The G Protein-Coupled Receptor GPR30Mediates the Proliferative Effects Induced by 17?-Estradiol and Hydroxytamoxifen in Endometrial Cancer Cells
Adele Vivacqua,*Daniela Bonofiglio,*Anna Grazia Recchia,Anna Maria Musti,Didier Picard,Sebastiano Ando `,and Marcello Maggiolini Departments of Pharmaco-Biology (A.V.,D.B.,A.G.R.,A.M.M.,M.M.)and Cellular Biology (S.A.),University of Calabria,87030Rende (Cosenza),Italy;and Department of Cellular Biology (D.P.),University of Geneva,1211Geneva,Switzerland
The growth of both normal and transformed epi-thelial cells of the female reproductive system is stimulated by estrogens,mainly through the acti-vation of estrogen receptor ?(ER ?),which is a ligand-regulated transcription factor.The selective ER modulator tamoxifen (TAM)has been widely used as an ER antagonist in breast tumor;how-ever,long-term treatment is associated with an increased risk of endometrial cancer.To provide new insights into the potential mechanisms in-volved in the agonistic activity exerted by TAM in the uterus,we evaluated the potential of 4-hy-droxytamoxifen (OHT),the active metabolite of TAM,to transactivate wild-type ER ?and its splice variant expressed in Ishikawa and HEC1A endome-trial tumor cells,respectively.OHT was able to antagonize only the activation of ER ?by 17?-es-tradiol (E2)in Ishikawa cells,whereas it up-regu-lated c-fos expression in a rapid manner similar to E2and independently of ER ?in both cell lines.This stimulation occurred through the G protein-cou-pled receptor named GPR30and required Src-re-lated and epidermal growth factor receptor ty-rosine kinase activities,along with the activation of both ERK1/2and phosphatidylinositol 3-kinase/AKT pathways.Most importantly,OHT,like E2,stimulated the proliferation of Ishikawa as well as HEC1A cells.Transfecting a GPR30antisense ex-pression vector in both endometrial cancer cell lines,OHT was no longer able to induce growth effects,whereas the proliferative response to E2was completely abrogated only in HEC1A cells.Furthermore,in the presence of the inhibitors of MAPK and phosphatidylinositol 3-kinase path-ways,PD 98059and wortmannin,respectively,E2and OHT did not elicit growth stimulation.Our data demonstrate a new mode of action of E2and OHT in endometrial cancer cells,contributing to a better understanding of the molecular mechanisms in-volved in their uterine agonistic activity.(Molecular Endocrinology 20:631–646,2006)
E
STROGENS ARE PLEIOTROPIC hormones that regulate the growth and differentiation of many tissues.Acting as mitogens,they also promote the development of breast and endometrial tumors,which cumulatively account for about 40%of cancer inci-dence among women (1).The classical mechanism of action of 17?-estradiol (E2)(1)involves binding to es-trogen receptors (ER)?and ?,which interact with the
estrogen response elements (EREs)located in the reg-ulatory region of target genes (2,3).It is well known that the ERs contain two main transcription activation functions (AF):the N-terminal AF1and the C-terminal AF2,which is associated with the ligand-binding do-main responsible for hormone-dependent transactiva-tion (4,5).The involvement of both AF1and AF2in ER-mediated transcriptional activity depends on the cell and promoter examined,because in a certain con-text an individual domain functions as the only deter-minant,whereas in other cases AF2acting in combi-nation with AF1elicits the maximal potency in the ER response (4,6,7).
Approximately 50%of all breast cancers express elevated levels of ER ?(8),and prolonged exposure to E2is a major risk factor for breast tumor progression (9).Consequently,ER ?antagonists such as tamoxifen (TAM),ICI 182,780(ICI),and raloxifene have been used in the front-line pharmacological management of ER ?-positive breast cancers to inhibit the mitogenic action of E2(10–14).Although originally considered an antiestrogen,TAM is now classified as a selective ER modulator because in the breast it functions as an
First Published Online October 20,2005
*A.V.and D.B.contributed equally to this work.
Abbreviations:AF,Activation function;AP1,activating pro-tein 1;AS-ODN,antisense oligonucleotide;Cx,cyclohexi-mide;DEX,dexamethasone;DHT,5?-dihydrotestosterone;DN,dominant negative;E2,17?-estradiol;EGF,epidermal growth factor;EGFR,epidermal growth factor receptor;ER,estrogen receptor;ERE,estrogen-responsive element;FBS,fetal bovine serum;GPCR,G protein-coupled receptor;ICI,ICI 182,780;OHT,hydroxytamoxifen;PD,PD 98059;PI3K,phosphatidylinositol 3-kinase;PR,progesterone receptor;PRG,progesterone;PT,pertussis toxin;SRE,serum re-sponse element;TAM,tamoxifen;WM,wortmannin.
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Copyright ?2006by The Endocrine Society
doi:10.1210/me.2005-0280
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antagonist,whereas in the uterus,bone,and the car-diovascular system it displays agonistic activity(15, 16).Furthermore,TAM can induce uterine cell growth in vivo in animal models(17)as well as in humans(18, 19).Also,the active metabolite of TAM,4-hydroxyta-moxifen(OHT),stimulates the proliferation of both hu-man endometria(20,21)and cultured human endo-metrial carcinoma cells(21,22).Many molecular mechanisms have been proposed to explain this phe-nomenon;however,it still remains to be completely elucidated.
In normal and neoplastic uterine tissue,the expres-sion of full-length ERa mRNA is generally greater than the expression of a given splice variant(23,24).Oc-casionally,ER?mRNA splice variants are translated into proteins that may act either as dominant-positive or dominant-negative(DN)vs.the wild-type ER?(25). In addition,an altered expression of ER coregulators has been associated with the progression of breast cancers toward a more malignant and TAM-resistant phenotype(26).The involvement of coactivators to promote TAM agonism may represent a relevant hy-pothesis;however,alteration of their expression pat-terns did not parallel the development of endometrial malignancy(27).The tissue-specific effects of TAM may follow the activation of cellular signaling path-ways,including ER?phosphorylation at both serine 118and167,as well as the MAPK kinase kinase 1-dependent increase in TAM agonistic activity(28).
A large number of studies have also been performed toward the identification of membrane-associated es-trogen signals that may alter gene expression inde-pendently of the nuclear ERs.Intriguingly,recent re-ports,including our own(29–31),have described the estrogen-binding and activation properties of a G pro-tein-coupled receptor(GPCR)named GPR30,which has therefore been proposed as a candidate for trig-gering a broad range of rapid E2activity at the plasma membrane level.In this respect,we have demon-strated that in breast cancer cells lacking ERs,GPR30 mediates up-regulation of the c-fos protooncogene by either E2or estrogen-like compounds,such as the phytoestrogens genistein and quercetin(29).c-fos is a classical early gene whose expression is rapidly and transiently induced by different extracellular stimuli, including mitogens and hormones(32–38).The nu-clear protein encoded by c-fos interacts with c-jun family members to form the heterodimeric activating protein1(AP1)transcription factor(39–42),which reg-ulates a series of genes as well as interacting proteins (43–48).The transcription of c-fos is controlled by multiple cis elements,such as the cAMP response element(49),the signal transducers and activators of transcription group of transcription factors(50),and the serum response element(SRE),which recruits the ternary complex factors,including Elk-1and serum-response factor accessory proteins1and2(51–55). As it concerns ER?,binding to the imperfect palin-dromic ERE sequence within the c-fos promoter is not sufficient for transactivation because it requires inter-action of ER?with a downstream Sp1site(56).Be-sides,it has been reported that E2signaling may con-verge on the SRE target sequence through a nongenomic ER?pathway leading to MAPK-depen-dent phosphorylation and binding of Elk-1to the SRE sequence(57).
In the present study we show that E2and OHT up-regulate c-fos expression in a rapid manner through the membrane-associated GPR30protein in Ishikawa and HEC1A human endometrial cancer cells. Our results provide new insights into the molecular mechanisms by which OHT exerts a growth stimula-tory action in these uterine tumor cells.
RESULTS
E2,But Not OHT,Transactivates and Down-Regulates ER?in Ishikawa Cells
Considering the controversies regarding the ability of E2and OHT to activate the endogenous and/or over-expressed ER?in Ishikawa cells(25,58–60),we first examined whether a transiently transfected ER re-porter gene responds to both ER ligands.E2induced a dose-dependent luciferase expression,whereas OHT as well as ICI were not able to activate ER?in the concentration range used(Fig.1A).However,10?M of both ER antagonists repressed the luciferase activity elicited by100n M E2(Fig.1B).An additional hallmark of ER?activation by an agonist is represented by reduction of its expression consequent to the high turnover of the E2-activated receptor protein and the transcription inhibition of its own gene(61).Hence,we investigated whether the levels of ER?contained in Ishikawa cells could be regulated by E2and OHT. Western analysis of ER?performed with an anti-ER?antibody raised against the C-terminus region recog-nized a protein corresponding to the size of wild-type ER?(66kDa;Fig.1,C and E).In addition,the ER?antibody recognized a protein with a smaller size(?48 kDa;Fig.1,C and E),which was no longer detected using an anti-ER?antibody raised against the N-ter-minus domain(data not shown).Short treatments did not modify the levels of ER?and its variant(Fig.1,C and D),whereas a12-h exposure to E2markedly re-duced the content of wild-type ER?(Fig.1,E and F). As it concerns ER?expression,no changes were ob-served in either of the ligands used(Fig.1,C and D, and E and F).Overall,these findings indicate that in Ishikawa cells the transcriptional activation of endog-enous ER?gene is dependent on E2,whereas OHT exerts an antagonist activity.
E2and OHT Neither Transactivate Nor Regulate the ER?Splice Variant Expressed in HEC1A Next,we evaluated the potential transcriptional effects of E2and OHT in HEC1A human endometrial cancer cells,because ER expression and E2responsiveness
632Mol Endocrinol,March2006,20(3):631–646Vivacqua et al.?GPR30Function in Endometrial Cancer Cells
are still controversial in these cells (62–67).In agree-ment with previous reports (63,65,67),we did not detect any ERE-mediated transcriptional activity on E2,OHT,or ICI in the concentration range used (Fig.2A).Furthermore,E2and OHT had no effect on the protein levels of ER ?or the ER ?splice variant
con-
Fig.1.E2,But Not OHT,Activates and Down-Regulates Wild-Type ER ?in Ishikawa Cells
Ishikawa cells were transfected with the luciferase reporter plasmid XETL and treated with increasing concentrations of E2,OHT,and ICI (A)or with 100n M E2,10?M OHT,and ICI alone or in combination as indicated (B).The luciferase activities were normalized to the internal transfection control,and values of untreated cells (?)were set as 1-fold induction upon which the activity induced by treatments was calculated.Each data point represents the mean ?SD of three independent experiments performed in triplicate.Ⅺ,P ?0.05for untreated (?)vs.E2-treated cells;f ,P ?0.05for E2-treated cells vs.those treated with E2and OHT or ICI.C and E,Immunoblots of ER ?and ER ?from Ishikawa cells treated with 1?M E2or OHT for the indicated times.?-Actin serves as a loading control.D and F,Quantitative representations of data (mean ?SD )from three independent experiments including those in C and E,respectively.Ⅺ,P ?0.05for untreated (?)vs.E2-treated cells.
