incubated with F.solani spores. These results indicate that the inhibition of FAK signaling alters cell-spore interaction.
Western blot analysis of the integrin-FAK signaling pathway in HCECs: The FAK proteins are the key proteins of the FAK
signaling pathway, which is phosphorylated and subsequently
Figure 1. Comparison of the number of F. solani spores adhered to HCECs. After pretreatment with/without 200 μM genistein for 1 h, cells were incubated with F. solani spores for different times (0 to 24 h). In the F. solani and HCEC co-incubated group, the number of adhered spores began to rise at 6 h after incubation, peaked at 8 h, and maintained at over 10 h (A -D ). In the genistein treated group, the adhesiveness was reduced compared with the genistein non-treated HCECs (E -H
).
Figure 2. The comparison of optical density (IOD) levels between the F.solani and HCEC co-incubated group and the genistein treated group. Spore adhesion assays were performed by measuring the IOD. Statistical significance was tested by one-way ANOVA and Student-Newman-Keul's test. The p values indicated significant differences between the data in the experimental groups (6, 8, and 10 h) and their corresponding treated groups.**p<0.001.
activated. To further understand the state of activity of the integrin-FAK signal cascade as a key position of the adherence of F. solani spores to the HCECs, we compared the expression levels of the p-FAK, p-PAX, and β1integrin proteins in the genistein pretreatment group and the group incubated with F. solani spores. Incubation of HCECs with
F. solani spores stimulates FAK tyrosine phosphorylation at
7 h after incubation. The expression reached a peak after 8 h
Figure 3. Changes in the ultrastructure after HCECs were coincubated with F. solani spores. A-D: Scanning electron micrographs of HCECs coincubated with F. solani spores. After coincubation for 6 h, the F. solani spores (arrow) began to attach to the surface of the HCECs as seen in panels (A) and (D). The number of adhered cells increased at 8 h after coincubation (B, arrow). Most of the cells presented a great number of spores congregated to the projections of plasma membrane (A-E, arrow). Damage to the HCECs can be seen in panels B, E, C, and F. Ruptured and extensively destroyed membrane with spores adhering to it (C, arrow) and characteristics of dead cells (F, arrow) at 10 h after coincubation. Original magnification: (A, B, and C) 2,000×; (D, E, and F) 800×. G-L: Transmission electron micrographs of cells incubated with HCECs and F. solani spores. Photograph shows F. solani spores attached to the plasma membrane, followed by the subsequent formation of cell projections around it (8 h, G, and J, arrow). Then, spores are internalized into the cell cytoplasm and cell organelles appeared to be destroyed at 10 h (H and I). The ultrastructures of most HCECs show confused organelle structure degeneration of the nucleus (H, arrow), and vacuolization of the mitochondria (K, arrow) at 10 h after incubation. Original magnification: (G) 8,000×; (I and J) 5,000×; (H) 3,000×; (K) 20,000×; (L) 12,000×.
incubation and decreased at 9 h. Pretreatment of cells with genistein downmodulated FAK tyrosine phosphorylation induced by the spores’ interaction with the cells (Figure 6A,B). The data also show that the expressions of the p-PAX and β1 integrin proteins both increase at 6 h in the experimental groups and downregulation of p-FAK inhibits p-PAX expression in the HCECs (Figure 6C,D,E).
DISCUSSION
Keratitis caused by F. solani usually occurs following corneal injury. Pioneering work identified that injury predisposes the cornea to infection by permitting this organism to adhere to it. Adherence is not immediate and requires that the organisms remain on the corneal surface for some time [1,6].Furthermore, sparse information is available regarding the signaling events triggered by the contribution of F. solani
to
Figure 4. Flow cytometry analysis results for the expression of β1 integrin.The data evaluated the expression of β1integrin on permeabilized HCECs by flow cytometry. As shown in the figure,the anti-β1 integrin, mAb, positively stained cells both in the group incubated with F. solani spores and in the genistein pretreated groups (97.97% and 83.60%,respectively), while 56.43% of the cells were positive in the negative controls.Significant differences were observed between the above two groups. The results were representative of one of
three separate experiments.
Figure 5. Immunofluorescence and confocal microscopy were used to detect the expression of PAX and F-actin. F-actin was stained with FITC (A , D , and G ) and PAX was stained with Texas red (B , E , and H ). The areas of co-localization appear yellow in the merged sections (C , F , and I ). When the cells were pretreated with genistein, the expression of PAX decreased (B , E , and H ). Incubation with F. solani spores induced alterations in the F-actin microfilaments of the HCECs (A , D , and G ). These results showed that the untreated HCECs exhibited normal morphology, while an actin rearrangement was noted in cells incubated with the F. solani spores. A combined treatment with genistein and the spores decreased the polymerization of actin, and the HCECs became more spreading than in the group incubated with F. solani spores.
Figure 6. Involvement of FAK phosphorylation and integrin signaling with adhesion of HCECs to F. solani spores. After pretreatment with/ without genistein, HCECs were exposed to F. solani spore suspensions. Western blot assay showed that 5 h after incubation, p-FAK production was significantly increased (A). The graph (B) compares scanning signal intensity of p-FAK expression by ImageJ software. The expression of p-FAK greatly increased (p<0.001) in all treated groups (7 and 8 h). There were significant differences for the phosphorylation levels of FAK in all treated groups. *p<0.01, **p<0.001. The β1 integrin and p-PAX from cells incubated with F. solani spores were also analyzed by western blot (C). The graph (D) compared scanning signal intensity of β1 integrin expression by ImageJ software and indicated the significant overexpression (p<0.001) of β1 integrin protein in all treated groups (6, 7, and 8 h). The data showed no significant differences (p<0.05) between the genistein treated and non-treated groups. The expression of p-PAX was significantly increased (p<0.001) in all treated groups
and when the cells were pretreated with genistein, the expression of p-PAX was significantly lower (p<0.001) than in the no-genistein treated
the pathogenesis of corneal infection. Here, we provide the first evidence of the presence of a focal adhesion kinase (FAK) protein and its involvement in the control of integrin-mediated F. solani spore adhesion.
Tyrosine phosphorylation of cellular proteins is a primary response to integrin stimulation and the role of the PTKs belonging to the FAK family in the control of cellular adhesion and migration is documented [18,19,23]. Our findings suggest that the enhancement of adhesion of F. solani to HCECs is dependent on the presence of β1 integrin and tyrosine phosphorylation of FAK, which is obviously blocked by using the PTK inhibitor, genistein, pretreatment [9]. These results are in line with previous evidence in other cells where cell adhesion to ECM or clustering of β3 and β5 integrins triggered p125 FAK tyrosine phosphorylation [24, 25].
