Healthcare

a Cyclodextrin extracts diacylglycerol from insect high density lipoproteins

Description
a -Cyclodextrins are water-soluble cyclic hexa- mers of glucose units with hydrophobic cavities capable of solubilizing lipophiles. Incubating a -cyclodextrin with high density lipophorin from Manduca sexta or Bombyx mori re- sulted in a cloudy,
Categories
Published
of 7
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
Share
Transcript
  Journal of Lipid Research  Volume 41, 2000  933    -Cyclodextrin extracts diacylglycerol from insecthigh density lipoproteins  Zeina E. Jouni,  1  Jorge Zamora, Marcus Snyder, William R. Montfort, Andrzej Weichsel,and Michael A. Wells  Department of Biochemistry and Center for Insect Science, Biological Sciences West, University of Arizona, Tucson, AZ 85721   Abstract      -Cyclodextrins are water-soluble cyclic hexa-mers of glucose units with hydrophobic cavities capable of solubilizing lipophiles. Incubating    -cyclodextrin with highdensity lipophorin from Manduca sexta    or Bombyx mori    re-sulted in a cloudy, turbid solution. Centrifugation separated   a pale yellowish precipitate. Thin-layer chromatography anal-ysis of the lipid extract of the precipitate showed that themajor lipid was diacylglycerol, while KBr density gradientanalysis of the supernatant demonstrated the presence of a   lipid-depleted very high density lipophorin. Transfer of diacyl-glycerol from lipophorin to cyclodextrin was specific to    -cyclodextrin and was not observed with    - or    -cyclodextrins.pH had no effect on diacylglycerol transfer to    -cyclodextrin.However, the transfer was strongly dependent on the con-centration of    -cyclodextrin and temperature. Increasing   the concentration of    -cyclodextrin in the incubation mixturewas associated with the formation of increasingly higherdensity lipophorins. Thus, at 20, 30, and 40 m   M      -cyclodex-trin, the density of B. mori    lipophorin increased from 1.107g/ml to 1.123, 1.148, and 1.181 g/ml, respectively. At con-centrations greater than 40 m   M   ,    -cyclodextrin had no fur-ther effect on the density of lipophorin.    -Cyclodextrin re-moved at most 83–87% of the diacylglycerol present inlipophorin. Temperature played an important role in alteringthe amount of diacylglycerols transferred to    -cyclodextrin.At 30 m   M      -cyclodextrin, the amount of diacylglycerol trans-ferred at different temperatures was 50% at 4      C, 41% at15      C, 20% at 28      C, and less than 3% at 37      C. We proposethat diacylglycerol transfers to    -cyclodextrin via an aque-ous diffusion pathway and that the driving force for thetransfer is the formation of an insoluble    -cyclodextrin–diacylglycerol complex.   —Jouni, Z. E., J. Zamora, M. Snyder,W. R. Montfort, A. Weichsel, and M. A. Wells.      -Cyclodex-trin extracts diacylglycerol from insect high density lipopro-teins.  J. Lipid Res.   2000. 41:   933–939.  Supplementary key words  lipophorin •  fluid-phase transfer •    Man-duca sexta   •    Bombyx mori   Cyclodextrins are produced from starch by the actionof cyclodextrin glycosyltransferase. Structurally, cyclodex-trins are cyclic oligosaccharides consisting of 6, 7, or 8   D   -glucopyranosyl units connected by    -(1   →   4) glycosidiclinkages and are referred to as    -,    -, and    -cyclodextrins,   respectively (1). In the donut-shaped cyclodextrins, thepyranosyl rings of the cyclodextrins are arranged so thatthe hydroxyl groups point outward creating a hydropho-bic inner surface. The number of glucose units in the cy-clodextrin determines the size of this hydrophobic pocketand, in turn, which lipids the cyclodextrin solubilizes (2).Many factors affect the solubilization of lipids by cyclodex-trins, including the concentration of cyclodextrins, thecharacteristics of the guest molecules (hydrophobicity andsize), and the reaction conditions. The inclusion complexescan contain one or more molecules of cyclodextrins and cancontain one or more entrapped “guest” molecules. As partof an ongoing study of lipid transport in insects, we inves-tigated whether    -,    -, or    -cyclodextrins could removelipids from the insect lipoprotein, lipophorin. Lipophorinis