Vivacqua et al.?GPR30Function in Endometrial Cancer Cells Mol Endocrinol,March 2006,20(3):631–646633
tained in HEC1A cells (Fig.2,B and C,and D and E).Collectively,these results suggest that the ER ?splice variant expressed in the endometrial cancer cells used is not able to induce ERE-mediated transcriptional activity or to act as a DN form of wild-type ER ?.E2and OHT Transactivate c-fos Promoter Constructs in Ishikawa Cells
Having established that OHT acts as an antagonist of the classical ER ?-ERE pathway in Ishikawa cancer cells,we examined its ability to activate a transiently transfected full-length human c-fos promoter (?2.2kb),which contains several target sequences re-sponding to a variety of extracellular stimuli (32–38).Interestingly,E2and OHT,but not ICI,were able to transactivate c-fos in an ERE-independent manner,because an ERE deletion mutant (?1172bp)still elic-ited the transcriptional response (Fig.3A).The ternary complex factor member Elk-1is crucial for the ERK-dependent activation of the c-fos gene promoter
(52);
Fig.2.The Splice Variant of ER ?Expressed in HEC1A Cells Is Neither Activated Nor Modulated by E2and OHT
A,HEC1A cells were transfected with the luciferase reporter plasmid XETL and treated with increasing concentrations of E2,OHT,and ICI.The luciferase activities were normalized to the internal transfection control,and values of untreated cells were set at 1-fold induction upon which the activity induced by treatments was calculated.Each data point represents the mean ?SD of three independent experiments performed in triplicate.B and D,Immunoblots of ER ?and ER ?from HEC1A cells treated with 1?M E2or OHT for the indicated times.?-Actin served as a loading control.C and E,Quantitative representations of data (mean ?SD )from three independent experiments including those in B and D,respectively.
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therefore,we investigated the effects of E2and OHT on Elk-1-mediated transcriptional activity.As shown in Fig.3B,both E2and OHT induced Elk-1transactiva-tion,which was abrogated by the MAPK kinase-spe-cific inhibitor PD 98059(PD)and or the phosphatidyl-inositol 3-kinase (PI3K)inhibitor wortmannin (WM).In addition,Elk1activity was no longer noticeably over-expressing the oncosuppressor protein menin,previ-
ously shown by us to inhibit the ERK-dependent acti-vation of Elk-1(68).Hence,distinct signal transduction pathways converge to regulate c-fos expression,which could be considered an additional sensor of estrogen signaling.
E2and OHT Rapidly Induce c-fos mRNA Expression in Ishikawa Cells
It is well established that c-fos gene expression is promptly stimulated by a variety of extracellular sig-nals,including E2(29,60,69).To investigate whether OHT is also able to induce in a rapid manner the expression of c-fos or two estrogen target genes such as the well known progesterone (PRG)receptor (PR)and pS2(70),we performed semiquantitative RT-PCR experiments comparing the mRNA levels after stan-dardization on a housekeeping gene encoding the ri-bosomal protein 36B4.A short exposure (1h)of Ish-ikawa cells to E2and OHT up-regulated c-fos expression (Fig.4,A and B),which was no longer noticeable after 12h of treatment (Fig.4,C and D).
The
Fig.3.Transcriptional Activation of c-fos and Gal4-Elk1Re-porters by E2and OHT in Ishikawa Cells
A,The luciferase reporter plasmid c-fos encoding a ?2.2-kb-long upstream region of human c-fos as well as the de-letion mutant c-fos ?ERE lacking the ERE sequence and en-coding a ?1172-bp upstream fragment of human c-fos are activated by 1?M E2and OHT in Ishikawa cells.B,The luciferase reporter plasmid for the fusion protein consisting of Elk1and the Gal4DNA binding domain is activated by 1?M E2and OHT in Ishikawa cells.A concentration of 10?M PD,10?M WM,and the expression vector for the menin gene reversed the response.The luciferase activities were stan-dardized to the internal transfection control,and values of untreated cells (?)were set at 1-fold induction upon which the activity induced by treatments was calculated.Each data point represents the mean ?SD of three independent exper-iments performed in triplicate.Ⅺand f ,P ?0.05for un-treated cells (?)vs.
treatments.
Fig.4.Rapid Induction of c-fos mRNA by E2and OHT in Ishikawa Cells
A and C,c-fos ,pS2,and PR semiquantitative RT-PCR from Ishikawa cells treated with 1?M E2and OHT for the indicated times;36B4mRNA levels were determined as a control.
B and D,Quantitative representation of data (mean ?SD )from three independent experiments after densitometry and correction for 36B4expression including those of A and C,respectively.Ⅺ,f ,and E ,P ?0.05for untreated cells (?)vs.treatments.
Vivacqua et al.?GPR30Function in Endometrial Cancer Cells Mol Endocrinol,March 2006,20(3):631–646635
latter time was also required for E2to increase the mRNA levels of pS2and PR and for OHT to enhance PR levels(Fig.4,C and D).These results indicate that in Ishikawa cells,exposure time and ligand specificity regulate the expression of estrogen target genes.
E2and OHT Up-Regulate c-fos Protein Levels in Ishikawa and HEC1A Cells
To ascertain the transduction pathways involved in c-fos regulation by E2and OHT,we first investigated whether c-fos protein expression mimics the mRNA accumulation upon these ligands.In Ishikawa cells,a 2-h exposure to E2and OHT up-regulated c-fos ex-pression,which was no longer noticeable after12h of treatment(Fig.5,A and B).Next,we analyzed c-fos protein levels after treating cells for2h with E2and OHT in the presence of specific inhibitors of distinct metabolic cascades.The protein synthesis inhibitor cycloheximide(Cx)abolished the effects of both li-gands(Fig.5C),indicating that the up-regulation of c-fos is strictly dependent on new protein synthesis. ICI slightly inhibited the increase in c-fos after E2and OHT treatment(Fig.5D),whereas the MAPK inhibitor PD(Fig.5E)and the ectopically expressed DN form of the ERK protein(Fig.5F)completely blocked c-fos induction.The latter effect was also obtained using the Src family tyrosine kinase inhibitor PP2(AG1879)(Fig. 5G),the epidermal growth factor(EGF)receptor (EGFR)kinase inhibitor tyrphostin AG1478(Fig.5H), the PI3K inhibitor WM(Fig.5I),and the GPCR inhibitor pertussis toxin(PT;Fig.5J).On the contrary,the pro-tein kinase A inhibitor H-89did not repress the c-fos response to E2and OHT at either the mRNA or protein level(data not shown).
To evaluate these results in a different cellular con-text,we analyzed HEC1A endometrial cancer cells, which contain only an ER?splice variant,as docu-mented in the present study(Fig.2,B and D).Also in HEC1A cells,E2and OHT rapidly(2h)up-regulated c-fos protein levels(Fig.6,A and B),which were de-pendent on new protein synthesis(Fig.6C)and were not altered in presence of ICI(Fig.6D).The c-fos response displayed the same sensitivity to inhibitors used in Ishikawa cells(Fig.6,E–J).The steroids dexa-methasone(DEX),PRG,and5?-dihydrotestosterone (DHT)did not enhance c-fos content(Fig.6K),as also observed after17?-estradiol treatment(data not shown),indicating that a ligand specificity is required for the rapid response observed.Cumulatively,our data suggest that diverse transduction pathways are triggered by E2and OHT to regulate c-fos expression in endometrial cancer cells.
GPR30and MAPK Activation Mediate the
Up-Regulation of c-fos by E2and OHT in Ishikawa and HEC1A Cells
On the basis of the results described above and con-sidering that diverse extracellular factors signal through GPCRs and result in ERK1/2activation(71–73),we investigated the involvement of this transduc-tion pathway in c-fos regulation by E2and OHT in Ishikawa and HEC1A cells.As shown in Fig.7,A and B,and E and F,in both endometrial cancer cell lines a specific GPR30antisense oligonucleotide(GPR30/ AS-ODN)abrogated the response to the ligands used, whereas a control scrambled oligonucleotide had no effect.GPR30/AS-ODN efficiently silenced GPR30 protein expression(Fig.7,C and G),whereas ER?levels(Fig.7,D and H)as well as those of PR(data not shown)were not altered.Interestingly,in human cer-vical HeLa cancer cells,which express very low levels of GPR30(Fig.8C),both ligands failed to up-regulate c-fos expression.However,c-fos was induced by E2-and OHT-transfecting cells with a plasmid encoding GPR30,but not in control cells transfected with an empty vector(Fig.8).Moreover,HeLa cells engineered to overexpress GPR30were growth stimulated by the treatments,although not in a significant manner com-pared to control cells(data not shown).
Because GPCR signals result in ERK1/2activation (71–73),and in the present study both PD and DN/ERK inhibited c-fos induction(Figs.5and6),we evaluated MAPK activation in Ishikawa and HEC1A cells.A5-min treatment with E2and OHT induced ERK1/2phos-phorylation without changes in total ERK2(Fig.9), indicating that both ligands signal through the MAPK pathway in the endometrial cancer cells used.
E2and OHT Stimulate the Proliferation of Ishikawa and HEC1A Cells
These findings were also evaluated in a more complex physiological response such as cell proliferation.Of note,either E2or OHT induced growth effects in Ish-ikawa and HEC1A cells(Fig.10A).However,transfect-ing a GPR30/AS expression vector,the stimulatory action of OHT was no longer noticeable in either cell line,whereas the proliferation produced by E2was abrogated completely in HEC1A cells and partially in Ishikawa cells(Fig.10,B–D).Finally,the proliferative effects elicited by E2or OHT were abrogated in both endometrial cancer cell lines in presence of the MAPK inhibitor PD and the PI3K inhibitor WM(Fig.10E). Hence,cell growth assays confirmed the involvement of GPR30and the transduction pathways studied in mediating the stimulatory action of both ligands in Ishikawa and HEC1A cells.
DISCUSSION
It has been largely documented that treatment with TAM,widely used in women to prevent breast cancer recurrence,is associated with an increased incidence of proliferative and neoplastic endometrial changes (74).In this regard,previous studies have demon-strated that TAM is converted into reactive species
636Mol Endocrinol,March2006,20(3):631–646Vivacqua et al.?GPR30Function in Endometrial Cancer Cells
Fig.5.Immunoblots of c-fos from Ishikawa Cells
Ishikawa cells were treated for the indicated times with 1m M E2and OHT (A and B)and in combination with 50?M protein synthesis inhibitor Cx (C),1m M ICI (D),10?M of the MAPK inhibitor PD (E),the expression vector for the DN/ERK2(F),10?M of the Src family tyrosine kinase inhibitor PP2(G),10?M of the EGFR kinase inhibitor tyrphostin AG 1478(H),10?M of the PI3K inhibitor WM (I),and 100ng/ml of the G protein inhibitor PT (J).?-Actin was used as a loading control.The side panels show the quantitative representations of data (mean ?SD )from three independent experiments performed for each condition.f ,P ?0.05for untreated cells (?)vs.treatments.
Vivacqua et al.?GPR30Function in Endometrial Cancer Cells Mol Endocrinol,March 2006,20(3):631–646637
Fig.6.Immunoblots of c-fos from HEC1A Cells
HEC1A cells were treated for the indicated times with 1?M E2and OHT (A and B)and in combination with 50?M of the protein synthesis inhibitor Cx (C),1m M ICI (D),10?M of the MAPK inhibitor PD (E),the expression vector for the DN/ERK2(F),10?M of the Src family tyrosine kinase inhibitor PP2(G),10?M of the EGFR kinase inhibitor tyrphostin AG 1478(H),10?M of the PI3K inhibitor WM (I),and 100ng/ml of the G protein inhibitor PT (J).Besides,cells were treated with 1?M DEX,1?M PRG and 1?M DHT (K).?-actin was used as a loading control.The side panels show the quantitative representations of data (mean ?SD )from three independent experiments performed for each condition.f ,P ?0.05for untreated cells (?)vs.treatments.
638Mol Endocrinol,March 2006,20(3):631–646Vivacqua et al.?GPR30Function in Endometrial Cancer Cells
forming DNA adducts,leading,in turn,to endometrial carcinogenesis (75–78).As it concerns the mechanism through which TAM acts as an estrogen agonist in the uterus,it has been suggested that both the AF1do-main of ER ?(62)as well as cell-and promoter-specific factors may be involved (28,79,80).Furthermore,it has been shown that coregulator proteins facilitating ER ?interaction with the general transcriptional
ma-
Fig.7.c-fos Induction by E2and OHT Is Abrogated by Transfecting Endometrial Cancer Cells with a GPR30AS-ODN Oligonucleotide
Ishikawa (A)and HEC1A (E)cells transfected with control scrambled (CS-ODN)or GPR30/AS-ODN oligonucleotides were treated with 1?M E2and OHT.B and F,Quantitative representation of data (mean ?SD )from three independent experiments including those in A and E,respectively.GPR30and ER ?protein expression in Ishikawa (C and D,respectively)and in HEC1A (G and H,respectively)cells transfected with CS-ODN or GPR30/AS-ODN.?-Actin was used as a loading control.f .P ?0.05for untreated cells (?)vs.treatments.
Vivacqua et al.?GPR30Function in Endometrial Cancer Cells Mol Endocrinol,March 2006,20(3):631–646639
chinery and chromatin could be responsible for the differential ability of partial agonists/antagonists to modify gene expression (81,82).
Considering that recent studies have revealed a functional interaction between either TAM or E2with GPR30in triggering rapid cellular responses to estro-gen signals at the plasma membrane level (29–31),in the present study we have evaluated and demon-strated that in endometrial cancer cells,TAM retains the ability to antagonize ER ?-dependent signaling,yet acquires that to induce c-fos expression and cell pro-liferation in a GPR30-mediated fashion.Agents able to block G protein and GPR30signaling,such as PT and the GPR30/AS-ODN,both prevented the enhance-ment of c -fos in a similar manner as the specific in-hibitors of the EGFR,Src,PI3K,and MAPK pathways (Figs.5and 6).Given that EGFR tyrosine kinase ac-tivity is required for GPR30-dependent ERK1/2acti-vation (29,71),our results suggest novel molecular mechanisms that may serve as a unique signaling prototype leading to gene expression in various cell
types.It is worth noting that our findings recall previ-ous observations indicating the existence of sequen-tial and even reciprocal cross-talk among the above-mentioned transduction pathways in diverse cellular contexts (83–85).For instance,in hepatocyte growth factor-treated myogenic cells,the specific PI3K inhib-itor WM abolished MAPK/ERK and Elk-1phosphory-lations,which,in turn,stimulated cell proliferation (86).The mechanistic differences among antiestrogens have already revealed the complex molecular pharma-cology of ER ?,which is now paralleled by the mem-brane estrogen-binding protein GPR30.Although E2and the ER antagonists TAM and ICI bind to GPR30directly (31),in our previous investigation we demon-strated that GPR30mediates c-fos expression only after E2treatment in breast cancer cells (29)and also after OHT treatment in endometrial tumor cells as shown in the present study.Collectively,our observa-tions suggest that GPR30function may be dependent on the cellular context and the specific binder used.In contrast,TAM acts differentially on GPR30-and ER ?-mediated signals,and it even activates GPR30in a spatially different manner from E2(31).Interestingly,the above-mentioned findings were recapitulated in a complex physiological response such as proliferation of Ishikawa and HEC1A cells engineered to abolish GPR30expression.E2can induce growth effects through at least two diverse mechanisms:one
involv-
Fig.8.c-fos Is Up-Regulated by E2and OHT in HeLa Cells Transfected with a Plasmid Encoding GPR30
HeLa cells were transfected in 10-cm dishes with 5?g empty vector (v;A)or 5?g expression plasmid encoding GPCR30(pGPR30;B)and were treated with 1?M E2and OHT.The side panels are the quantitative representation of data (mean ?SD )from three independent experiments in-cluding those in A and B,respectively.C,GPR30protein expression in HeLa cells transfected with an empty vector (v)or pGPR30.?-Actin was used as a loading control.f ,P ?0.05for untreated cells (-)vs.
treatments.
Fig.9.ERK1/2Phosphorylation in Endometrial Cancer Cells Ishikawa (A)and HEC1A (B)cells were treated for 5min with 1?M E2and OHT.Total ERK2proteins were used to normalize ERK1/2expression.The side panels show the quantitative representations of data (mean ?SD )from three independent experiments including those in A and B,respec-tively.Ⅺand f ,P ?0.05for untreated cells (?)vs.treat-ments.
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ing the wild-type ER ?and a second involving the GPR30-MAPK pathway,which are crucial for the OHT-induced effects or those exerted by E2in an ER ?-independent manner.
To provide new criteria for the selection of thera-peutic interventions in endocrine-related disorders,the potential of distinct selective ER modulators to signal through GPR30should be investigated at the level of distinct tissues.For instance,the widespread use of raloxifene in the prevention-therapy of osteo-porosis in postmenopausal women should be carefully considered,because it could act to prevent or pro-mote the development of estrogen-sensitive tumors.This scenario is particularly poignant,because the beneficial effects of raloxifene on osteoporosis re-quires continuous treatment for decades.In this con-text,it should be taken into account that abundant phytoestrogens,such as genistein and quercetin,in-duce gene expression by both ER ?as well as GPR30(29,87).The environmental contaminant ortho,para -dichlorodiphenyldichoroethylene activates ER ?(88)and binds to GPR30(31).Hence,estrogen signaling has become increasingly complex,showing multi-faceted biological actions that may have profound implications in the pathophysiology of breast,endo-metrial,ovarian,placenta,prostate,neural,and vas-cular tissues.
As mentioned above,controversial results have been reported on the ability of antiestrogens to stim-ulate transcription in endometrial cancer cells through promoters containing EREs or AP1-binding sites (12-O-tetradecanoyl phorbol 13-acetate response ele-ments)regardless of ER content and the expression of splice variants (24–25,58–60,64,89).Using two hu-man-derived endometrial tumor cells that present im-portant differences in ER ?expression,we ascertained the ability of E2and OHT to transactivate distinct reporter constructs as well as to induce gene expres-
untreated cells was set at 100%upon which cell growth induced by treatments was calculated.Each data point is the mean ?SD of three independent experiments performed in triplicate.B,Ishikawa and HEC1A cells were transfected with an empty vector (v)or an expression vector for GPR30/AS (see Materials and Methods ),treated as described in A,then counted on d 6.Proliferation of untransfected and untreated cells was set at 100%upon which cell growth induced by treatments in presence of the empty vector or the expression vector for GPR30/AS was calculated.Each data point is the mean ?SD of three independent experiments performed in triplicate.GPR30protein expression was determined in Ish-ikawa (C)and HEC1A (D)cells transfected with an empty vector (v)or GPR30/AS.?-Actin was used as a loading con-trol.E,Ishikawa and HEC1A cells were treated with 1?M E2and OHT or in combination with 10?M of the MAPK inhibitor PD and 10?M of the PI3K inhibitor WM and counted on d 6.Proliferation of untreated cells was set at 100%upon which cell growth induced by treatments was calculated.Each data point is the mean ?SD of three independent experiments performed in
triplicate.
Fig.10.E2and OHT Stimulate the Proliferation of Endome-trial Cancer Cells
A,Ishikawa and HEC1A cells were treated with 1?M E2and OHT in medium containing 5%charcoal-stripped FBS (medium was refreshed,and treatments were renewed every 2d),then counted on the indicated day.Proliferation of
Vivacqua et al.?GPR30Function in Endometrial Cancer Cells Mol Endocrinol,March 2006,20(3):631–646641
sion.In line with the findings of others(60,89,90), prolonged exposure to these treatments was required to up-regulate pS2and PR,whereas c-fos was rapidly enhanced,suggesting that independent mechanisms are involved in such genomic responses.
Notably,the data we report suggest that c-fos in-duction is a GPR30-ERK1/2-dependent transcrip-tional effect that involves Elk-1signaling to c-fos pro-moter,as also demonstrated in breast cancer cells(29, 57).Indeed,our model system,designed to highlight the transduction pathways contributing to such mo-lecular events in endometrial cancer cells,has outlined the complex cascade described in Fig.11.
c-fos belongs to a family of transcription factors that,alone and/or after heterodimerization with jun family members generates the AP1transcription fac-tor,binds to cognate sites at the regulatory sequences of genes involved in relevant cellular functions such as proliferation(32–48).Interestingly,c-fos was shown to be a transcriptional regulator of cyclin D1(91),which translocates from the cytoplasm to the nucleus in the mouse uterine epithelium undergoing OHT-induced cell cycle progression(92).Hence,the up-regulation of c-fos by extracellular mitogens and growth factors could represent a unique early molecular sensor to evaluate the genomic responses leading to growth stimulation in endometrial cancer cells.
One major concern with TAM therapy for breast cancer is the tumorigenic action in the endometrium. The present study provides new insights into the mo-lecular mechanisms by which TAM may elicit unde-sired side effects that if selectively abrogated could endorse the benefits of long-term treatment alongside its prophylactic use in breast tumor disease. MATERIALS AND METHODS
Reagents
E2,OHT,Cx,WM,LY294002,PT,PD,DEX,PRG,and DHT were purchased from Sigma-Aldrich Corp.(Italy).ICI was obtained from Tocris Chemicals(Bristol,UK),tyrphostin AG 1478(AG1478)was purchased from Biomol Research Lab-oratories,Inc.(Milan,Italy),and H-89and PP2were obtained from Calbiochem(VWR International,Milan,Italy).All com-pounds were solubilized in dimethylsulfoxide,except E2, OHT,PD,and WM,which were dissolved in ethanol. Plasmids
The firefly luciferase reporter plasmid for ERs was EREtkLuc (also called XETL)(29),which presents the following features: the ERE from the Xenopus vitellogenin A2gene(nucleotides ?334to?289),the herpes simplex virus thymidine kinase
promoter region(nucleotides?109to52),the firefly lucif-erase coding sequence,and the simian virus40splice and polyadenylation sites from plasmid pSV232A/L-AA5?.Re-porter plasmids for c-fos and its deletion mutant c-fos DERE (which lacks the ERE sequence)encode?2.2and?1.172kb 5?upstream fragments of human c-fos,respectively(gifts from K.Nose,Showa University,Tokyo,Japan)(93).The reporter plasmid Gal4-luc was described together with the expression vectors for Gal4-Elk1and menin in our previous study(68).The plasmids encoding GPR30(pGPR30),GPR30/ AS,and DN ERK2were provided by R.Weigel(Thomas Jefferson University,Philadelphia,PA),E.R.Prossnitz(Uni-versity of Albuquerque,Las Cruces,NM),and M.Cobb(Uni-versity of Texas,Dallas,TX),respectively.The Renilla lucif-erase expression vector pRL-thymidine kinase(Promega Corp.,Milan,Italy)was used as a transfection standard. Cell Culture
Ishikawa human endometrial cancer cells were obtained from D.Picard(University of Geneva,Geneva,Switzerland)and HEC1A human endometrial cancer cells were a gift from A. Peri(University of Florence,Florence,Italy),respectively. HeLa human cervical cancer cells were purchased from American Type Culture Collection(Manassas,VA).Ishikawa and HeLa cells were maintained in DMEM without phenol red supplemented with10%fetal bovine serum(FBS;Invitrogen Life Technologies,Inc.,Milan,Italy),whereas HEC1-A cells were maintained in RPMI1640without phenol red supple-mented with10%FBS(Invitrogen Life Technologies,Inc.). Cells were switched to medium without serum48h before RT-PCR,immunoblots,and evaluation of ERK1/2 phosphorylation.
Transfections and Luciferase Assays
A total of100,000Ishikawa or HEC1A cells were plated into 24-well dishes with500?l regular growth medium/well the day before transfection.The medium was replaced with that lacking serum on the day of transfection,which was per-formed using FuGene6reagent as recommended by the manufacturer(Roche,Indianapolis,IN)with a mixture con-taining0.5?g reporter plasmid,0.1?g effector plasmid where applicable,and2ng pRL-thymidine kinase.After4h, we renewed the serum-free medium containing the indicated treatment,and then cells were incubated for about12
h. Fig.11.Model of the Potential Signal Transduction Path-ways Triggered by E2and OHT for c-fos Expression in En-dometrial Cancer Cells
SRF,Serum-response factor.
642Mol Endocrinol,March2006,20(3):631–646Vivacqua et al.?GPR30Function in Endometrial Cancer Cells
Luciferase activity was measured with the dual luciferase kit (Promega Corp.)according to the manufacturer’s recommen-dations.Firefly luciferase values were normalized to the in-ternal transfection control provided by the Renilla luciferase activity.The normalized relative light unit values obtained from untreated cells were set at1-fold induction,upon which the activity induced by treatments was calculated.
RT-PCR
The evaluation of gene expression was performed by semi-quantitative RT-PCR as we have previously described(94). For c-fos,pS2,PR,and the acidic ribosomal phosphoprotein P0(36B4),which was used as a control gene,the primers were,respectively,5?-AGAAAAGGAGAATCCGAAGGGAAA-3?(c-fos forward),5?-ATGATGCTGGGACAGGAAGTC-3?(c-fos reverse),5?-TTCTATCCTAATACCATCGACG-3?(pS2forwa-rd),5?-TTTGAGTAGTCAAAGTCAGAGC-3?(pS2reverse),5?-TAGTGAGGGGGCAGTGGAAC-3?(PR forward),5?-AGGAG-GGGGTTTCGGGAATA-3?(PR reverse),5?-CTCAACATCTC-CCCCTTCTC-3?(36B4forward),and5?-CAAATCCCATATC-CTCGTCC-3?(36B4reverse),to yield products of420,210, 442,and408bp,respectively,with20,15,20,and15PCR cycles.
Western Blotting
Cells were grown in10-cm dishes,exposed to ligands,and then lysed in500?l50m M HEPES(pH7.5),150m M NaCl,1.5
m M MgCl
2,1m M EGTA,10%glycerol,1%Triton X-100,1%
sodium dodecyl sulfate,and a mixture of protease inhibitors
containing1m M aprotinin,20m M phenylmethylsulfonylfluo-
ride,and200m M sodium orthovanadate.The protein con-
centration was determined using Bradford reagent according
to the manufacturer’s recommendations(Sigma-Aldrich
Corp.).Equal amounts of whole protein extract were resolved
on a10%sodium dodecyl sulfate-polyacrylamide gel,trans-
ferred to a nitrocellulose membrane(Amersham Biosciences,
Arlington Heights,IL),probed overnight at4C with the anti-
bodies against ER?(F-10and D-12),c-fos(D-1),PR(B-30),?-actin(all purchased from Santa Cruz Biotechnology,Inc., Milan,Italy),ER?and GPR30(Serotec and Lifespan Bio-sciences,respectively,Milan,Italy),pERK1/2and ERK2(Cell
Signaling Technology,Inc.,and Cellbio,Milan,Italy)and then
revealed using the enhanced chemiluminescence system
(Amersham Biosciences).Five micrograms of DN/ERK ex-
pression plasmid was transfected using FuGene6reagent as
recommended by the manufacturer(Roche)for24h before
treatments.
AS-ODN Experiments
AS-ODNs were purchased from MWG(Florence,Italy)and
synthesized as previously described(95,96).The ODNs used
were5?-TTGGGAAGTCACATCCAT-3?for GPR30and5?-
GATCTCAGCACGGCAAAT-3?for the scrambled control.For
AS experiments,a concentration of200n M of the indicated
ODNs was transfected using FuGene6reagent as recom-
mended by the manufacturer(Roche)for4h,before treat-
ment with ligands.
Proliferation Assays
For quantitative proliferation assays,10,000cells were
seeded in24-well plates in regular growth medium.Cells
were washed extensively once they had attached and then
were incubated in medium containing5%charcoal-stripped
FBS with the indicated treatments;medium was renewed
every2d(with treatments),and cells were trypsinized and
counted in a hemocytometer.Two hundred nanograms of GPR30/AS or200ng empty vector was transfected using FuGene6reagent as recommended by the manufacturer (Roche)every2d.
Statistical Analysis
Statistical analysis was performed using ANOVA,followed by Newman-Keuls testing to determine differences in means. P?0.05was considered statistically significant.
Acknowledgments
Received July8,2005.Accepted October11,2005.
Address all correspondence and requests for reprints to: Dr.Marcello Maggiolini,Department of Pharmaco-Biology, University of Calabria,87030Rende(CS),Italy.E-mail: marcellomaggiolini@yahoo.it.
This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro,the Ministero dell’Universita`e Ricerca Scientifica e Tecnologica,the Re-gione Calabria and Swiss National Science Foundation, Krebsforschung Schweiz,and the Canton of Gene`ve(to D.P.).
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添加本地打印机与共享网络上的打印机 一.如何添加本地打印机 *.为确保安装过程正常，请关闭杀毒软件和防火墙 *.在安装过程中如果系统出现“驱动程序没有通过徽标测试……”的提示框，选择‘仍然继续’即可。请放心，这没有任何问题，不会对系统造成任何问题。只要选择“仍然继续”，系统就会开始正常的复制驱动程序安装文件。 *.下载相应驱动 1.点击电脑屏幕右下角的“开始”菜单\“设置”\“控制面板”\“打印机”，会打开“打印机和传 真”文件夹； 2.在“打印机和传真”文件夹中，双击“添加打印机”，进入“添加打印机向导”，点击“下一步”； 3.选择“连接到这台计算机的本地打印机”，并且将“自动检测并安装我的即插即用打印机”前面的 勾去掉，点击“下一步”； 4.在“使用以下端口”中选择“LPT1：打印机端口”(根据实际情况选择)，点击“下一步”； 5.请注意，此时一定要选择“从磁盘安装”； 6.选择“浏览”，然后选择到下载的驱动程序解压所在的文件夹中，然后选择“确定”； 7.此时，在“打印机”列表中，会自动显示出来打印机的型号，点击“下一步”继续安装驱动； 8.在“打印机名”中，不用修改打印机的名称，使用默认的名称即可；同时，可以根据自己的需要选 择是否将打印机设置成为默认打印机（推荐选择“是”）。然后，点击“下一步”； 9.选择“不共享这台打印机”，点击“下一步”； 10.根据自己的需要选择是否“要打印测试页”，点击“下一步”； 11.点击“下一步”，将会出现“正在完成添加打印机向导”的提示界面。此时，选择“完成”。 本地打印机添加成功。 二．如何添加网络打印机（6楼阅览室的打印机59.77.57.163） ?首先准备好相关打印机驱动程序及记住所安装打印机的IP地址 ?下面演示的是网络打印机HP Lj4250（IP地址：59.77.57.163 主楼3#607资料室）的驱动程序下载及 安装过程（其他安装步骤类同，不同之处在于IP地址。 ?主楼3#605HPLj5100 IP地址：59.77.57.164 ?人文楼B#308 HP LJ4250 IP地址：59.77.49.2 第一步：下载驱动并保存驱动程序 学院有提供该驱动程序位置，下载地址： HP https://www.doczj.com/doc/521099368.html,/public/net/lj4250.exe https://www.doczj.com/doc/521099368.html,/public/net/lj5100.exe 单击下载地址会弹出如下对话框，选择［运行］--［运行］
1、圆锥破碎机工作原理 圆锥破碎机工作时，电动机的旋转通过皮带轮或联轴器、圆锥破碎机传动轴和圆锥破碎机圆锥部在偏心套的迫动下绕一周固定点作旋摆运动。从而使破碎圆锥的破碎壁时而靠近又时而离开固装在调整套上的轧臼壁表面，使矿石在破碎腔内不断受到冲击，挤压和弯曲作用而实现矿石的破碎。电动机通过伞齿轮驱动偏心套转动，使破碎锥作旋摆运动。破碎锥时而靠近又时而离开固定锥，完成破碎和排料。支撑套与架体连接处靠弹簧压紧，当破碎机内落入金属块等不可破碎物体时，弹簧即产生压缩变形，排出异物，实现保险，防止机器损坏。中鑫圆锥式破碎机在不可破异物通过破碎腔或因某种原因机器超载时，圆锥式破碎机弹簧保险系统实现保险，圆锥式破碎机排矿口增大。异物从圆锥破碎机破碎腔排出，如异物卡在排矿石可使用清腔系统，使排矿继续增大，使异物排出圆锥破碎机破碎腔。圆锥破碎机在弹簧的作用下，排矿口自动复位，圆锥式破碎机机器恢复正常工作。破碎腔表面铺有耐磨高锰钢衬板。排矿口大小采用液压或手动进行调整。 2、圆锥式破碎机工作原理图 3、圆锥碎石机性能特点 1.破碎力大、效率高、处理量高、动作成本低、调整方便、实用经济 2.零件选材与结构设计合理，使用寿命长
3.破碎产品的粒度均匀，减少了循环负荷 4.密封采用润滑脂密封，避免了给水及排水系统堵塞4、圆锥破碎机技术参数： 型号破碎 头底 部直 径 (mm) 最大 进料 料度 (mm) 出料 调整 范围 (mm) 破碎 产量 (t/h) 电机 功率 (KW) 偏心轴 转速 (r/min) 重量 (t)外形尺寸(mm) PYB6006007512-25403035652234×1370×1675 PYD600600402-1312-2330356 5.52234×1370×1675 PYB90090011515-5050-905533311.22692×1640×2350 PYZ900900605-2020-655533311.22692×1640×2350 PYD900900503-1315-505533311.32692×1640×2350 5、圆锥破碎机结构组成 圆锥破碎机主要由机架、定锥总成、动锥总成、弹簧机构、碗型轴架部以及传动等部分组成。另外圆锥破碎机辅助部分由电气系统、稀油润滑系统、以及液压清腔系统组成。
打印控件安装指南 如何取消窗口拦截？ 1.当点击“导出”或“打印”按钮后，系统会弹出一个窗口进行下一步的导出或打印操作，如果IE 浏览器设置了窗口拦击功能，需要将拦截功能取消： 关闭IE的弹出窗口拦截功能。打开IE，在“工具”下拉菜单中，选择“弹出窗口阻止程序”，关闭弹出窗口阻止程序；如果您还安装了其他带有窗口拦截功能的插件（如百度Toolbar、Google 工具栏等），请暂时关闭这些插件的拦截功能，具体关闭方法请查看插件的操作说明书。 如何自动安装控件？ 1.如果您使用的是windows xp系统(sp2)，IE浏览器会屏蔽上面的弹出窗口，这时候您只要按照下 面的步骤安装就可以了： 2.第一步：登录交易系统时，IE窗口会出现下图所示的提示，此时点击“单击此处” 提醒：请不要关闭此提示框，否则，您就不能正常安装我们的插件了！ 3.第二步：选择“安装ActiveX控件”： 提醒：选择并点击“安装ActiveX控件”，我们保证选择此控件对您的计算机没有任何风险！ 4.第三步：弹出如下图所示安全警告窗口，选择“安装”
为什么我按上面的操作还是无法安装，或是安装后也无法正常使用？ 1.请检查您的电脑是否安装了3721或其他拦截广告的插件，如果是，请将其设置为不要拦截ActivX 控件！ 2.请检查您的IE设置，并按以下步骤修改您的IE设置:
1.第一步：打开IE浏览器，选择“工具”-〉“Internet选项”，出现如下窗口： 2.第二步：按一下“设置”按钮，出现如下画面，将“检查所存网页的较新版本”设置为 “每次访问此页时检查（E）”，然后按“确定”按钮。
打印机常见硬件故障及处理方法 症状一：打印机不装纸，. 装纸后还出现缺纸报警声，装一张纸胶辊不拉纸，需要装两张以上的纸胶辊才可以拉纸。原因分析一般针式或喷墨式打印机的字辊下都装有一个光电传感器，来检测是否缺纸。在正常的情况下，装纸后光电传感器感触到纸张的存在，产生一个电讯号返回，控制面板上就给出一个有纸的信号。如果光电传感器长时间没有清洁，光电传感器表面就会附有纸屑、灰尘等，使传感器表面脏污，不能正确地感光，就会出现误报。以上这几种现象基本是同一问题，光电传感器表面脏污所致。 解决方法拔掉电源，打开机盖。对于大多数针式打印机（如EPSON LQ- 1600K、STAR-3200等），拿掉上盖板后，要先把与打印头平行*在打印头下部的一块小盖板拿掉（大约有20多厘米长1厘米宽），把打印头推至这中间（喷墨式打印机一般没有此小盖板），这样可以拿掉上盖，否则是拿不下来的。卸掉打印机周边的螺丝（大多数在打印机底部），慢慢活动就可以将外壳拆开，然后卸下黑色的进纸胶辊，要注意进纸辊两边是由一个切槽通过两个铁片卡住，只要用力推它，使它绕纸辊旋转，就可以拿下纸辊。此时能看见纸辊下有一小光电传感器，清除周围灰尘，用酒精棉轻拭光头，擦掉脏污，重新安装好纸辊、机盖等，通电开机，问题解决。 症状二：打印时字迹一边清晰，而另一边不清晰。原因分析此现象一般也是出现在针式打印机上，喷墨打印机也可能出现，不过几率较小，主要是打印头导轨与打印辊不平行，导致两者距离有远有近所致。解决方法可以调节打印头导轨与打印辊的间距，使其平行。具体做法是：分别拧松打印头导轨两边的螺母，在左右两边螺母下有一调节片，移动两边的调节片，逆时针转动调节片使间隙减小，顺时针可使间隙增大，最后把打印头导轨与打印辊调节平行就可解决问题。要注意调节时找准方向，可以逐渐调节，多试打几次。症状三：打印时感觉打印头受阻力，打印一会就停下发出长鸣或在原处震动。原因分析打印头导轨长时间滑动会变得干涩，打印头移动时就会受阻，到一定程度就可以使打印停止，严重时可以烧坏驱动电路。 解决方法在打印导轨上涂几滴仪表油，来回移动打印头，使其均匀。重新开机，如果还有此现象，那有可能是驱动电路烧坏，需要拿到维修部了。症状四：打印字符残缺不全，并且字符不清晰。原因分析对于喷墨打印机，可能有两方面原因：1. 墨盒墨尽；2. 打印机长时间不用或受日光直射，墨嘴堵塞。对于针式打印机，可能是以下几方面原因：1. 打印色带用得时间过长；2. 打印头长时间没有清洁，脏污太多；3. 打印头有断针；4. 打印头驱动电路有故障。 解决方法对于喷墨打印机，可以换新墨盒或注墨水，如果墨盒未用完，可以断定是墨嘴堵塞：拿下墨盒（对于墨盒喷嘴非一体的打印机，需要拿下喷嘴，但需要仔细），把喷嘴放在温水中浸泡一会，注意，一定不要把电路板部分也浸在水中，否则后果不堪设想，用吸水纸吸走沾有的水滴，装上后再清洗几次喷嘴就可以了。对于针式打印机，可以调节一下打印头与打印辊间的间距（在打印头移动线的周围可以找到），如果不行，可以换新色带，如果还不行，就需要清洁打印头了，方法是：卸掉打印头上的两个固定螺钉，拿下打印头，用针或小钩清除打印头前、后夹杂的脏污，一般都是长时间积累的色带纤维等，再在打印头的后部看得见针的地方滴几滴仪表油，以清除一些顽污，不装色带空打几张纸，再装上色带，这样问题基本就可以解决，如果是打印头断针或是驱动电路的问题，那只好拿到维修部去了。 症状五：打印机开机后没有任何反应，根本就不通电。原因分析打印机都有过电保护装置，当电流过大时就会引起过电保护，此现象出现基本是打印机保险管烧坏。解决方法
1 绪论 1.1引言 随着社会的进步，原材料消耗不断增加，导致富矿资源日益枯竭，矿石品位日趋贫化。以我国冶金矿山为例，铁矿石平均品位31%、锰矿石品位22%。绝大多数的原矿需要破碎和选矿处理后才能成为炉料。破磨作业是选矿的龙头，也是能耗、钢耗的大户。因此，节能、降耗是破磨设备研究的主题，“多碎少磨”是节能、降耗的重要措施，其关键问题是降低破碎产品的最终粒度。圆锥破碎机的生产效率高，排料粒度小而均匀，可将矿岩从350mm破碎到10mm以下的不同级别颗粒，可以满足入磨粒度的需要，成为金属矿山选矿厂的主要破碎设备。 破碎机的发展与人类社会的进步和科学技术的水平密切相关。随着科学技术的发展，各学科间相互渗透，各行业间相互交流，广泛使用新结构、新材料、新工艺，目前破碎机正向着大型、高效、可靠、节能、降耗和自动化方向发展。 1.2历史发展 圆锥破碎机诞生于20世纪初叶。弹簧式圆锥破碎机是由美国密尔沃基城西蒙斯（Symons）兄弟二人研制的，故称之为西蒙斯圆锥破碎机。其结构为主轴插入偏心套，用偏心套驱动动锥衬板，从而使矿岩在破碎腔内不断地遭到挤压和弯曲而破碎。破碎效果差，振动大，弹簧易损坏。用大型螺旋套调整排矿口大小，调整困难，过载保护用弹簧组，可靠性差。多年来，虽然不断改进，结果日趋完善，但其工作原理和基本构造变化不大。 20世纪40年代末，美国Allis Chalmers公司首先推出底部单缸液压圆锥破碎机，是在旋回式破碎机基础上发展起来的陡锥破碎机。该机采用液压技术，实现了液压调整排矿口和过载保护，简化了破碎机结构，减轻了重量，提高了使用性能。 20世纪50-60年代，法国Dragon公司的子公司Babbitless公司和日本神户制钢有限公司等推出上部单缸、周边单缸液压圆锥破碎机。 20世纪70-80年代，美国Allis Chalmers公司在底部单缸液压圆锥破碎机的基础上推出高能液压圆锥破碎机；Nordberg公司推出旋盘式圆锥破碎机，适用于中硬物料的破碎，其给料粒度小，偏心距小，破碎力不大。之后，相继又推出超重型短头圆锥破碎机。
打印控件安装异常处理指引 CRM项目组
1.1 打印控件自动安装失败处理 通常情况下，可以根据网运部提供的自安装文件执行，浏览器设置异常或者杀毒软件的设置都可能到导致自动注册失败。请按照以下方式尝试手工注册： 1、在执行注册命令regsvr32 出现错误，处理步骤： A.关闭所有浏览器。 B.请杀毒软件内配置允许修改注册表。 C.重新执行注册命令。 2、确保本地电脑中已有打印组件Print_For_Siebel.dll 3、在DOS命令行中注册该打印组件。 注册命令regsvr32 绝对路径\组件名称。 例如： regsvr32 C:\bin\Print_For_Siebel.dll 注册命令绝对路径组件名称 1.2 浏览器调用打印模板失败处理 在浏览器调用打印模板的出现错误，处理步骤： A．查看Internet选项中ActiveX控件和组件应该全部启用。 B．查看Internet选项中脚本应该全部启用。 C．杀毒软件中不要设置禁用activeX控件。 1.2.1 浏览器设置 在登录平台前，需要对浏览器进行设置： 第一步：选择“工具”中的“Internet选项”
第二步：选中“Internet”后，单击“子定义级别”
第三步：ActiveX控件和组件，全部选为“启用”。
第四步：脚本，全部选为“启用”。 客户端系统配置要求： Windows 2000及以上Microsoft操作系统 1.3 打印机失败处理 A.检查是否正常安装打印机驱动。 B.建议使用打印机型号：四通5530 C.清空打印作业队列中堵塞的作业，重新打印。 D.是否正常设置默认打印机。
喷墨_打印的图片中含有水平条纹或线条，怎么办？ 打印输出存在条纹或线条。线条方向垂直于纸张在打印机内的移动方向，平行于打印过程中的墨盒移动方向。 图 1: 水平条纹或线条 注意事项 如果您使用了非惠普（HP）原装墨盒或者自行灌注墨水的墨盒，建议您更换该墨盒。 ?对于使用非惠普（HP）原装墨盒，惠普不提供质量或可靠性担保。为了保证良好的打印质量，建议使用惠普（HP）原装墨盒。 ?对于使用自行灌注墨水的墨盒，无法正确检测墨水量，会提示“墨水量不足”。建议使用惠普（HP）原装墨盒。灌墨造成的不良后果请参考以下文档： HP 单功能喷墨打印机 - 灌墨或者连续供墨系统有哪些危害？ 解决方法
请按照以下步骤操作，解决该问题： 步骤一：等待 30 分钟 如果可能，让打印机闲置 30 分钟，然后再次尝试打印。某些情况下，设备经过 一段时间的闲置后可解决部分打印问题。 步骤二：查看墨水量，更换墨水不足的墨盒或空 墨盒 如果一个或两个墨盒的墨水量较低，可能会导致打印输出中含有条纹或线条。使 用 HP 工具箱检查墨水量，确定墨盒的墨水量是否不足，以及是否需要更换。 请按照以下步骤操作，使用 HP 工具箱查看估计墨水量。 1.确保打印机已开启并连接至电脑。 2.根据不同的操作系统做以下操作： o在 Windows XP 操作系统中，依次点击“开始”→“打印机和传真”。在“打印机和传真”窗口中，找到打印机的图标。 o在 Windows Vista 操作系统中，依次点击“开始 （）”→“控制面板”，在“控制面板”窗口中，点击 “打印机”选项。在“打印机”窗口中，找到打印机的图标。 o在 Windows 7 操作系统中，依次点击“开始 （）”→“所有程序”→“HP”→“HP 解决方案中 心”。在“HP 解决方案中心”软件中，点击“打开工具箱”按 钮。 注 : 在 Windows 7 操作系统中，使用“HP 解决方案中心”软件打开工具后，请按照第五步续往下操作。 3.在“打印机和传真”窗口中，右键点击“HP Deskjet D2500 series” 图标，选择“打印首选项”菜单项。 注 : 本文以 Windows XP 操作系统中的操作方法为例，其他操作系统的操方法可作参考。 4.在“HP Deskjet D2500 series 打印首选项”窗口中，选择“功能”选
PC机、打印机、复印机常见故障问题解决方法一．PC机常见故障问题解决方法 电脑常见死机故障分析 一、硬件故障引起 1、散热不良 2、移动不当 3、灰尘 4、软、硬件不兼容 5、内存条故障 6、硬件资源冲突 7、内存容量不够 二、软件故障引起 1、病毒感染 2、系统文件的误删除 3、硬盘剩余空间太少或碎片太多 4、非法卸载软件 5、启动的程序太多 6、非法操作 7、非正常关闭计算机电脑不启动常见故障分析 一、听声音，判故障： 电脑出现启动故障时都会发出不同的鸣叫声提示故障的部位，下面列举 出 AMI BIOS 和 Award BIOS 鸣叫声的含义： AMI BIOS 1 短内存刷新失败 2 短内存 ECC 校验错误 3 短系统基本内存(第一个 64K)自检失败 4 短系统时钟出错 5 短 CPU 出错 6 短键盘控制器错误 7 短系统实模式错误，不能进入保护模式 8 短显示内存错误 9 短主板 Flash ROM 或 EPROM 检验错误 1长3短内存错误(如内存芯片损坏)
Award BIOS： 1 短系统正常启动 2 短常规错误。应进入 CMOS Setup，重新设置不正确的选项 1长1短内存或主板出错 1长2短显示器或显卡错误 1长3短键盘控制器出错 1长9短主板 FlashRAM 或 EPROM 错误(如 BIOS 被 CIH 破坏) 不间断长鸣内存未插好或芯片损坏 不停响声显示器未与显卡连接 重复短鸣电源故障 二、电脑黑屏 1，显示器断电或显示器数据线接触不良； 2，主板没有供电； 3，显卡接触不良或损坏； 4，CPU 接触不良； 5，内存条接触不良； 6，机器感染 CIH 病毒，BIOS 被破坏性刷新。 电脑自动重新启动故障 一、软件 1．病毒破坏2．系统文件损坏 二、硬件 1．市电电压不稳 2．插排或电源插座的质量差，接触不良 ①不要图省钱而购买价廉不物美的电源排插，购买一些名牌的电源插排，因为其内部都是机器自动安装压接的，没有采用手工焊接。 ②对于是否属于墙壁插座内部虚接的问题，我们可以把主机换一个墙壁插座试一试，看是否存在同样的自动重启问题。
打印机驱动常见问题 一.安装前的问题 1.打印机后台程序有没有开。方法如下： 点击“我的电脑”（win7.8.10为计算机），鼠标右键管理会进入到计算机管理界面，点击“服务和应用程序”在点击“服务” 找到“Print Spooler”点击右键启动，如果后面不是自动请点击右键属性将手动改为自动即可。 二.安装方法 1.光盘进行安装 这个大家应该都会，就不一一说了，但是小编提醒的是在出现让打印机和电脑连接时若果已经连接，但是没有检测到时将USB先拔掉重新插下即可。
2.通过手动指定安装 ⑴有光盘的就不说了，没有的话先在官网网站进行下载对应系统的驱动程序，下载好后放到桌面上新建一个文件夹进行解压。（32或64分清） ⑵将打印和电脑连接好后开机，电脑发现新硬件，点击“我的电脑或计算机”右键管理，设备管理器，右侧找到打着“？”的其他设备。
⑶点开将看到打印机型号，点住打印机型号右键，更新驱动程序，手动选择光盘位置或者下载好的驱动程序位置。
点击确定，下一步，打印机进入安装步骤，等待一分钟就OK了。 （如在安装过程中出现0*000000002或0*000000003错误时请更换电脑系统或换台电脑） 三.因驱动出现的常见问题 1.打印机驱动已经安装，但是却打印不了 ⑴确认打印机是否有打印动作，打印机是否报错，有打印动作或报错则不需要考虑驱动。 ⑵打印机任务是否暂停：点击开始菜单，设备和打印机，找到驱动，看看是不是暂停状态如图 暂停打印时将不能打印，点击驱动右键恢复打印即可。针式打印机打印机面板上也有暂停键，亮灯则不能打印机，按下熄灭即可 ⑶打印机驱动图标是否显示脱机状态，脱机状态则不能打印，驱动一般脱机状 态都是打印机和电脑没有连接好，检查连接，如果已连接好就将USB口换个口，
课程设计 GP200圆锥破碎机 结构参数和性能参数选择及计算 学院: 专业： 学生姓名： 学号 指导教师：
结构参数选择与计算 1.1 分矿盘与接矿漏斗 矿石从晃动的分矿盘落下时，不允许矿石直接落入给矿口中，而使其落到接矿漏斗上。分矿盘的高度，从它的顶面到动锥球面中心的距离，一般为400?600mm 。 对于中碎机，分矿盘与定锥形成的空间不应影响矿石进入给矿口，更不能产生大块矿石楔在此空间的现象 接矿漏口的锥角应按K 述要求确定；应使落到接矿漏斗 斜面上的矿石，能沿斜面顺利地滑到动锥上部的衬板上，其 下滑的速度足够使其越过张开的给矿口，然后调转方向缓慢 地滑向给矿口 1. 2给矿口与排矿口宽度 圆锥破碎机给矿M 的宽度B ，用动锥接近定锥时，两锥体 的上端距离表示。排矿口宽度b 用动锥靠近定锥时，两锥体的下端的距离表示。B 和b 的选择给矿和排矿粒度有关，一般情况下，B=（1.2?1.25）Dmax 给矿粒度Dmax 根据选矿流程 决定。 取B=220mm 排矿口宽度b 取决于所要求的产品粒度。对于每一种破 碎机，b 值都冇-定范围，以供破碎各种硬度矿石的需要。对 于不同硬度的矿石，其排矿的过大颗粒系数K= dmax/b(dmax 是产品的最大颗粒）不同。对中碎机来说，破碎硬矿石K=2.8?3.0、中硬矿石尺=2?2.2、软矿石K=1.6□因此设计与使用中碎机时，决定排矿口宽度，就必须考虑产品中过 大颗粒对细碎机给矿粒度的影响，这主要是中碎机一般不设检杳筛分。由于细碎机一般都有检査筛分，它的排矿口宽度 平常就等于所要求的产品粒度，而不必考虑产品的过大颗粒影响。 1.3 啮角 动锥与定锥衬板之间的夹角称为啮角，并用0α表示。它 的作用是保证破碎腔两衬板有效地咬住矿石，不许向上滑动。 给矿口处啮角，必须小于矿石与定锥衬板以及矿石与动锥衬板的摩擦角之和（下图） 啮角可按下式计算：)(0010γααα±-= 式中，“+”号用于计算开口边啮角；“－”号用于计算闭口边啮角。 啮角过太，矿石将在破碎腔内打滑，降低生产能力，增加衬板磨损和电能消耗；啮角过小，则破碎腔过长，增加破碎机的高度。通常啮角为21°≤ 0α ≤23°，max 0α =26°。
打印机无法打印解决方法： 一、使打印机处于联机状态。 如果打印机没有处于联机状态，自然是无法打印了。 二、重新开启打印机。 如果打印机处于联机状态仍无法打印文档，此时你可以重新开启打印机，不仅清除了打印机内存，还能解决不少的打印故障。 三、将打印机设置为默认打印机。 步骤如下: 1.单击Windows“开始”菜单，指向“设置”，单击“打印机”，打开“打印机”窗口。 2.右键单击打印机图标，系统弹出快捷菜单，单击其中的“设为默认值”。 四、取消暂停打印。 方法是:在“打印机”窗口，右键单击在用的打印机图标，然后单击以清除“暂停打印”选项前的对号“√”。 五、使硬盘上的可用空间不低于10MB。 如果可用硬盘空间小于10MB，必须释放更多的空间系统才能完成打印任务。这时，请单击“磁盘清理”按钮，然后在“要删除的文件”列表框中，选中要删除的文件类型，单击“确定”按钮。 六、增加打印机的超时设置。 检查或更改超时设置，步骤如下: 1.在“打印机”窗口，右键单击打印机图标，再单击“属性”。 2.单击“详细资料”选项卡，在“超时设置”下增加各项超时设置。“未选定”项是指定Window s 等待打印机进入联机状态的时间，超过指定时间之后就将显示错误消息。 七、确保打印到合适的本地打印机端口。 步骤如下:
1.在“打印机”窗口，右键单击打印机图标，再单击“属性”。 2.单击“详细资料”选项卡，在“打印到以下端口”框中，确保已将打印机设置到适当的端口。最常用的端口设置为“LPT1”，也有打印机使用USB端口。 八、程序生成的输出不正确。 要确定程序生成的输出是否正确，可以采用通过其他程序打印文档的方法验证。我们以“记事本”打印测试文档，步骤如下: 1.单击“开始”，指向“程序”，指向“附件”，单击“记事本”，打开“记事本”窗口。 2.键入几行文字，然后在“文件”菜单中，单击“打印”命令。 如果能够打印测试文档，就是原来你使用进行打印的程序有问题，请重新安装程序。 九、重新安装打印机驱动程序。 有时，打印机驱动程序可能被损坏，从而引发无法打印文档的错误。我们可以重新安装合适的驱动程序，然后再打印。 1.在“打印机”窗口，右键单击打印机图标，再单击“删除”，然后单击“是”按钮。如果系统提示“删除这台打印机的专用文件”，请单击“是”按钮。如果系统提示删除默认打印机，请单击“确定”按钮。 2.在“打印机”窗口，双击“添加打印机”图标，打开“添加打印机向导”，单击“下一步”按钮，然后执行屏幕指令。 十、确保端口与打印机电缆工作正常。 进行了上述九项工作之后，以下的方法能够帮助你进一步发现问题之所在: 1.打印机电缆连接是否牢靠如果计算机直接与打印机相连，要确保连接计算机和打印机的电缆两端都插对插牢。如果使用打印切换设备，请先绕过切换设备，将打印机直接与计算机相连，然后尝试进行打印。 2.测试端口连接将打印机连接到另一个可用端口，重试打印文档，如果能够打印则表明原端口损坏。 3.测试打印机电缆换上另一根打印机电缆，然后重试打印文档，如果能够打印则表明原电缆
圆锥破碎机适用于冶金、建筑、筑路、化学及硅酸盐行业中原料的破碎，根据破碎原理的不同和海量产品颗粒大小不同，又分为很多型号。圆锥破碎机破碎比大、效率高、能耗低，产品粒度均匀，适合中碎和细碎各种矿石，岩石。 随着矿山技术的不断发展，圆锥破碎机也分为好几种，按照种类包括弹簧圆锥破碎机、轧臼式圆锥破、液压圆锥破碎机以及复合圆锥破碎机4大类；按照型号分为普通的PY圆锥破碎机、西蒙斯圆锥破碎机、复合圆锥破碎机、标准液压圆锥破碎机、单缸液压圆锥破碎机以及多缸液压圆锥破碎机等诸多型号。液压式破碎机是在消化吸收了各国具有80年代国际先进水平的各类型圆锥破碎机的基础上研制成的。它与传统的圆锥破碎机的结构在设计上显然不同，并集中了迄今为止已知各类型圆锥破碎机的主要优点。它适用于细破碎和超细破碎坚硬的岩石、矿石、矿渣、耐火材料等。 根据破碎设备工厂店的资料显示，圆锥破碎机结构简介：圆锥破碎机其结构主要有机架、水平轴、动锥体、平衡轮、偏心套、上破碎壁(固定锥)、下破碎壁(动锥)、液力偶合器、润滑系统、液压系统。 圆锥破碎机工作原理：在圆锥破碎机的工作过程中，电动机通过传动装置带动偏心套旋转，动锥在偏心轴套的迫动下做旋转摆动，动锥靠近静锥的区段即成为破碎腔，物料受到动锥和静锥的多次挤压和撞击而破碎。动锥离开该区段时，该处已破碎至要求粒度的物料在自身重力作用下下落，从锥底排出。 根据粉碎设备工厂店内数据显示，圆锥破碎机中无论是访问量，咨询量还是采购量最多的都是弹簧圆锥破碎机，那么弹簧圆锥破碎机到底有什么特点呢？ 弹簧圆锥破碎机性能特点： 1. 增大偏心距，提高处理能力，增大功率，提高破碎细度; 2. 关键部件采用高强度材质，适当增加重量，可靠性更好; 3. 衬板加厚，延长寿命，产量和粉矿含量高，产品料度较均匀; 4. 物料夹在两锥体之间，受到挤压、弯曲和剪切作用，破碎较容易，动力消耗较低; 5. 动锥连续转动，物料的破碎过程和卸料过程沿工作表面交替连续进行，生产率高。 弹簧圆锥破碎机分粗碎弹簧圆锥破碎机、中碎弹簧圆锥破碎机和细碎弹簧圆锥破碎机三种。其中粗碎用弹簧圆锥破碎机又叫旋回式破碎机；中碎弹簧圆锥破碎机为标准型圆锥破碎机；细碎圆锥破碎机又叫短头型圆锥破碎机或者叫“圆磨”。 圆锥破碎机的发展前景： 矿山开力度逐渐加大，人工或机械对有利用价值的天然矿物资源的开采，最关键的是矿石破碎，加工成品矿石用于冶金、建筑、化工等多种行业。随着国家建设扩大内需，基础设施建设加大，于是对砂石料及相关破碎机械需求剧增，圆锥破碎的市场前景广阔。
奔图打印机常见故障及 解决方法 YKK standardization office【 YKK5AB- YKK08- YKK2C- YKK18】
奔图打印机常见故障及解决方法 如何应对打印机卡纸？ 卡纸是激光打印机用户最常遇到的问题，造成卡纸的原因有很多，那么如何能够避免卡纸或降低卡纸的机率呢，我们需要注意以下几个方面： 尽量不要二次用纸，因为打印过一次的纸经过加热会造成纸张柔韧性下降，更加易断易碎容易造成卡纸。 使用平整的纸张，褶皱或折角的纸更容易卡在机器内部造成卡纸。使用质量较好的纸张，一些质量不好的复印纸容易产生大量的纸屑长时间使用会造成搓纸轮故障而卡纸。 一旦发生卡纸要注意在取出卡纸时一定要尽量轻的取出整张纸，不要在机器内部残留碎片或使某些零件脱落，如果机器内部有碎片残留或某些零件脱落将会造成频繁的卡纸。 ? 为什么有时候打印颜色浅？ 造成激光打印机打印浅的原因有很多，其中主要有以下几方面， 打印机粉量低、缺粉，打印机墨粉量低会造成打印偏浅或部分区域偏浅，所以我们要及时补充打印机耗材 纸张受潮容易造成打印颜色浅，一些地区空气潮湿或某个时期潮湿会造成纸张受潮而打印颜色偏浅，更换干燥的纸张就可以打印正常了。
感光鼓损耗严重，感光鼓是一个容易消耗的部件，使用一段时间后损耗严重造成打印颜色偏浅，所以我们要定期更换感光鼓等易消耗部件。 打印机激光器故障，打印机的激光器故障同样会造成打印颜色偏浅，如果发生这种情况则需要专业的维修人员进行维修。 为什么打印的文件会出现周期性斑点或线条？ 造成周期性故障的原因主要是打印机内部的一些辊、轮状的部件发生故障而产生的，例如，如果感光鼓有划伤则会造成90MM左右的周期性黑色印记。 ? 为什么会出现打印重影? 打印机打印重影的情况主要有以下几方面: 硒鼓重影硒鼓重影一般是表面清理不干净或漏电形成。另外粉仓刮板也会导致重影象，在刮板受损后磁辊吸粉会很厚导致刮不均匀而导致重影。加热组件重影由于打印机加热组件出现故障或定影膜破坏，在给带着字体纸张加热过程温度不正确导致加热辊沾粉，从而导致重影。另外某些打印纸张也会造成打印重影,例如:铜板纸，硫酸纸，要比普通纸要厚导致碳粉溶解后不能完全进入纸的纤维，容易出现重影现象。
一、产品简介 1.易普泰克H系列单缸液压圆锥破碎机是经过吸收世界先进破碎技术研制出的具有先进水平的圆锥破碎机，广泛应用于冶金、建筑、水电、交通、化工、建材工业中，适合破碎坚硬、中等硬度以上的各种矿石和岩石。 2.易普泰克H系列单缸液压圆锥破碎机达到了国际领先的技术水平，实现了完全智能化控制，单缸液压机构使得设备的调节十分方便，即使在设备运行的过程中，也能够轻松的实现排料口的任意调节。智能型的自动化控制系统使得破碎机始终处于最佳工作状态，并实现内外锥衬板磨损的自动补偿功能。我们从操作面板就能准确设定和调整排料口大小，也可以从操作面板轻松地自动完成磨损件的磨损量补偿。同时，由于采用了单缸的机械结构，整个机型十分紧凑，大量减少了油管油路和外挂调节机构。因此，我公司单缸液压圆锥破外形简洁流畅，体积小，重量轻，生产能力却大幅度获得了提升，开创了中国圆锥破碎机发展的新纪元。单缸液压圆锥破碎机可广泛应用于各黑色、有色、非金属矿山及砂石料等工业领域。易普泰克机械设备(上海)有限公司专业生产矿山设备，提供完善的服务。 二、产品优势 1.易损件消耗少、运行成本低、结构合理、圆锥破碎机原理及技术参数先进、运转可靠、运行成本低、破碎机的所有部件均有耐磨保护； 2.稀油润滑、可靠先进、提高使用寿命独特的稀油润滑系统设计大大提高了设备使用寿命； 3.单缸液压圆锥破碎机破碎比大、生产效率高； 4.液压保护及液压清腔、自动化程度高、减少停机时间液压调节排料口和过载保护使圆锥破碎机运转水平得到很大提高使维修更简单、操作更方便、停机时间更短。 三、工作原理 电机带动破碎机的小齿轮，小齿轮带动大齿轮，大齿轮组件(大齿轮、大齿轮架、偏心钢套)带动偏心套组件(偏心缸套、偏心铜套)和主轴组件(主轴、内锥、内锥衬板)以理论垂直线为中心，在铜衬套内公转，主轴组件在偏心铜套内以主轴的中心线可以实现自转。空机运行的时候，偏心套组件“抱着”主轴组件和随大齿轮一同公转，当物料加入到破碎腔后，主轴总成(主轴、内锥)在物料的阻力下在偏心铜套内缓慢的自转。内锥的运行轨迹看起来是在破碎腔内来回摆动，同时缓慢的旋转。物料被摆动的内锥挤压破碎。支撑套与架体连接处靠液压缸压紧，当破碎机内落入金属块等不可破碎物体时，单缸的动锥由底部液压活塞托起，起到排放口调整和过铁保护、反复起落排除堵矿的作用。 四、技术参数 c 功率 (kw) 腔 形 最大 进料 (MM) 紧边排料口下的标准产能(TPH) 6 8 10 13 16 19 22 25 32 38 44 51 ED44 220 EC 217 ————115-202 123-279 132-297 140-316 160-360 177-399 ——C 177 ———102 110-220 118-295 126-316 135-335 153-382 169-339 ——MC 142 ———98-124 107-266 115-286 122-305 130-325 148-333 163-245 ——M 112 ———119-190 128-283 138-303 148-324 157-345 179-286 198 —— MF 87 ——117 127-232 137-251 147-269 157-289 167-306 190-254 ——— F 72 — 93-139 99-181 107-196 115-212 123-227 133-243 141-258 160-214 ———
安装打印控件 1. 此处下载 jatoolsPrinter 免费版软件包。 2. 确定打印页面在服务器中目录位置。 3. 将jatoolsPrinter.cab复制到打印页面的同一目录. 比如将要打印的页面是: https://www.doczj.com/doc/521099368.html,/print/getPages.jsp，那么，jatoolsPrinter.cab的位置必须在https://www.doczj.com/doc/521099368.html,/print/jatoolsPrinter.cab 这样，当打开getPages.jsp，浏览器可以自动提示用户是否安装。 第一个打印页面：Hello world: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
一、打印大文件死机： 有的激光打印机在打印小的文件时正常，但打印大文件时会死机。 处理方法是：查看硬盘上的剩余空间。删除一些无用文件，或者查询打印机内存数量，是否可以扩容？ 二、打印时出现一个红叉： 一般用EPSON－MJ1500打印机，经常发生打印时出现一个红叉的现象。 处理方法是：看进纸是否顺利，使用的纸张是否符合要求。因为一些打印机要80克或75克纸。而有的打印纸的正反面放反了也会造成进纸不畅而出现红叉。如果是水平进纸，应使光滑面朝下；如果是垂直方向进纸，应使光滑面朝前。 三、不定时的不打印： EPSON 440打印机在驻留VRV杀毒软件时不一定什么时候就不能打印。 处理方法：依次将频幕右下角任务栏上的图标挂起，看结果如何。多可以使用鼠标右键单击图标，选择“挂起”功能即可。如KILL 98 的实时监测软件，可以选择“挂起实施监控器”，但若选择“关闭实施监控器”则可能使KILL 98 很难恢复，即使重装KILL 98 。 四、打印机信号不同步： 有的电脑使用EPSON打印机可以正常输出，但使用HP LASER JET 1100就不正常了，打印测试页异常：标题右偏，打印的文件少几个。HP打印机自检是正常使用。使用Windows 98 的HP LASER JET 驱动可以正常打印测试页，但字的质量不高。 处理方法：尝试安装Windows 9X 光盘上的HP LASER JET 打印驱动程序，然后再改回HP LASER JET 打印驱动程序，然后再改回HP LASER JET 1100 的打印驱动。 五、打印机打印过程中出现乱码的解决： 可依据以下步骤来判断故障 （1）使用KV300启动电脑，执行KV300。检查/清除病毒。 （2）打印机自检。（判断打印机本身是否存在硬件故障） （3）检测打印机电缆：（判断打印电缆是否存在故障）在MS-DOS下，执行以下命令：dir>prn。 （4）在Windows 95下，打印测试页：（判断打印机驱动程序是否正确）“我的电脑”>>“打印机”>>找到对应的打印机>>右击鼠标右键>>弹出菜单，选择“属性”>>选择“常规”>>单击“打印
单缸液压圆锥破碎机，如图中A处所示，就是源于此类圆锥破碎机底部采用了一个液压缸。这个液压缸具有调节排料口尺寸大小、防过铁、快速清腔的综合性功能。 弹簧圆锥破和多缸圆锥破的排料口调节比较复杂和繁琐，需要停机后对上壳体进行大螺母的转动，其过程原理和螺帽在螺栓中旋转一样的原理。而单缸液压圆锥破碎机采用了底部大油缸之后，可以非常便捷的实现这一功能。 单缸液压圆锥破碎机结构原理： 如图中所示，A处为底部液压大油缸。油缸承受了主轴总成的所有重量和破碎过程中形成的巨大向下压力。转子总成在C处的偏心套、B出的悬点、A处的油缸的综合制约之下，以及在物料的阻滞下，只能形成缓慢的自转和水平摆动，摆动的幅度就是偏心套的偏心量。 如图所示，中部红色区域就是单缸液压圆锥破碎机的破碎腔，内外深红色的部件就是匝臼壁和破碎壁。我们从解剖图中可以看到，匝臼壁和破碎壁的两边的排料口尺寸不同，这就是由于偏心套造成的偏心状态。单缸液压圆锥破碎机运转的时候，我们可以看到转子总成以B点位悬点左右摆动，匝臼壁和破碎壁之间的间隙犹如颚破一样的变化，从而实现对物料的挤压破碎。 图中所示，OSS代表的就是单缸圆锥破碎机的开口边，CSS代表的就是单缸圆锥破碎机的闭口边。我们在设置排料口的尺寸的时候，通常以CSS闭口边为参数对象。 单缸圆锥破的结构与特点： 单缸液压圆锥破碎机主要在于单缸这个概念，整个设备的控制和调节就在于破碎机底部的液压缸。
从示意图中我们可以看到，单缸液压圆锥破主要是由内锥衬板、外锥衬板、底部油缸、传动大小齿轮组成破碎系统。红线以上的部分被安装在上壳体上，红线以下的部分被安装在下壳体上，上下壳体之间用螺栓进行连接，从图中我们可以看到基本的结构。 当单缸圆锥破碎机工作时，电机带动破碎机的小齿轮，小齿轮带动大齿轮，大齿轮组件（大齿轮、大齿轮架、偏心钢套）带动偏心套组件（偏心缸套、偏心铜套）和主轴组件（主轴、内锥、内锥衬板）以理论垂直线为中心，在铜衬套内公转，主轴组件在偏心铜套内以主轴的中心线可以实现自转。空机运行的时候，偏心套组件“抱着”主轴组件和随大齿轮一同公转，当物料加入到破碎腔后，主轴总成（主轴、内锥）在物料的阻力下在偏心铜套内缓慢的自转。内锥的运行轨迹看起来是在破碎腔内来回摆动，同时缓慢的旋转。物料被摆动的内锥挤压破碎。
单缸液压圆锥破碎机用途： 圆锥破广泛应用于矿山、水泥厂、砂石行业中，用于中、细破碎压强在360兆帕以下的各种矿山岩石，如铁矿石、有色金属矿石、玄武岩、花岗石、石灰岩、沙岩、鹅卵石等。 圆锥破碎机工作原理： 方特远圆锥破碎机采用的是物料分层选择破碎。破碎腔填满给料，物料在破碎腔中承受全方位的挤压、剪切和揉搓后起到破碎、和自碎目的，使破碎腔壁避免了直接接触,有效的防止衬板互相磨损，以及避免物料被金属污染物的污染，从而使该机的易损易耗件的程度降低，有效延长了耐磨件的使用寿命，破碎比是其它同类设备的一倍以上。 液压圆锥破碎机优势和特点： 1.破碎比大、生产效率高 方特远圆锥破碎机的破碎比率增多，产品粒度随意性很强，大小可任意调节。圆锥破的破碎力与该机的偏心静力矩及转速有着一定的关系。调节破碎腔的间隙，可很方便地调节所需的破碎比 2.应用灵活，适用性强 方特远FTCH/CS系列圆锥破碎机的设计特点在于使破碎机发挥出更好性能以适用于各种破碎工艺：从中碎到细碎，从固定式破碎场到移动式破碎站。FTCH/CS系列圆锥破碎机允许在同一主机结构上更换不同的破碎腔型，满足不同生产情况的要求。 3.易损件消耗少，运行成本低 方特远FTCH/CS系列圆锥破碎机破碎腔型采用了优化设计，衬板均匀磨损，磨耗成本更低，将维修费用降低到更低限度，一般使用寿命可提高35%以上。 4.先进的自动化控制 方特远自动控制装置已成为FTCH/CS系列圆锥破碎机的主要特点。液压调整系统提供了安全保护和排矿口设置的调整功能，同时也可以提供自动过载保护，是不可破碎物料安全通过后主轴自动恢复原位。自动控制系统全程监控破碎机的运行状况，并自动记录生产数据和破碎机性能，提供设备的运行曲线，提高了信息记录能力。 5.层压破碎，粒形优良 方特远FTCH/CS系列圆锥破碎机，采用并实现了层压破碎：颗粒的破碎不仅发生在颗粒与衬板之间，而且还发生在颗粒与颗粒之间。颗粒之间相互挤压，扁平长条状的颗粒延其薄