To examine the role of the integrin-FAK signaling pathway in the adhesion of HCECs, we examined the signaling molecules that were involved in mediating spore-induced effects on cells and investigated whether β1 integrin is physically associated with FAK/PAX or whether the activation of β1 integrin-mediated signaling by fungus is sufficient to activate FAK/PAX in HCECs. We proved that FAK phosphorylation correlated with the activation of its downstream PAX signaling pathway. Our data also indicates that the fungus-induced phosphorylation of FAK correlated with the physical association of β1 integrin with subsequent activation of the PAX signaling pathway. When FAK tyrosine phosphorylation was blocked with genistein, adherence was lower in the blockade group than in the non-blockade group. Recent reports demonstrate the involvement of PAX with FAK signaling pathway activation, cell migration, and signal spreading [26,27]. The interaction of FAK and PAX produces a molecular switch resulting in tyrosine phosphorylation of the remaining PAX and determines the fate of downstream signaling events [28]. So, we suspect that F. solani infection is associated with rapid and transient phosphorylation of PAX, followed by phosphorylation of FAK. Fusarium solani-induced activation of FAK is an early event and possibly a prerequisite for complete activation of FAK.
Previous data have shown that the interaction of fungus with epithelial cells results in actin rearrangement in the host cells, membrane ruffling, and cellular motility, the effects of which are both dose and time dependent [29]. We studied the effects of the FAK/PAX and FAK inhibitor, genistein, on actin rearrangement in our model system and attempted to explore the putative action mechanism of fungus. Our observations indicate that the interaction of F. solani with HCECs causes actin rearrangement in HCECs. The activity of the F. solani in altering actin arrangement was decreased by the supplementation of FAK inhibitor and the interaction of spores with the cells was reduced. This may possibly be explained through the perturbation caused by genistein to the actin polymerization.
Our results indicate that once F. solani spores attach to the ECM, integrin stimulation of FAK/PAX promotes adhesion and enters the cells by a process of triggered ruffling and internalization. Although the results obtained in this study showed the consequence of the interaction between mammalian cells with F. solani, the mechanisms of this process and its implications in the infection process still require further investigation. Further elucidation of the molecular interactions that trigger the uptake of host cells will be important in understanding this mode of pathogenesis.
ACKNOWLEDGMENTS
The authors thank Yao Wang, Lingling Yang, Hongmei Yin, and Ting Liu for their technical assistance, and Xiaoguang Dong and Weiyun Shi for their valuable discussions. Supported by the National Natural Science Foundation of China (30630063).
REFERENCES
1.Xie L, Zhong W, Shi W, Sun S. Spectrum of fungal keratitis in
north China. Ophthalmology 2006; 113:1943-8.[PMID:
16935335]
2.Sun XG, Zhang Y, Li R, Wang ZQ, Luo SY, Jin XY, Zhang
WH. Etiological analysis on ocular fungal infection in the
period of 1989 - 2000. Chin Med J (Engl) 2004;
117:598-600. [PMID: 15109456]
3.Guo H, Wu X, Yu FS, Zhao J. Toll-like receptor 2 mediates the
induction of IL-10 in corneal fibroblasts in response to
Fusarium solu. Immunol Cell Biol 2008; 86:271-6. [PMID:
18195725]
4.Kruppa M, Calderone R. Two-component signal transduction
in human fungal pathogens. FEMS Yeast Res 2006;
6:149-59. [PMID: 16487338]
5.Menon V, Li D, Chauhan N, Rajnarayanan R, Dubrovska A,
West AH, Calderone R. Functional studies of the Ssk1p
response regulator protein of Candida albicans as determined
by phenotypic analysis of receiver domain point mutants. Mol
Microbiol 2006; 62:997-1013. [PMID: 17038117]
6.Dong X, Shi W, Zeng Q, Xie L. Roles of adherence and matrix
metalloproteinases in growth patterns of fungal pathogens in
cornea. Curr Eye Res 2005; 30:613-20. [PMID: 16109640] 7.Sanders MA, Basson MD. Collagen IV-dependent ERK
activation in human Caco-2 intestinal epithelial cells requires
focal adhesion kinase. J Biol Chem 2000; 275:38040-7.
[PMID: 10986280]
8.Sawai H, Okada Y, Funahashi H, Matsuo Y, Takahashi H,
Takeyama H, Manabe T. Activation of focal adhesion kinase
enhances the adhesion and invasion of pancreatic cancer cells
via extracellular signal-regulated kinase-1/2 signaling
pathway activation. Mol Cancer 2005; 4:37.[PMID:
16209712]
9.Santoni G, Lucciarini R, Amantini C, Jacobelli J, Spreghini E,
Ballarini P, Piccoli M, Gismondi A. Candida albicans
expresses a focal adhesion kinase-like protein that undergoes
increased tyrosine phosphorylation upon yeast cell adhesion
to vitronectin and the EA.hy 926 human endothelial cell line.
Infect Immun 2002; 70:3804-15. [PMID: 12065524]
10.Sundstrom P. Adhesion in Candida spp. Cell Microbiol 2002;
4:461-9. [PMID: 12174081]
11.Yang YL. Virulence factors of Candida species. J Microbiol
Immunol Infect 2003; 36:223-8. [PMID: 14723249]
12.Whiteway M, Oberholzer U. Candida morphogenesis and host-
pathogen interactions. Curr Opin Microbiol 2004; 7:350-7.
[PMID: 15358253]
13.Calderone RA, Braun PC. Adherence and receptor relationships
of Candida albicans. Microbiol Rev 1991; 55:1-20. [PMID:
2030668]
14.Upadhyay SK, Mahajan L, Ramjee S, Singh Y, Basir SF, Madan
T. Identification and characterization of a laminin-binding
protein of Aspergillus fumigatus: extracellular thaumatin
domain protein (AfCalAp). J Med Microbiol 2009;
58:714-22. [PMID: 19429746]
15.Bergmann S, Lang A, Rohde M, Agarwal V, Rennemeier C,
Grashoff C, Preissner KT, Hammerschmidt S. Integrin-linked
kinase is required for vitronectin-mediated internalization of
Streptococcus pneumoniae by host cells. J Cell Sci 2009;
122:256-67. [PMID: 19118218]
16.Hostetter MK. RGD-mediated adhesion in fungal pathogens of
humans, plants and insects. Curr Opin Microbiol 2000;
3:344-8. [PMID: 10972491]
17.Rudrabhatla RS, Sukumaran SK, Bokoch GM, Prasadarao NV.