a high density lipoprotein found in the hemolymph of insects and functions as a reusable shuttle that transportslipids through insect hemolymph (3, 4). The major trans-ported lipid is diacylglycerol but little is known about themechanism by which diacylglycerol is transferred betweenlipophorin and tissues. We report in this paper that    -cyclodextrin is able to remove diacylglycerol from lipo-phorin. The ability of    -cyclodextrin to modify the diacyl-glycerol content of lipophorin may be an important tool ininvestigating the mechanism of diacylglycerol transfer be-tween lipophorin and tissues. METHODS   Materials   2-[Morpholino]-ethanesulfonic acid (MES) and 3-[N-mor-pholino]-propanesulfonic acid (MOPS) were from Amersham(Piscataway, NJ).    -Cyclodextrin, hydroxypropyl    -cyclodextrin,  Abbreviations: MES, 2-[morpholino]-ethanesulfonic acid; MOPS, 3-[N-morpholino]-propanesulfonic acid; PMSF, phenylmethylsulfonicacid; K   eq  , equilibrium constant defined in terms of concentrations of reactants and products present at equilibrium; DPG, dipalmitoyldiacylglycerol.   1  To whom correspondence should be addressed.   b  y  g u e s  t   , onF  e b r  u ar  y  3  ,2  0 1  6 www. j  l  r . or  gD  ownl   o a d  e d f  r  om    934Journal of Lipid Research  Volume 41, 2000  [   3   H]8,9 oleic acid, phenylmethylsulfonyl fluoride (PMSF), andGrace’s medium were from Sigma (St. Louis, MO).   Insects    Bombyx mori   of the polyvoltine race N   4   (a gift from Dr. Yamashita,Nagoya University) were fed on an artificial diet prepared frommulberry leaves (Yakult Corp., Tokyo, Japan).  Manduca sexta   were fed on an artificial diet prepared from wheat germ (5). Allinsects were reared at 25–27      C with an 18-h light, 6-h dark cycle.   Lipophorin isolation   High density lipoproteins were isolated from the hemolymphof third or fourth day fifth instar  M. sexta   or  B. mori   by densitygradient ultracentrifugation (5, 6). Briefly, the hemolymph den-sity was adjusted to 1.31 g/ml (8.9 g KBr in 20 ml), placed in aQuick-Seal centrifuge tube, and overlaid with 20 ml of 0.9%NaCl. The tubes were centrifuged at 50,000 rpm in a VTi 50 ro-tor at 5      C for 18 h. One-ml fractions were collected, absorbancyat 280 nm and 450 nm was determined, and the densities of thefractions were measured. Fractions containing lipophorin weredialyzed against PBS buffer (150 m   M   NaCl, 2 m   M   EDTA, and 0.5m   M   benzamidine hydrochloride, 50 m   M   sodium phosphate, pH6.5). The solution was then concentrated using a 100 kD molec-ular mass cut centricon filters (Amicon, Beverly, MA). The purityof lipophorin preparations was confirmed by SDS-PAGE analysis,using either a linear (7.5%) or gradient (4–15%) SDS-PAGEprepared as described by Laemmli (7). All gels were stained withCoomassie Brilliant Blue R-250.   Transfer studies   Lipophorin was incubated with    -cyclodextrin on an orbitalshaker at 100 rpm for 15 min at room temperature. The turbidreaction mixture was centrifuged at 10,000 rpm for 10 min. Asample of the supernatant was subjected to density gradient ultra-centrifugation and analyzed as described above. Another sampleof the supernatant was extracted 3 times with Folch (methanol–chloroform 1:2 (v:v)) and the combined lipid extracts were sepa-rated on TLC plates. Diacylglycerol spots were identified with io-dine vapor, collected and extracted with Folch, dried under N   2   gas, resuspended in hexane, and used for the determination of diacylglycerols. A vanillin-based neutral lipid assay was used tomeasure diacylglycerol content, using diolien as a standard (8).The precipitate was washed several times with PBS and extractedthree times with dichloromethane and the extracts were used forthe determination of diacylglycerols. When mentioned, organicphases were separated on TLC plates and diacylglycerols werecollected and used as mentioned above.   Effect of pH and temperature   For these studies, 30 m   M      -cyclodextrin and 3 mg/ml of  M.sexta   lipophorin were used. To study the effect of pH, lipophorinwas dialyzed into a buffer containing MES (0.01 M), MOPS (0.01   M   ), Trizma base (0.5 m   M   ), and NaCl (0.15 M   ) of different pHvalues between 5 and 7. For the effect of temperature on transferstudies, stock solutions of    -cyclodextrin and lipophorin (PBSbuffer) were pre-equilibrated to temperatures ranging from 4      Cto 37      C, then mixed to start the experiment.   