Modulation of myosin light-chain phosphorylation by p21-
activated kinase 1 in Escherichia coli invasion of human brain
microvascular endothelial cells. Infect Immun 2003;
71:2787-97. [PMID: 12704153]
18.Yee KL, Weaver VM, Hammer DA. Integrin-mediated
signalling through the MAP-kinase pathway. IET Syst Biol
2008; 2:8-15. [PMID: 18248081]
https://www.doczj.com/doc/0b12621817.html,czko R, Szauter KM, Jansen MK, Hollosi P, Muranyi M,
Molnar J, Fong KS, Hinek A, Csiszar K. Active lysyl oxidase
(LOX) correlates with focal adhesion kinase (FAK)/paxillin
activation and migration in invasive astrocytes. Neuropathol
Appl Neurobiol 2007; 33:631-43. [PMID: 17931358]
20.Dorfleutner A, Hintermann E, Tarui T, Takada Y, Ruf W.
Cross-talk of integrin alpha3beta1 and tissue factor in cell
migration. Mol Biol Cell 2004; 15:4416-25.[PMID:
15254262]
21.Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H,
Tano Y, Handa H. An SV40-immortalized human corneal
epithelial cell line and its characterization. Invest Ophthalmol
Vis Sci 1995; 36:614-21. [PMID: 7534282]
22.Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S,
Itoh N, Shibuya M, Fukami Y. Genistein, a specific inhibitor
of tyrosine-specific protein kinases. J Biol Chem 1987;
262:5592-5. [PMID: 3106339]
23.Shikata Y, Rios A, Kawkitinarong K, DePaola N, Garcia JG,
Birukov KG. Differential effects of shear stress and cyclic
stretch on focal adhesion remodeling, site-specific FAK
phosphorylation, and small GTPases in human lung
endothelial cells. Exp Cell Res 2005; 304:40-9.[PMID:
15707572]
24.Kokkinos MI, Brown HJ, de Iongh RU. Focal adhesion kinase
(FAK) expression and activation during lens development.
Mol Vis 2007; 13:418-30. [PMID: 17417603]
25.Robles E, Gomez TM. Focal adhesion kinase signaling at sites
of integrin-mediated adhesion controls axon pathfinding. Nat
Neurosci 2006; 9:1274-83. [PMID: 16964253]
26.Yamaoka Y, Kwon DH, Graham DY. A M(r) 34,000
proinflammatory outer membrane protein (oipA) of
Helicobacter pylori. Proc Natl Acad Sci USA 2000;
97:7533-8. [PMID: 10852959]
27.Yamaoka Y, Kikuchi S, el-Zimaity HM, Gutierrez O, Osato
MS, Graham DY. Importance of Helicobacter pylori oipA in
clinical presentation, gastric inflammation, and mucosal
interleukin 8 production. Gastroenterology 2002;
123:414-24. [PMID: 12145793]
28.Tabassam FH, Graham DY, Yamaoka Y. OipA plays a role in
Helicobacter pylori-induced focal adhesion kinase activation
and cytoskeletal re-organization. Cell Microbiol 2008;
10:1008-20. [PMID: 18067607]
29.Sandovsky-Losica H, Berdicevsky I, Tsarfaty I, Segal E. Effect
of Candida albicans metabolite(s) on cellular actin. FEMS
Microbiol Lett 2002; 215:57-62. [PMID: 12393201]
Articles are provided courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China. The print version of this article was created on 2 March 2011. This reflects all typographical corrections and errata to the article
降尿酸的药物及分类 第一类为抑制尿酸合成的药。其代表药是别嘌醇(别嘌呤醇)。别嘌醇可用于各种年龄的原发性和继发性痛风病人，不受肾功能的限制，故痛风病人合并肾功能不全、肾结石及尿酸排出过多应首选本药。与促尿酸排泄药合用有协调作用。因药源充足、廉价，是较理想的降尿酸药之一。 别嘌醇为黄嘌呤氧化酶抑制剂，其结构类似次黄嘌呤，有较强的抑制黄嘌呤氧化酶作用，从而阻断次黄嘌呤向黄嘌呤、黄嘌呤向尿酸的代谢转化，可减少尿酸的生成，降低血尿酸浓度。该药能在PRPP(5-磷酸核糖-1-焦磷酸合成酶)存在时转变成相对应核苷酸，消耗PRPP使IMP(次黄嘌呤核苷酸)合成减少，从而可迅速降低血尿酸值，抑制痛风石和肾结石形成，并促进痛风石溶解。 适应证：⑴尿酸合成过多而导致的高尿酸血症。⑵肾功能严重损害而不能使增大的尿酸负荷排出者。⑶大剂量尿酸排泄促进剂无效或过敏，或不能耐受者。⑷肾尿酸结石反复形成者。⑸每日尿酸排泄超过5.9毫摩尔(1000毫克)者，易发生尿酸性肾结石的危险。⑹有较大的多部位痛风结节者(需要两种药联用以阻断尿酸的产生和增加尿酸的排泄)。⑺继发于骨髓增殖性疾病的高尿酸血症，特别是细胞毒制剂治疗前的患者，否则大量尿酸从肾排出，可发生急性肾小管阻塞。 用法：别嘌醇开始每天100毫克，每日2～3次口服，可逐渐增至每次200毫克，每日3～4次，每日最大剂量不超过600毫克为宜。血尿酸浓度正常后逐渐减至维持量，每次100毫克，每日1～2次。其副作用为过敏性皮疹、药物热、肠胃不适、白细胞及血小板减少、肝功能损害等。 注意事项：①需从小剂量开始，逐渐增加剂量，以免血尿酸浓度急剧下降而诱发痛风急性发作。②定期复查周围血象、肝功能等。 第二类是促进肾脏排泄尿酸药。主要用于无明显肾功能损害、60岁以下的痛风或高尿酸血症病人，尤适用于痛风结节较多者。用药后尿液酸碱度(pH值)迅速下降者要大量饮水并同服碱性药，以减少尿酸盐在肾脏沉积，防止结石形成。常用的促肾排尿酸药有苯溴马隆(痛风利仙)、丙磺舒(羧苯磺胺)，属磺胺类药，对磺胺过敏者禁用，长期应用要定期查全血细胞，防止骨髓抑制现象发生。磺吡酮(苯磺唑酮)，可作为磺胺过敏者丙磺舒的替代物。 适用于尿酸排泄低下型高尿酸血症，如果肾功能有轻度损害也可应用，有时在促进尿酸排泄增多后，肾功能也可得到改善。该类药物主要通过抑制近端肾小管对尿酸的重吸收而促进尿酸的排泄。 药物和用法： (1)丙磺舒(羧苯磺胺)：是一种有效的尿酸排泄促进剂,每天1克可使肾对尿酸的排泄平均增加50％，血尿酸平均下降30％。开始时每次0.25克，每日2次，2周内递增至每次O.5克，每日2～3次，如果血尿酸明显高于正常，可每1～2周再增加0.5克，直至血尿酸降至正常水平。每日最大剂量2克以下。高尿酸血症控制后可再逐渐减量维持。约5％患者有皮疹、发热、胃肠刺激、肾绞痛及激发急性痛风发作等副作用。
铜线检验标准 本标准是对～《电工圆铜杆》标准的修订。与原标准相比，本标准作了如下修改：？ 1.将标准名称改为《电工用铜线坯》。？ 2.在标准结构上，将原来的四项分标准合并编写，不再设立分标准，取消了很多重复性的内容。？ 3.主要技术参数和技术内容有较大进步。本标准中铜线坯的公称直径及其答应偏差与ISO4738；1982《铜线坯》及BS6926-1988《电工用铜--高导铜线坯》标准的要求等效；化学成分要求是参考了美国ASTMB49-92《电工用再拉铜线坯》标准，并依据GB/T467《阴极铜》及GB/T468《电工用铜线锭》标准而修订的；热态铜线坯力学性能的低限值35%与德国DIN17652-82《铜线坯》标准的要求等同，高于其他国外标准中30%的要求；硬态铜线坯的力学性能指标较原标准有所进步；检验组批及各种性能测试的取样方法和取样数目要求是参照英国BS6926-1988标准修订的，其中本标准对取样数目的规定严于BS6926-1988标准的要求。？ 4.注重了与其他相关国家标准的一致性，标准编写格式和编写方法遵守GB/T 标准的规定。？ 本标准与国外先进国家标准水平相比，达到了国际先进水平。？ 本标准自实施之日起，同时代替～。？ 本标准由中国有色金属产业总公司提出。？ 本标准由北京铜厂、中国有色金属产业总公司标准计量研究所、铜陵有色金属公司负责起草。？
本标准由北京铜厂、中国有色金属产业总公司标准计量研究所、铜陵有色金属公司、常州东方鑫源铜业有限公司、沈阳冶炼厂、云南冶炼厂共同起草。？ 本标准主要起草人：何新宇、尧川、陈明勇、陈彪、刘婉容、赵华、李晓丽、温晓云、孙励筠、邢伟。？ 1范围？ 本标准规定了电工用铜线坯的要求、试验方法、检验规则及标志、包装、运输和贮存。？ 本标准适用于直径为～36.0mm、供进一步拉制线材或其他电工用铜导体的圆形截面铜线坯。？ 2引用标准？ 下列标准所包含的条文，通过在本标准中引用而构成为本标准的条文。本标准出版时，所示版本均为有效。所有标准都会被修订，使用本标准的各方应探讨使用下列标准最新版本的可能性。裸电线试验方法尺寸丈量？ 裸电线试验方法拉力试验？ 裸电线试验方法扭转试验？ GB/电线电缆金属导体材料电阻率试验方法？ GB/～铜及铜合金化学分析方法？ GB/T13293-91高纯阴极铜化学分析方法？ 3订货单（或合同）内容？ 本标准所列产品的订货单（或合同）应包括下列内容？ 产品名称。？ 牌号、状态、规格？
普乐可复 【适应症】 普乐可复适用于治疗肝脏或肾脏移植术后应用其他免疫抑制药物无法控制的移植物排斥反应。 普乐可复适用于预防肝脏或肾脏移植术后的移植物排斥反应。 【规格】 普乐可复胶囊:5mg/粒;1mg/粒; 0.5mg/粒； 普乐可复针剂：1ml:5mg 【用法用量】 每日服普乐可复两次（早晨和晚上），最好用水送服。建议空腹，或者至少在餐前1小时或餐后2-3小时服用。如必要可将胶囊内容物悬浮于水，经鼻饲管给药。若患者临床状况不能口服，首剂须静脉给药。 【不良反应】 由于患者疾病非常严重，且经常是多药合用，与免疫抑制剂相关的不良反应通常难以确定。有证据表明普乐可复下述的多种不良反应均为可逆性，减量可使其减轻或消失。与静脉给药相比，口服给药的不良反应发生率较低。 常见不良反应有： 1）增加了对病毒、细菌、真菌和/或原虫感染的易感染性。 2）肾功能异常（血肌酐升高、尿素氮升高、尿量减少）。 3）神经系统症状：震颤，头痛，感觉异常和失眠，上述症状可单独出现或同时出现。 4）高血压，胃肠道症状，脱发，多毛等。 【注意事项】 他克莫司免疫抑制作用比环孢素强100倍，具有活性强、抗排异效果好、患者细菌和病毒感染率低、亲肝性强、不良反应低、高移植存活率等优点，是目前临床疗效最好的免疫抑制剂。使用他克莫司时需注意以下事项： 1. 使用他克莫司前注意：是否已怀孕或准备怀孕；是否母乳喂养；是否对他克莫司和辅助剂成分过敏。 2. 饮食注意：进食可影响药物吸收，有一定的脂肪食物可降低该药的吸收。建议在空腹下口服，在饭前一小时或饭后2-3小时口服，服用他克莫司时饮酒会增加视觉和神经系统不良反应。 3. 患者不可自行改变他克莫司的剂量或停药，任何剂量的调整都应该由您的移植医生进行。 4. 常见副反应及处理：副反应常常为震颤、头痛、失眠、眼部疾患者视线模糊、白内障、恶心、高血糖等。出现副反应立即与医生联系。一般与浓度过高有关，大多数发生在服药一个月以内。一般减药 或降低浓度，副作用即缓解或消失。 【禁忌】 妊娠、对他克莫司或其他大环内酯类药物过敏者、对胶囊中其他成份过敏者。 【药物相互作用】 当普乐可复与具有潜在神经毒性的化合物合用时，如阿昔洛韦或更昔洛韦，可能会增强这些药物的神经毒性。 应用普乐可复可能导致高钾血症，或加重原有的高钾血症，应避免摄入大量的钾或服用留钾利尿剂（如氨氯吡咪、氨苯喋啶及安体舒通）。 与血浆蛋白结合的相互作用：本品与血浆蛋白广泛结合。因此，应考虑可能与血浆蛋白结合率高的药物发生相互作用（如口服抗凝剂，口服抗糖尿病药等）。 影响特殊器官或身体机能的相互作用： 在使用普乐可复时，疫苗的效能会减弱，应避免使用减毒活疫苗。 与已知有肾毒性的药物联合应用时应注意，如氨基糖甙、二性霉素B，旋转酶抑制剂、万古霉素、复方新诺明和非甾体类抗炎药。 普乐可复与含有中等脂肪饮食一起服用会显著降低其生物利用度和口服吸收率。因此，为达到最大口服吸收率，须空腹服用或至少在餐前1小时或餐后2-3小时服用。
Designation:D3359–02 Standard Test Methods for Measuring Adhesion by Tape Test1 This standard is issued under the?xed designation D3359;the number immediately following the designation indicates the year of original adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.A superscript epsilon(e)indicates an editorial change since the last revision or reapproval. This standard has been approved for use by agencies of the Department of Defense. 1.Scope 1.1These test methods cover procedures for assessing the adhesion of coating?lms to metallic substrates by applying and removing pressure-sensitive tape over cuts made in the?lm. 1.2Test Method A is primarily intended for use at job sites while Test Method B is more suitable for use in the laboratory. Also,Test Method B is not considered suitable for?lms thicker than5mils(125μm). N OTE1—Subject to agreement between the purchaser and the seller, Test Method B can be used for thicker?lms if wider spaced cuts are employed. 1.3These test methods are used to establish whether the adhesion of a coating to a substrate is at a generally adequate level.They do not distinguish between higher levels of adhesion for which more sophisticated methods of measure-ment are required. N OTE2—It should be recognized that differences in adherability of the coating surface can affect the results obtained with coatings having the same inherent adhesion. 1.4In multicoat systems adhesion failure may occur be-tween coats so that the adhesion of the coating system to the substrate is not determined. 1.5The values stated in SI units are to be regarded as the standard.The values given in parentheses are for information only. 1.6This standard does not purport to address the safety concerns,if any,associated with its use.It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 2.Referenced Documents 2.1ASTM Standards: D609Practice for Preparation of Cold-Rolled Steel Panels for Testing Paint,Varnish,Conversion Coatings,and Related Coating Products2 D823Practices for Producing Films of Uniform Thickness of Paint,Varnish,and Related Products on Test Panels2 D1000Test Method For Pressure-Sensitive Adhesive-Coated Tapes Used for Electrical and Electronic Applica-tions3 D1730Practices for Preparation of Aluminum and Aluminum-Alloy Surfaces for Painting4 D2092Guide for Preparation of Zinc-Coated(Galvanized) Steel Surfaces for Painting5 D2370Test Method for Tensile Properties of Organic Coatings2 D3330Test Method for Peel Adhesion of Pressure-Sensitive Tape6 D3924Speci?cation for Standard Environment for Condi-tioning and Testing Paint,Varnish,Lacquer,and Related Materials2 D4060Test Method for Abrasion Resistance of Organic Coatings by the Taber Abraser2 3.Summary of Test Methods 3.1Test Method A—An X-cut is made through the?lm to the substrate,pressure-sensitive tape is applied over the cut and then removed,and adhesion is assessed qualitatively on the0 to5scale. 3.2Test Method B—A lattice pattern with either six or eleven cuts in each direction is made in the?lm to the substrate,pressure-sensitive tape is applied over the lattice and then removed,and adhesion is evaluated by comparison with descriptions and illustrations. 4.Signi?cance and Use 4.1If a coating is to ful?ll its function of protecting or decorating a substrate,it must adhere to it for the expected service life.Because the substrate and its surface preparation (or lack of it)have a drastic effect on the adhesion of coatings, a method to evaluate adhesion of a coating to different substrates or surface treatments,or of different coatings to the 1These test methods are under the jurisdiction of ASTM Committee D01on Paint and Related Coatings,Materials,and Applications and are the direct responsibility of Subcommittee D01.23on Physical Properties of Applied Paint Films. Current edition approved Aug.10,2002.Published October2002.Originally published as D3359–https://www.doczj.com/doc/0b12621817.html,st previous edition D3359–97. 2Annual Book of ASTM Standards,V ol06.01. 3Annual Book of ASTM Standards,V ol10.01. 4Annual Book of ASTM Standards,V ol02.05. 5Annual Book of ASTM Standards,V ol06.02. 6Annual Book of ASTM Standards,V ol15.09. 1 Copyright?ASTM International,100Barr Harbor Drive,PO Box C700,West Conshohocken,PA19428-2959,United States. Copyright ASTM International Reproduced by IHS under license with ASTM Licensee=daimlerchyrsler account/5957216001 Not for Resale, 12/09/2005 00:34:54 MST No reproduction or networking permitted without license from IHS --` ` , , , , ` , , , ` ` ` ` ` ` , , ` ` ` , , , ` , ` ` ` -` -` , , ` , , ` , ` , , ` ---
第二军医大学学报Acad J Sec M il M ed Univ 2006Feb ;27(2) 189 论 著 迷迭香酸对黄嘌呤氧化酶的抑制作用 尚雁君1,黄才国1*,蒋三好2,朱大元2,魏善建1,焦炳华1 (1.第二军医大学基础医学部生物化学和分子生物学教研室,上海200433,2.中国科学院上海药物研究所,上海201203)[摘要] 目的:研究迷迭香酸对黄嘌呤氧化酶的抑制作用。方法:将20、40、60μg /ml 迷迭香酸或1μg /ml 阳性对照别嘌呤醇,分别加入黄嘌呤溶液(测尿酸生成量:1mmol /L ;测超氧离子:50μmol /L )和0.1U /ml 黄嘌呤氧化酶中,用生化仪测定5min 尿酸生成量和超氧离子生成(NBT 显色法)。在1ml 2×105/ml HL -60细胞悬液中加入100μl 6mo l /L 黄嘌呤、100μl 0.1U /ml 黄嘌呤氧化酶、500μg /ml 迷迭香酸,分别以Annexin Ⅴ-P I 双标试剂盒法(以1μg /ml 别嘌呤醇为阳性对照)或细胞周期法(以100U /ml SO D 为阳性对照)测定细胞凋亡率。结果: 迷迭香酸显著抑制尿酸生成和超氧离子引起的N BT 显色,两种方法测得其IC 50分别为56μg /ml 和21μg /ml ;对细胞凋亡的抑制率均在40%以上。 结论: 迷迭香酸是黄嘌呤氧化酶的竞争性抑制剂。 [关键词] 迷迭香酸;黄嘌呤氧化酶;尿酸;超氧离子;细胞凋亡 [中图分类号] R 285.5 [文献标识码] A [文章编号] 0258-879X (2006)02-0189-03 Inhibition of xathine oxidase by rosmarinic acid S HA N G Y an -jun 1,HU A NG Cai -guo 1*,JI AN G San -hao 2,Z H U Da -y uan 2,W EI Shan -jian 1,JIAO Bin -hua 1(1.Depar tme nt of Bio chemistry and M o lecular Bio log y ,Co llege of Basic M edical Science s ,Second M ilitary M edical U niver sity ,Shanghai 200433,China ;2.Shanghai I nstitute of M ateria M edica ,Shanghai 201203) [ABSTRACT ] Objective :T o study the inhibito ry effect of rosmarinic acid on x anthine ox idase.Methods :Xanthine ox idase (0.1U /ml )w as incuba ted with xa nthine (1mmol /L for determining for ma tion of uric acid ;50μmo l/L fo r de te rmining super -o xide anions )in the presence of 20,40and 60μg /ml rosmarinic acid o r allo purino l as positiv e contro l.T he forma tion o f uric acid w as deter mined by automatic bio chemical analyzer 5min after reactio n and the production of supero xide anio ns w as meas -ured by N it ro Blue Btetr azo lium (NBT )reduction.HL -60cells (1ml ,2×105/ml )wer e pretrea ted w ith xanthine (100μl ,6mol /L )and xanthine o xidase (100μl ,0.1U /ml ),then ro smarinic acid (500μg /ml )o r allopurinol (1μg /ml ,a s positive con -trol )(A nnexin Ⅴ-P I kit )was added to de te rmine the cell a po pto sis rate.H L -60cells (1ml ,2×105/ml )w ere also pre treated with xanthine (100μl ,6mo l/L )and x anthine ox idase (100μl ,0.1U /m l ),then ro smarinic acid (500μg /ml )or SO D (100U /ml ,a s positive contro l )(cell cycle me tho d )was added to de te rmine the cell apopto sis rate.Results :Ro smarinic acid obvio usly inhibited the production of uric acid and supero xide anion -induced reaction in N BT assay ,with their IC 50being 56μg /ml and 21μg /ml ,respec tively.T he rates of apoptosis inhibitio n by ro sma rinic acid w ere bo th o ver 40%by Annexin Ⅴ-PI kit and cell cy -cle me tho d.C onclusion :Rosmarinic acid is a competitive inhibito r of x anthine o xidase.