Lipophorin concentration dependence   The effect of increasing concentrations of  M. sexta   lipophorin(1.5, 3, 5, and 7 mg/ml) was determined using a constant con-centration of    -cyclodextrin (30 m   M   ).   Transfer of [   3   H]diacylglycerol to    -cyclodextrin    B. mori   larvae were starved for 1 h then fed a cube of diet (0.5cm    0.5 cm    0.5 cm) containing [   3   H-n-8,9]oleic acid. Afterconsumption of the labeled diet, the insects were switched to anormal diet for 1 h. Then hemolymph was collected in a solu-tion containing phenylthiourea, benzamidine (0.5 m   M   ), andPMSF (0.5 m   M   ) in PBS (9). [   3   H]diacylglycerol-labeled  B. mori   li-pophorin was isolated as described above and incubated with 40m   M      -cyclodextrin on an orbital shaker for 15 min, then the tur-bid reaction mixture was centrifuged at 10,000 rpm for 10 min.A sample of the supernatant was subjected to density gradient ul-tracentrifugation and density, radioactivity, and absorbances at280 and 452 nm along the gradient were determined. Anothersample of the supernatant was extracted 3 times with methanol–chloroform 1:2 (v: v) and the extract was used for the determina-tion of diacylglycerols, as described above. The pellet was washedseveral times with PBS and then extracted 3 times with dichlo-romethane for the determination of diacylglycerols.   Analysis of the composition of the    -cyclodextrin–diacylglycerol complex   To isolate the complex, 5 mg/ml  M. sexta   HDLp was incu-bated in 40 m   M      -cyclodextrin at room temperature on an or-bital shaker for 15 min, then the turbid reaction mixture wascentrifuged at 10,000 rpm for 10 min. The pellet was washed sev-eral times with PBS, resuspended in 1 ml PBS, and a 300-      l sam-ple was extracted three times with dichloromethane. The or-ganic phases were combined, dried under nitrogen gas, andused for the determination of diacylglycerols, as describedabove. An aliquot of the aqueous phase was used to determine    -cyclodextrin concentration using anthrone (10) and    -cyclodextrinwas used as a standard. Data are reported as the ratio of    molesof    -cyclodextrin/       moles of diacylglycerols.   Modeling the structure of the    -cyclodextrin–diacylglycerol complex   A model of dipalmitoyl diacylglycerol (DPG) solvated by    -cyclodextrin was built using the coordinates for dipalmitoyl di-acylglycerol (11) and    -cyclodextrin (12). An initial model wasbuilt by hand on an SGI graphics computer, using the Insight IIsoftware package MSI (Molecular Simulations Inc., San Diego,CA). Three rings of cyclodextrin were sufficient to cover theDPG molecule while stacking atop one another. The complexwas energy-minimized using the Discover module in Insight IIand the cvff forcefields, resulting in a model with reasonable ste-reochemistry, extensive van der Waals contacts and hydrogenbonds, but no disallowed high-energy contacts.   Analyses of data   Transfer studies are reported as    g diacylglycerol transferredto    -cyclodextrin or were calculated as the percentage of diacyl-glycerol transferred to    -cyclodextrin. In some cases, the datawere analyzed by linear or non-linear regression using GraphPadPrism (GraphPad Software, Inc).   Protein analysis   Protein concentrations were determined using a modifiedLowry method with bovine serum albumin as a standard (13).   Statistical analysis   For statistical analysis, Student’s unpaired t    -tests were used todetermine the significance of differences between means.  RESULTS AND DISCUSSION      -Cyclodextrin removes diacylglycerol from lipophorin   Addition of 40 m   M    -cyclodextrin to  B. mori  [ 3 H]diacyl-glycerol-labeled lipophorin resulted in a turbid reaction   b  y  g u e s  t   , onF  e b r  u ar  y  3  ,2  0 1  6 www. j  l  r . or  gD  ownl   o a d  e d f  r  om    Jouni et al.  -Cyclodextrin and lipophorin935 mixture with the spontaneous formation of a pale yellow-ish precipitate. This precipitate was recovered by centri-fugation at 10,000 g  for 10 min. TLC analysis of the lipidextract of the precipitate showed that diacylglycerols ac-counted for the majority of the lipid present, along withsmall amounts of carotenoids and phospholipids (lessthan 7% combined). About 67% of the radiolabeled di-acylglycerols present in the srcinal lipophorin preparationwere precipitated with  -cyclodextrin. Incubation with  -cyclodextrin caused a shift in the density of the radioac-tive peak (diacylglycerol) relative to control ( Fig. 1 ). Bothpeaks shown in Fig. 1 corresponded to lipophorin as con-firmed by SDS-PAGE and immunoblotting using anti-apolipophorin-I and -II antibodies (data not shown).  -Cyclodextrin concentration dependenceFig. 2  shows the effect of increasing concentrations of   -cyclodextrin on the density of  B. mori  lipophorin. In-creasing the concentration of  -cyclodextrin in the incu-bation mixture was associated with production of a higherdensity lipophorin. Untreated  B. mori  lipophorin has adensity of 1.107 g/ml and addition of 10 m M    -cyclodextrindid not cause a significant density shift. However, at 20,30, and 40 m M    -cyclodextrin, the density of lipophorinprogressively increased to 1.123, 1.148, and 1.181 g/ml,respectively. At the higher  -cyclodextrin concentrations,the peak of lipophorin was broader, suggesting the pres-ence of subfractions of lipophorin with a wide range of densities. No significant differences in the absorbances at452 nm in the untreated and  -cyclodextrin-treated lipo-phorin were observed, indicating that  -cyclodextrin doesnot extract carotenoids from lipophorin. In addition, noloss of lipophorin protein was observed at any  -cyclodex-trin concentration used. This observation is consistentwith the work of Ohtani et al. (14), who showed that  -,  -,and  -cyclodextrins failed to bind to polypeptides in sig-nificant amounts. Similar results were obtained using  M.sexta  HDLp (data not shown).The amount of diacylglycerol removed from either  B.mori  or  M. sexta  lipophorin by increasing concentrationsof  -cyclodextrins is presented in Table 1 . Although at 10m M    -cyclodextrin, lipophorin lost 4–7% of its diacylglyc-erol content, this amount was not enough to cause a den-sity shift of the particle (Fig. 2). This observation is consis-tent with the report of Soulages, van Antiwerpen, andWells (6), which showed that at least a 10% change in di-acylglycerol content was required to obtain a noticeableshift in the density of the particle. At all concentrations of   -cyclodextrin there was not a significant difference in theamount of diacylglycerol removed from the two differentsources of lipophorin. The large amount of diacylglyceroltransferred to  -cyclodextrin indicates that most of thetransfer is from the core diacylglycerol pool because thesurface pool of diacylglycerol is less than 5% of the di-acylglycerol in lipophorin (6). At this time, it is not clear Fig.1. Effect of  -cyclodextrin on the density of  B. mori  lipo-phorin. Lipophorin was incubated with 40 m M    -cyclodextrin. Afterremoval of the precipitate, the lipophorin in the supernatant wassubjected to KBr density gradient ultracentrifugation. Fractionsfrom the KBr gradient were analyzed for radioactivity and density;lipophorin before incubation (dashed line); lipophorin after incu-bation with  -cyclodextrin (solid line). Fig.2. Effect of  -cyclodextrin concentration on diacylglycerolremoval from lipophorin.  B. mori  lipophorin (5 mg) was incubatedwith increasing concentrations of  -cyclodextrin. After removal of the precipitate, the lipophorin in the supernatant was subjected toKBr density gradient ultracentrifugation. KBr gradient fractionswere analyzed for protein at 280 nm; (  ) control; (  ) 10 m M ; (  )20 m M ; (  ) 30 m M ; and (  ) 40 m M    -cyclodextrin. Table1.Effect of increasing  -cyclodextrin (  -CD) concentrationon transfer of diacylglycerol (DG)  -CDAmount of DG in  M. sexta  HDLpAmount of DG in  B. mori  HDLp m  M    g   g 03421.4   239.13268.2   255103287.2   162.13030.4   147202335.2   25.4 a 2296.4   53 a 40561.1   53.2 a 622.7   71 a 60342.8   117.3 a 326.3   11 a Five mg of either  B. mori  or  M. sexta  lipophorin (HDLp) wastreated with the indicated concentration of  -cyclodextrin and theamount of diacylglycerol remaining in solution was determined. a Significantly different from 0 m M  and 10 m M    -cyclodextrin.   