[KEY WORDS ] rosmarinic acid ;xanthine oxidase ;uric acid ;super oxide anio ns ;apo pto sis [A cad J Sec M il M ed U niv ,2006,27(2):189-191] [基金项目] 国家自然科学基金(29632050).S upported b y National Natural Science Foundation of China (29632050).[作者简介] 尚雁君,硕士生.E -m ail :syjsmm u @https://www.doczj.com/doc/0b12621817.html, *Corres ponding autho r.E -mail :hu angcaig @h https://www.doczj.com/doc/0b12621817.html, 丹参是临床上常用的活血化瘀药,是唇形科植 物丹参Salvia miltiorrhiza Bung e 的根,常用于妇科病、冠心病、缺血性脑卒中、动脉粥样硬化等症的治疗。临床上丹参制剂对冠心病、脑血栓、肝炎、肝硬化等有显著的疗效。丹参的活性成分主要分为脂溶性和水溶性两类。中医传统用药方法是用其水煎剂,即丹参的水溶性部位,所以研究丹参的水溶性成分更有意义[1] 。研究表明其水溶性成分主要是丹参素、原儿茶醛、丹酚酸。丹酚酸是一类既有咖啡酰缩酚酸结构又有新木脂素骨架的水溶性成分。丹酚酸类化合物包括丹酚酸A 、B 、C 、D 、E 、F 、G 、H 、I ,迷迭香酸(ro smarini acid ),紫草酸(litho spermic acid ) 等,其中迷迭香酸是由1分子丹参素和1分子咖啡 酸缩合而成。黄嘌呤氧化酶是人体内产生尿酸过程中的关键酶,同时也是治疗痛风时药物的作用靶点,本文研究其对黄嘌呤氧化酶的抑制作用。1 材料和方法 1.1 试剂 H L -60细胞株,购自上海中国科学院细胞所;Annexin Ⅴ-PI 双标试剂盒购自晶美公司;黄
目录 一.导体（线）束、绞工序产品过程检验规程 (2) 二.紧压圆形导体绞制工序产品过程检验规程 (4) 三.耐火云母带绕包工序产品过程检验规程 (5) 四.塑料绝缘、护套工序产品过程检验规程 (7) 五.橡皮绝缘、橡皮护套及硫化工序产品过程检验规程 (9) 六.三层挤出工序产品过程检验规程 (11) 七.低压线缆成缆工序产品过程检验规程 (14) 八.高压交联成缆工序产品过程检验规程 (17) 九.金属屏蔽工序产品过程检验规程 (19) 十.绕包工序产品过程检验规程 (20) 十一.装铠工序产品过程检验规程 (21) 十二.成圈和包装工序产品过程检验规程 (23) 十三.温水交联工序产品过程检验规程 (24) 十四.编织工序产品过程检验规程 (25) 十五.附录 (26)
一、导体（线）束、绞工序产品过程检验规程 1适用范围 本标准适用于电线电缆用导电线的束丝、绞线工序，裸铝（铜）绞线及钢芯铝绞线、圆线同心绞架空导线工序检验（除紧压圆形导电线芯）。 2本规范依据 GB/T3953-2009 电工圆铜线 GB/T3955-2008 电工圆铝线 GB/T1179-2008 铝绞线及钢芯铝绞线 GB/T1179-2008 圆线同心绞架空导线 GB/T3956-2008 电缆的导体 3质量要求 3.1束、绞前后的圆铜线外径，应符合相关产品工艺卡片及导体丝径检验规范的规定。 3.2束、绞导电线芯节径比和绞向应符合表1规定，裸铝（铜）绞线及钢芯铝绞线、圆线同心绞架空导线节径比和绞向应符合表2规定。 3.3在同一层内，相邻两个接头之间的距离应不小于300mm。 3.4束、绞以后的导电线芯表面应光滑，无三角口、裂纹、毛刺、油污等。 3.5束、绞以后的导电线芯应紧密，不得缺股，断线、跳滨，允许轻微擦伤、擦毛等现象。
免疫抑制剂的用药护理 免疫抑制剂定义 是一类通过抑制细胞及体液免疫反应，而使组织损伤得以减轻的化学或生物物质。其具有免疫抑制作用，可抑制机体异常的免疫反应，目前广泛应用于器官移植抗排斥反应和自身免疫性疾病的治疗。 免疫抑制剂的分类 1、钙调素抑制剂类：环孢菌素CsA类、他克莫司（FK506） 2、抗代谢类：硫唑嘌呤、霉酚酸脂（MMF） 3、激素类：甲强龙、醋酸泼尼松 4、生物制剂：抗T细胞球蛋白（ATG）、抗淋巴细胞球蛋白（ALG） 免疫抑制剂用药原则 1、预防性用药：环孢素A、FK506、霉酚酸脂（MMF）等。 2、治疗/逆转急性排斥反应（救治用药）：MP（甲基强的松龙）、ALG或ATG、霉酚酸脂（MMF）、FK506等。 3、诱导性用药（因急性肾小管坏死而出现延迟肾功能、高危病人、二次移植、环孢素肾毒性病人）：ATG、ALG等。 4、二联：激素（醋酸泼尼松）+抗代谢类（骁悉） 三联：激素（醋酸泼尼松）+抗代谢类（骁悉）+环孢素A（新山地明） 激素（醋酸泼尼松）+抗代谢类（骁悉）+FK506（他克莫司） 常用免疫抑制剂 1、环孢素(CsA)：新山地明（进口）田可、赛斯平（国产） 作用机理
属于钙神经蛋白抑制剂，可以选择性抑制免疫应答，通过破坏使T细胞活化的细胞因子的表达，阻断参与排斥反应的体液和细胞效应机制，防止排斥反应的发生。 药物的吸收和代谢 新山地明受进食和昼夜节律的影响较山地明小，所以服药时间不必将用餐考虑在内。 环孢素A依靠胆汁排泄，肝功能障碍，胆汁淤积症或严重胃肠功能障碍都会影响环保素A的吸收和代谢。只有极少部分药物经肾脏排出，且不能经透析去除，所以对于肾脏功能不全者和需透析治疗的患者，均不需调整药物浓度。 副作用 （1）肾毒性：血清肌酐、尿素氮增高；肾功能损害。个体差异大，临床表现不典型，与其他原因引起的移植肾损害很难鉴别。且发生肾损害时，血药浓度可能正常，甚至偏低。 （2）接近半数的患者会出现肝脏毒性，其发生率与用药量密切相关。 （3）神经毒性：表现为肢体震颤、失眠、烦躁等。 （4）胃肠道反应：恶心、呕吐。 （5）其他并发证：高血压、高胆固醇血症、高钾血症、牙龈增生、糖尿病、多毛症。 用量 联合用药时：初始剂量为6～8mg/kg/d，Q12h，以后根据血药浓度调整。 注意事项 （1）严格按医嘱服药,定时服药,禁忌自行调整用药剂量。
标题涂层表面附着力测试标准 文件类别规范文件文件号目标[质]字05第10 版本号 1 修改标记无修改次数无 编制/日期审核/日期批准/日期 执行主体监督主体 1．目的：指导涂层表面附着力测试工作，规范和统一涂层表面附着力检验标准； 2．范围：应用?涂层厚度大于50μm； 3．定义：符合BS?3900-E6、ISO2409、DIN53?151和ASTM?D3359-B测试方法； 4．流程：无 5．内容： 设备要求：划线器刀口由碳钨合金材料制成，齿数x齿间距?6齿x2mm；胶带用3M 600号2cm宽胶带；操作步骤 -用划格器在涂层上切出十字格子图形，切口直至基材； -用毛刷对角线方向各刷五次，用胶带贴在切口上再拉开； -观察格子区域的情况，可用放大镜观察； 划格结果附着力按照以下的标准等级 ISO等级：0 ASTM等级：5B 切口的边缘完全光滑，格子边缘没有任何剥落 ISO等级：1 ASTM等级：4B 在切口的相交处有小片剥落，划格区内实际破损 不超过5% ISO等级：2 ASTM等级：3B 切口的边缘和/或相交处有被剥落，其面积大于 5%，但不到15% 版本号1实施日期页次：共 2 页第 1 页
ISO等级：3 ASTM等级：2B 沿切口边缘有部分剥落或整大片剥落，及/或者部 分格子被整片剥落。被剥落的面积超过15%，但 不到35% ISO等级：4 ASTM等级：1B 切口边缘大片剥落/或者一些方格部分部分或全 部剥落，其面积大于划格区的35%，但不超过65% ISO等级：5 ASTM等级：0B超过上一等级 ? 测试结果判定：如果没有客人特殊要求，目标的产品要求达到ISO等级：1、ASTM等级：4B以上级别可以接受。 签发人签名 部门，现将《涂层表面附着力测试标准》抄发你部门（组织），请严格执行。 签发人/日期： 执行人签名现收到签发的《涂层表面附着力测试标准》，本人明白制度的详细内容，并保证本部门（人）严格贯彻执行。 执行人/日期： 版本号 1实施日期页次：共 2 页第 2 页
黄嘌呤氧化酶抑制剂/超氧阴离子清除剂双靶点高通量筛选模型的建立 谢涛?, 秦至臻?, 周睿, 赵赢, 杜冠华* (中国医学科学院、北京协和医学院药物研究所, 北京市药物靶点研究与新药筛选重点实验室, 北京 100050) 摘要: 本文建立了适用于同时筛选黄嘌呤氧化酶抑制剂和超氧阴离子清除剂的双靶点高通量复合筛选模型。在黄嘌呤氧化酶超氧阴离子生成体系中,加入WST-1作为超氧阴离子生成量的探针,以反应体系中标识性产 物尿酸为黄嘌呤氧化酶活性指示剂, 采用SpectraMax M5酶标测试仪, 同时检测两种指示剂的浓度变化, 通过 对反应体系中的影响因素进行优化建立双靶点HTS筛选模型, 并利用阳性药物对该模型进行评价。在反应体系 中, 反应终体积50 μL, 黄嘌呤氧化酶4 mU·mL?1、黄嘌呤250 μmol·L?1、WST-1浓度为100 μmol·L?1, 黄嘌呤氧 化酶抑制剂筛选模型的Z'-因子为0.5374, S/N为47.5199; 超氧阴离子清除剂筛选模型的Z'-因子为0.5074, S/N 为5.3889。结果表明, 本文建立的黄嘌呤氧化酶抑制剂/氧自由基清除剂高通量筛选模型具有稳定性好、成本较 低和重复性高等特点, 可以广泛应用于药物筛选。 关键词: 高通量筛选方法; 黄嘌呤氧化酶; 超氧阴离子清除剂; 抗氧化; 药物评价 中图分类号: R965 文献标识码:A 文章编号: 0513-4870 (2015) 04-0447-06 Establishment of double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers XIE Tao?, QIN Zhi-zhen?, ZHOU Rui, ZHAO Ying, DU Guan-hua* (Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China) Abstract: A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators’ concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU·mL?1, xanthine 250 μmol·L?1 and WST-1 100 μmol·L?1, separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.5374, S/N is 47.5199; the Z'-factor of model for superoxide anion scavengers is 0.5074, S/N is 5.3889. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research. Key words: high throughput screening method; xanthine oxidase; superoxide anion scavenger; antioxidant; drug evaluation 收稿日期: 2014-10-20; 修回日期: 2014-12-22. 基金项目: 重大新药创制科技重大专项 (2012ZX09101); 国际合作项目 (2012DFH30070). ?并列第一作者. *通讯作者 Tel / Fax: 86-10-63165184, E-mail: dugh@https://www.doczj.com/doc/0b12621817.html,
漆包线检验标准 This manuscript was revised on November 28, 2020
漆包线检验标准 1.外观检验： 表面光滑，色泽均匀，无漆瘤和白色润滑剂，表面绝缘漆膜无脱落、氧化、划痕、损伤，无打结现象 2.尺寸检验： 漆包线直径：标准参照IEC60317对照表，检验方法：千分尺 导体直径：标准参照IEC60317对照表，检验方法：千分尺 漆包线漆膜厚度：标准参照IEC60317对照表，检验方法：千分尺 导体误差值：标准参照IEC60317对照表 3.电性能： 电阻 取要检验的漆包线1m, 将两端的漆膜刮去，测量漆包线的电阻，电阻测量值要与IEC60317的要求电阻范围内；标准温度20度，换算公式：20度的电阻/+标准温度=实际测量电阻/+测量温度。 可焊性 ①剪取需要检验的铜线材料； ②根据材料可焊性条件范围设定锡炉温度； ③使用温度测量器对锡炉温度测量确认锡炉温度在材料承认书的可焊性条件范围内； ④焊锡条件依材料承认书，铜线上锡效果：当铜线浸锡后目检表面着锡面积95%以上,不遗留残渣。 ⑤.非直焊性线径剥皮处理后依(第①-④点)作业。 针孔及漏电流测试 配置溶液及接线 A. 配置盐水溶液：食盐30 克，清水10 公斤，浓度为3‰； B. 配置酚酞溶液：酒精100 克，酚酞3 克； C. 配置溶液：每10 公斤盐水溶液加入20 毫升酚酞溶液； D. 按图1 检查盐浴装置正负极接线是否正确。导电体接正极直接接入盐浴池，被测定转子接负极； E. 将电源正负极正确连接后，通直流电12V1000mA，每次做盐浴前要检查溶液的导电性能，将“+”和“-”极直接浸入盐浴池中，观察电流表的读数，导通电流是否达到标准值：500mA，当电流达到500mA及以上才可测试。 漏电流及针孔数测试标准 电流法测试标准 针孔数测试标准 如针孔数量少于五个且不在同一个部位30米之内，则可以接受，其它情形均不能接受。 击穿电压 取漆包线对折一次，将对折部位剪断并刮去四根线漆皮，然后再对折两次，并将对折后的漆包线扭成麻花状态,扭绞33圈，（两端各有两个线头）分别用高压仪测试同一端的两根线头的耐高压能力。（高压标准：参照附件IEC60317 Ⅱ级标准）。
体外筛选具黄嘌呤氧化酶抑制活性的天然产物近年来,随着人们生活水平的不断提高,痛风的发病率也呈逐渐上升趋势。痛风是由于体内嘌呤代谢紊乱,产生尿酸过多或尿酸排泄过少而导致血中尿酸升高,尿酸盐结晶沉积在组织中引起的反复发作性炎症疾病。 临床上治疗痛风的方法主要包括促进尿酸排泄和抑制尿酸生成。其中,通过抑制黄嘌呤氧化酶活性减少尿酸的生成被认为是治疗痛风的最有效的方法之一。 体内次黄嘌呤可在黄嘌呤氧化酶的催化下生成黄嘌呤,进一步生成尿酸,故抑制黄嘌呤氧化酶的活性可显著降低尿酸的生成。目前临床应用的抗痛风药物的不良反应越来越突出,人们开始倾向于从天然产物中筛选具有抗痛风活性的化学成分。 我国中药资源丰富、中药的化学成分结构多样,作用靶点众多,因此从中药中筛选高效、低毒的黄嘌呤氧化酶抑制剂具有良好的应用前景。黄嘌呤氧化酶抑制剂的体外筛选方法包括紫外分光光度法、电化学法、超高效液相色谱法等,但都存在不同的缺点,因此,本课题拟基于高效液相色谱(HPLC)建立一种简单、快捷、准确的体外筛选方法,并利用建立的方法筛选中药及天然产物中具有黄嘌呤氧化酶抑制活性的先导化合物,为抗痛风新药的研究提供理论依据。 目的建立基于HPLC体外筛选黄嘌呤氧化酶抑制剂的新方法;利用该方法考察具有抗痛风活性中药的黄嘌呤氧化酶抑制活性;选择活性最好的中药作为研究对象进行活性追踪分离,以期获得活性较好的先导化合物。方法通过HPLC测定酶促反应体系中底物黄嘌呤在反应前后含量的变化,建立体外筛选黄嘌呤氧化酶抑制剂的方法。 色谱条件如下:Agilent SB-C18柱,4.6 mm′250 mm,5mm;流动相,0.02