b  y  g u e s  t   , onF  e b r  u ar  y  3  ,2  0 1  6 www. j  l  r . or  gD  ownl   o a d  e d f  r  om   936Journal of Lipid Research Volume 41, 2000 whether diacylglycerol efflux from HDLp occurs from thecore pool via the surface pool of diacylglycerol that is inequilibrium with the core pool, or through a totally differ-ent location on HDLp. We consistently observed thatabout 15% of the lipophorin–diacylglycerol is not trans-ferred to  -cyclodextrin, which might suggest a “structural”role for this small pool of diacylglycerol. A structural rolefor diacylglycerol has been proposed previously (6). Specificity for  -cyclodextrin The  -,  -, and  -cyclodextrins have different cavity sizesof 5, 6, and 8 Å, respectively, and some specificity in the in-teraction of the different cyclodextrins with lipophorinsmight be expected (14). To determine transfer specificity,  M. sexta  lipophorin was incubated with 30 m M    -,  -, and  -cyclodextrins for 15 min and the extent of diacylglyceroltransfer was determined. Neither  - nor  -cyclodextrins wereable to cause any significant transfer of diacylglycerol fromlipophorin whereas under these conditions  -cyclodextrincaused a 28% transfer of diacylglycerol from lipophorin.The same results were obtained when longer incubationtimes of 2 h were used. The inability of  - and  -cyclodextrinsto extract diacylglycerol from HDLp was also confirmedusing up to 60 m M  concentrations of the correspondingcyclodextrins and longer incubation times of 2 h. Theseresults suggest that the cavity size is one important factorin determining diacylglycerol transfer to cyclodextrins. Lipophorin concentration dependence The amount of diacylglycerol transferred to  -cyclodex-trin increased with increasing the concentration of lipo-phorin in the incubation media ( Fig. 3 ). Nonlinear leastsquares analysis of the transfer data demonstrated a satu-ration behavior at high concentrations of lipophorin andthis was confirmed by linearity of the double reciprocalanalysis of the data (Fig. 3, inset). Effect of pH on diacylglycerol transfer pH values of the reaction mixture ranging from 5 to 7had no effect on the amount of diacylglycerol transferredto 30 m M    -cyclodextrin ( Fig. 4 ). The same results wereobtained when different concentrations of  -cyclodextrinwere used. Likewise, the ionic strength of the reactionmixture ranging from 0 to 2 m M  NaCl exhibited no effecton the ability of  -cyclodextrin to extract diacylglycerolfrom lipophorin (data not shown). Effect of temperature on diacylglycerol transfer Temperature played a critical role in determining theamount of diacylglycerol transferred to  -cyclodextrin( Table 2 ). At 30 m M    -cyclodextrin the amount of diacyl-glycerol transferred to  -cyclodextrin increased from lessthan 3% at 37  C to 50% at 4  C. Assuming that the reac-tion is, Lp-DG   CD i  Lp   CD-DG, where Lp-DG islipophorin–diacylglycerols, Lp is diacylglycerols-depletedlipophorin, CD is cyclodextrin, and CD-DG is cyclodextrin–diacylglycerols, the K eq  at 4, 15, and 28  C can be calcu-lated from the data in Table 2. A van’t Hoff plot of thesedata ( Fig. 5 ) has a slope of 4600   1100 and a Yinterceptof  20   3.8. These numbers translate into  H        38.2  9.1 kJ/mol and  S        166   31.6 J/   mol. The values for  H   and  S   are consistent with a reaction that proceedswell at low temperature and poorly at high temperature.However, if the equilibrium written above were the onebeing studied, one would expect hydrophobic interactionsto play a critical role in complex formation. Hydrophobic Fig.3. Effect of increasing concentration of  M. sexta  lipophorinon diacylglycerol transfer. Increasing concentrations of lipophorinwere incubated with 40 m M    -cyclodextrin and the amount of di-acylglycerol present in the precipitate was determined. In the mainfigure, the line was determined by non-linear least squares analysisassuming a simple saturable process. The inset shows a double re-ciprocal plot of the same data. From either plot, it was determinedthat the maximal amount of diacylglycerol transferred was 5,350  g. Values represent averages   standard deviation for four deter-minations. Standard deviations are smaller than the symbols. Fig.4. Effect of pH on diacylglycerol transfer.  -Cyclodextrin wasincubated with  M. sexta  lipophorin at pHs ranging between 5 and 7.After the reaction, the precipitate was removed and the amount of diacylglycerol remaining in solution was determined. Values repre-sent averages   standard deviation for six determinations from twoexperiments.   b  y  g u e s  t   , onF  e b r  u ar  y  3  ,2  0 1  6 www. j  l  r . or  gD  ownl   o a d  e d f  r  om    Jouni et al.  -Cyclodextrin and lipophorin937 interactions are usually not favored at low temperatureand favored at high ionic strength. As this is not the case,we assume that the equilibrium involved is the formationof the precipitated complex, which should indeed have anunfavorable  S  . The addition of  -cyclodextrin to a lipo-phorin solution is followed instantly by the formation of the precipitated inclusion complex, which would also beconsistent with the concept that the driving force for thisprocess is the formation of the precipitate.Previous studies have employed different techniquesfor the preparation of diacylglycerol depleted lipo-phorin. The in vitro incubation of insect hemolymphlipid transfer particle (LTP),  M. sexta  lipophorin, andhuman low density lipoprotein resulted in the forma-tion of a stable diacylglycerol-depleted very high densitylipophorin species (15). This is a laborious method toprepare diacylglycerol-depleted very high density lipo-phorin because it requires the purification of LTP andthe isolation of reaction products. Another techniqueused to manipulate the diacylglycerol content of lipo-phorin takes advantage of the actions of phospholipaseA 2  or triacylglycerol lipase on lipophorin (16). Themethod resulted in contaminated products and necessi-tated an additional purification step. Thus, it is clearthat  -cyclodextrin is a superior tool to manipulate dia-cylglycerol content of lipophorin. Model for the  -cyclodextrin–diacylglycerol complex Quantitative analysis of the  -cyclodextrin–diacylglyc-erol complex indicated the presence of a molar ratio of   -cyclodextrin:diacylglycerol of 3.3   1.3 (n   10). To ad-dress whether a chemically reasonable, water-soluble com-plex could form between  -cyclodextrin and diacylglyc-erol, we examined possible arrangements using computermodeling. After modeling and energy minimization to re-lieve bad contacts, a model was found with diacylglycerolcompletely surrounded by, and completely filling, three cy-clodextrin rings ( Fig. 6 ), which is consistent with the exper-imental results obtained. The computer-modeling complexhas no interior cavities, and no disfavored contacts be- Table2.Effect of temperature on the amount of diacylglycerolassociated with HDLp after treatment with  -cyclodextrin TemperatureDiacylglycerol Remaining in HDLp  C    g 4776.0   91.515915.5   205.9271122.7   92.1371540.3   26.5Three mg of  M. sexta  HDLp containing 1552  g of diacylglycerolin one ml were treated with 30 m M    -cyclodextrin at the indicated tem-peratures and the amount of diacylglycerol (DG) (  g) remaining in so-lution was determined. Fig.5. Van’t Hoff plot of the data in Table 2. The slope of the lineis 4600   1100 and the Yintercept is  20   3.8 and the correlationcoefficient (r 2 )   0.95. These numbers translate into  H        38.2  9.1 kJ/mol and  S        166   31.6 J/   mol. Fig.6. Model of the  -cyclodextrin–diacylglycerol complex. A:Side view of the complex with the diacylglycerol shown as a stickmodel and the  -cyclodextrin is represented by a sliced-throughsolid surface. The figure was made with program GRASP (20). B:End-on view with all non-hydrogen atoms of the complex shown asblack spheres for diacylglycerols and white spheres for cyclodex-trin. The figure was made with the program Insight II.   b  y  g u e s  t   , onF  e b r  u ar  y  3  ,2  0 1  6 www. j  l  r . or  gD  ownl   o a d  e d f  r  om 
Search
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks