Patent Application: US-8740202-A

Abstract:
the present invention provides novel isolated and purified nucleic acid encoding , or complementary to , a canine pept1 . the present invention also provide a method for determining canine pept1 - transportability of a peptide , or method for determining a peptide with beneficial nutritional property in an animal . the present invention further provides a dietary composition for an animal comprising a peptide identified by the method described above .

Description:
this invention relates to peptide amino acid absorption in the dog , and more particularly , to separate , whole or partial - length , complementary dna encoding putative canine low - affinity , high - capacity h + / peptide transport proteins ( cpept1 ), mrna transcripts corresponding to cpept1 , characterization of cpept1 by glycylsarcosine ( glysar ) uptake , identification of dipeptides , tripeptides , and tetrapeptides well recognized by cpept1 , and the effect of supplemental peptide substrate on the transport capacity of cpept1 . the invention also provides a pet food composition comprising at least one dipeptide , tripeptide , or tetrapeptide that provides enhanced uptake of amino acids by pept1 . a typical canine diet for use in the present invention may also , for example , contain about 20 to about 30 % crude protein , about 10 to about 20 % fat , and about 10 % total dietary fiber . however , no specific ratios or percentages of these or other nutrients are required . the inventors have discovered a method for identifying peptides ( e . g . dipeptides , tripeptides , or tetrapeptides ) that increase transport of amino acids by pept1 using mdck cells , particularly when incubated with lactalbumin hydrolysate and assayed at optimum time post - seeding , as indicated in example 2 . in order that the invention may be more readily understood , reference is made to the following examples which are intended to illustrate the invention , but not limit the scope thereof . initial attempts ( over 150 ) to partially clone the putative canine pept1 cdna by reverse transcriptase - polymerase chain reaction ( rt - pcr ) methodology failed . the source of mrna was canine liver tissue that had been frozen for about 6 months ( supplied by dr . randal buddington , mississippi state university ) and oligomer primers were based on the rabbit pept1 sequence . subsequently , frozen canine “ mid ” small intestine ( jejunal ) tissue segments became available ( supplied by dr . buddington ) and a partial length cdna of about 780 base pairs ( bp ) was cloned by rt - pcr . total rna was isolated from jejunal epithelium scraped from intestinal sections using a standard acidic phenol - chloroform protocol . one μg of mrna was isolated from total rna using poly a tract system ® ( promega , madison , wis .) and reversed transcribed using murine leukemia virus reverse transcriptase ( perkin elmer , foster city , calif .) and oligo ( dt ) primers ( gibco brl , grand island , n . y .). successful pcr reactions were 50 μl and contained 1 μm mgcl 2 and taq polymerase ( perkin elmer ). twenty - five thermal cycles of 94 ° c . for 1 min , 40 ° c . for 45 sec , and 72 ° c . for 1 min were used . the cycles were preceded by a 55 sec denaturization of the rt product at 95 ° c ., followed by a 10 min extension of rt - pcr products at 72 ° c . more than 150 rt - pcr reactions testing ten different primer sets were required to achieve this protocol . the resulting cdna using primer set 4 ( fig1 ) was ta - cloned into the pcr ® ii vector ( invitrogen , carlsbad , calif . ), plasmid - containing colonies selected by blue / white screening , and amplified following instructions of the manufacturer . restriction analyses of recovered pcr ® ii / cdna plasmids revealed that four of fifty - six clones contained cdna consistent with rabbit pept1 cdna ( fig2 ). northern blot analysis of cpept1 expression in dog tissue and mdck cells the potential expression of cpept1 mrna by canine kidney , small intestinal epithelium , and immortalized kidney distal tubule epithelial cells ( madin - darby canine kidney , mdck ) was evaluated by northern analyses using cdna derived from canine jejunal epithelium ( fig3 ). rna were subjected to 1 % gel electrophoresis in the presence of 0 . 02 m formaldehyde , transferred by downward capillary action to 0 . 45 - μm nylon membranes ( hybond - n , amersham , arlington heights , ill . ), and covalently cross - linked by ultra - violet light . cdna were randomly labeled with [ 32 p ]- ctp using a kit ( gibco brl ), purified through sephadex - 50 columns ( amersham pharmacia , piscataway , n . j . ), and hybridized with blots at 56 ° c . for 18 h . the blots were then washed 2 times at 56 ° c . for 15 min and once at 57 ° c . for 10 min . autoradiographs were exposed to blots at 80 ° c . for 24 h and the size of the transcript determined by regression of hybridized bands against the migration distance of 18s ( 1 . 9 kb ) and 28s ( 4 . 9 kb ) rna . each canine tissue - derived cdna ( ta - clone 26 , fig3 a ; ta - clone 6 , fig3 b ) hybridized to three mrna species in dog kidney , dog small intestinal epithelium , and mdck cells . to confirm identification of pept1 mrna by these canine cdnas , rna isolated from dog kidney and liver tissues were probed for expression of pept1 mrna using a full - length rabbit pept1 cdna ( fig4 ; rabbit pept1 cdna supplied by drs . f . leibach and v . ganapathy , medical college of georgia ). the results also demonstrated the expression of the same three pept1 mrna species by dog tissues , indicating that the full - length rabbit pept1 cdna and the cdna derived from canine tissue in the present study identified the same transcripts . the mean / sd of transcript sizes calculated from these three blots were 4 . 2 / 0 . 22 , 2 . 75 / 0 . 26 , and 1 . 46 / 0 . 42 kb , respectively . collectively , these data indicate that liver , intestinal epithelial , and mdck cells express the same size and number of pept1 transcripts . in comparison , various tissues of chicken , sheep , cow , pig , rabbit , rat , human , and caco2 cells are reported to express a single transcript , with the principle difference in size being between chicken ( 1 . 9 ) and mammalian species ( 2 . 8 , 2 . 8 , 2 . 9 , 2 . 9 , 3 . 0 , 3 . 1 , 2 . 9 , respectively partial cloning and sequence identification of canine pept1 ( cpept1 ) cdna from mdck cells to confirm the positive northern analysis , identification of pept1 mrna expression using cdna generated from dog small intestinal epithelium , rt - pcr methodologies were used to generate a pept1 cdna from mdck cells . the target cdna region was a subset of the cdna generated by rt - pcr from canine small intestine ( bp 83 to 887 of rabbit pept1 ). accordingly , pcr primers that corresponded to bp 259 to 619 of rabbit pept1 ( genbank acc . no . u06467 ) were used to generate a partial - length “ canine pept1 ” ( cpept1 ) cdna from mrna isolated from mdck cells . rna was collected from cells that were plated at 30 , 000 cm 2 on rat tail collagen - coated dishes and cultured for 3 days in 10 % fetal calf serum / dmem . reverse transcription of 5 μg of total rna by superscript ® ii reverse transcriptase ( gibco - brl ) was performed using random and oligo ( dt ) primers , per instructions of the manufacturer ( gibco - brl ). all pcr reactions contained 2 mm mgcl 2 and thermal cycling using taq polymerase included 30 cycles at 94 ° c . for 2 min , 55 ° c . for 1 min , and 72 ° c . for 2 min . the cycles were preceded by a 10 min denaturization of the rt product at 94 ° c ., followed by a 10 min extension of rt - pcr products at 72 ° c . more than one hundred rt - pcr reactions were required to achieve this protocol . the resulting cdna of about 380 bp was ta - cloned , into the site of pcr ® ii vector ( as described above ), amplified , bacterial colonies evaluated by blue / white screening , and pcr ® ii / cdna plasmids evaluated for cdna by eco ri / pst i restriction analysis ( as described above ). restriction analyses of recovered plasmids revealed that six of thirty - six clones contained cdna consistent with rabbit pept1 cdna . two of the confirmed plasmids were amplified in bacteria , recovered , and sent for sequencing by the university of florida dna sequencing core facility ( gainesville ). sequence comparisons of this 380 bp cdna ( fig5 ) to pept1 sequences of other species using blast 2 . 0 . 14 . software ( blast @ ncbi . nlm . nih . gov ) revealed that the canine sequence shares sequence homology of 79 % to rabbit ( bp 259 to 640 ; genbank acc . no . 473375 ), 83 % to rat ( bp 213 to 593 ; genbank acc . no . d50664 . 1 ), 83 % to mouse ( bp 213 to 589 ; genbank acc . no . af205540 ), and 87 % to human ( bp 285 to 665 ; genbank acc . no . 473375 and u13173 ) pept1 sequences . as seen in fig3 and 5 , mdck cells express a canine homolog of mammalian pept1 mrna . potential expression of pept1 transport activity ( h + - dependent , dipeptide inhibitable , low - affinity dipeptide transport ) by confluent mdck cells was evaluated using whole - cell transport techniques and glycylsarcosine ( glysar ) as a model dipeptide substrate . cells were seeded at 60 , 000 cells / cm 2 into 24 - well trays that had been coated with rat tail collagen or poly - l - lysine and cultured ( 95 % o 2 : 5 % co 2 at 37 ° c .) for 3 d in media consisting of dulbecco &# 39 ; s modified eagle medium / 10 % fetal calf serum / 1 % antimicrobial antibacterial medium . absorption ( pmols / mg protein ) of [ 3 h ]- glycyl - l - sarcosine ( glysar , 6 mci / ml , moravek biochemicals , brea , calif .) was determined using the 24 - well cluster tray method and representative scintillation counting . before transport , cells were incubated at 37 ° c . for 30 min in 25 mm hepes / tris ( ph 7 . 5 ), 140 mm nacl , 5 . 4 mm kcl , 1 . 8 mm cacl 2 , 0 . 8 mm mgso 4 , and 5 mm glucose ( uptake buffer ) to normalize intracellular amino acid and peptide pools . transport was initiated by the addition of 0 . 25 ml of uptake buffer that contained 2 . 88 μm glysar . after 30 min of uptake at 37 ° c ., transport was terminated by rapid washing of cells with 4 × 2 ml 4 ° c . uptake buffer . cellular protein was precipitated with 10 % trichloroacetic acid and the supernatant recovered and counted to determine radioactivity ( 3 h ) content . cellular protein was then solubilized in 0 . 2 n naoh and 0 . 2 % sds and quantified by the lowry procedure , using bovine serum albumin as a standard . the amount of h + - dependent glysar absorbed was calculated as the difference between uptake in ph 6 . 0 and ph 7 . 5 uptake buffers . the amount of competitor substrate - inhibitable glysar uptake was calculated as the quotient of glysar uptake in the absence and presence of 10 mm competitor substrate ( dipeptide or amino acid ) multiplied by 100 %. glysar uptake in the presence of an intracellularly h + gradient ( extracellular ph of 6 . 0 ) was 2 . 3 - fold higher in cells plated on collagen , and 1 . 7 - fold higher when grown on poly - l - lysine , than uptake in ph 7 . 5 medium ( table 1 ). h + - dependent uptake of glysar by mdck cells was inhibited by 88 or 92 % by the presence of 10 mm leutrp or trpleu when grown on collagen , and 87 or 92 % when grown on poly - lysine , respectively ( table 1 ). to preliminarily characterize the kinetic parameters of peptide transport by mdck cells , the uptake of glysar in media that contained ph 6 . 0 and 0 . 00064 , 0 . 0025 , 0 . 010 , 0 . 04 , 0 . 160 , 0 . 640 , 2 . 56 , or 10 . 2 mm of glysar was measured ( fig6 ). total glysar uptake was by a relatively low - affinity mechanism ( apparent k m of about 4 . 0 mm ) and high uptake velocity . collectively , these characteristics of glysar uptake are consistent with functional activity of pept1 expressed by other species , as opposed to high - affinity , h + - dependent uptake by pept2 ( μm k m ). accordingly , it is concluded that mdck cells possess pept1 - like activity , consistent with detection of pept1 mrna by rt - pcr ( fig1 , 5 ) and northern blot analyses ( fig3 ). separate partial - length canine pept1 cdnas ( cpept1 ) were generated by rt - pcr analyses from dog small intestinal epithelium ( n = 2 ; fig1 ) and immortalized canine kidney cells ( mdck cells , n = 1 ). the mdck cdna was sequenced ( fig5 ) and found to share 79 to 87 % sequence identity with pept1 mrna expressed by other mammalian species . northern blot analyses using the intestinal epithelium - derived rt - pcr cdna confirmed expression of canine pept1 ( cpept1 ) by dog tissues ( liver , n = 3 ; kidney , n = 3 ; small intestine n = 1 ) and mdck cells ( n = 2 ). the identification of mrna transcripts corresponding to pept1 using partial - length canine - derived pept1 cdna ( fig3 ) was confirmed by hybridization to full - length rabbit cdna ( fig4 ). characterization of glysar uptake by mdck cells demonstrated that mdck cells express pept1 - like activity ( table 1 , fig5 ), confirming detection of pept1 mrna expression by mdck cells and use of mdck cells as a model to characterize the function of canine pept1 . experimental model of mdck cells for evaluating the effects of various peptide and drug substrates , and hormones and / or growth factors , on the expression of pept1 activity example 1 above showed that ( 1 ) a canine homolog of pept1 ( cpept1 ) mrna cloned from epithelia of the mid small intestine ( jejunum ) shares high sequence identity with pept1 expressed by several other species , ( 2 ) canine liver , kidney , and jejunal epithelium express a similar pattern of cpept1 mrna , and ( 3 ) mdck cells are capable of h + - dependent peptide uptake . accordingly , mdck cells are an appropriate model to evaluate the biochemical characteristics of cpept1 . the specific goals of this research were to ( 1 ) characterize the functional activity of low - affinity h + - dependent glysar uptake ( pept1 activity ) by mdck cells and ( 2 ) identify di - and tripeptides that are well recognized by cpept1 ( cpept1 ), especially those that contain tryptophan and leucine . previous research ( brandsch et al ., 1994 , biochem j . 299 : 253 - 260 ) briefly reported that h + - dependent peptide uptake by mdck cells was greater when cells were grown in a medium that contained lactalbumin hydrolysate ( lhm ) versus one that contained free amino acids ( dmem ). therefore , in an attempt to establish the most sensitive model possible for evaluating peptide transport systems in mdck cells , the potential influences of lhm ( peptide - containing ) versus dmem ( peptide - lacking ) media , and subconfluent versus confluent initial cell plating densities were compared . mdck cells were seeded at either 60 , 000 cells / well ( subconfluent ) or 120 , 000 cells / well ( confluent ) in dmem and , after 1 d , cultured in dmem or lhm media for 1 , 2 , 3 , or 5 d . the amount of protein ( index of cell growth ) and glysar uptake ( index of peptide uptake capacity ) expressed by each well of cells was then determined . as seen in fig7 the amount of cellular protein increased ( p & lt ; 0 . 05 ) for both seeding densities and media with time of culture . a time × media interaction was observed , which reflects the greater protein content of cells grown in dmem at day 6 , as compared to those grown in lhm . at days 2 , 3 , or 4 , however , no difference in protein content was observed . the uptake of [ 3 h ]- glysar ( 2 . 88 μm , 5 μci / ml ) by the mdck cells described in fig7 was measured in the presence ( ph 6 . 0 uptake buffer ) and absence ( ph 7 . 4 uptake buffer ) of an extracellular - to - intercellular h + ( proton ) gradient . a representative graph ( fig8 ) compares the uptake of glysar by cells seeded at 60 , 000 / well and cultured in the lhm or dmem . for both culture media , glysar uptake in the presence of ph 6 . 0 was greater ( p & lt ; 0 . 01 ) than that in ph 7 . 4 buffer and displayed a quadratic ( p & lt ; 0 . 01 ) response to length of culture , reflecting a buffer × day of culture interaction ( p & lt ; 0 . 01 ). dmem - cultured cells seeded at 120 , 000 / well displayed almost identical uptake characteristics as just described for cells seeded at 60 , 000 / well . in contrast , glysar uptake in the presence of ph 6 . 0 buffer at day 3 by lhm - cultured cells was only 28 % larger ( quantitatively ) than that observed by dmem - cultured cells seeded at 60 , 000 / well . to further refine the analysis of media influence on the peptide transport capacity of mdck cells plated at 60 , 000 or 120 , 000 cells per well , the h + - dependent glysar uptake was calculated as the arithmetic difference between uptake in ph 6 . 0 and ph 7 . 4 buffers ( fig9 ). despite the comparable protein contents of cells observed at day 3 ( fig7 ), cells seeded at 60 , 000 and grown in lhm media demonstrated about 60 % greater capacity for glysar uptake as did cells grown in dmem ( fig9 ; day × media interaction , p & lt ; 0 . 01 ). for all cells , the capacity for glysar uptake per mg of cellular protein was decreased at day 6 . this difference was the result of a lesser uptake at ph 6 . 0 by the lhm - cultured cells , and not the result of a larger ph 7 . 4 uptake . the results of this experiment indicate that culturing cells in media that contains peptides does not increase growth rate but does increase the capacity for peptide uptake if cells are seeded at 60 , 000 / well and cultured for 2 days in lhm . as such , these data are consistent with the induction of pept1 expression by culture peptide - containing medium and describe an optimal set of culture conditions for characterizing h + - dependent peptide transport activity of the canine pept1 transporter . these data also confirm , and more thoroughly describe , the stimulating effect of lhm versus dmem media on peptide transport proteins that was initially reported by brandsch et al . ( 1994 ). using the maximal uptake - stimulating culture parameters determined in experiment 3 , the effect of an extracellular - to - intracellular ph gradient on glysar uptake was further evaluated to determine a ph level at which maximal glysar uptake could be achieved , but which would replicate physiologic conditions ( fig1 ). as expected , the presence of a ph gradient stimulated ( p & lt ; 0 . 001 ) h + - dependent glysar uptake , in a quadratic ( p & lt ; 0 . 01 ) fashion . uptake at ph 5 . 5 or 6 . 0 was about 2 . 7 times greater than that achieved at ph 7 . 5 . these results are consistent with the data in fig8 and 9 and known h + - dependence of mammalian peptide transport proteins . accordingly , the use ph 6 . 0 buffers for the characterization of h + - dependent glysar uptake was incorporated into the standard experimental conditions . to determine the appropriate time period to measure initial ( linear ) rates of glysar uptake , a by - minute time - course experiment was performed . as seen in fig1 , h + - dependent glysar ( 100 um ) uptake increased linearly for 1 h and then slowed ( quadratic response , p & lt ; 0 . 01 ). glysar uptake in ph 6 . 0 buffer at 3 . 75 , 7 . 5 , 15 , 30 , 60 and 120 min was about 2 , 2 . 1 , 2 . 25 , 2 . 65 , 2 . 79 , and 2 . 62 times more ( p & lt ; 0 . 001 ), respectively , than uptake from ph 7 . 4 buffer . because uptake was proportional to time of uptake through 1 h , future experiments were conducted using a 30 - min time period . to confirm that h + - dependent glysar uptake was saturable , and therefore mediated , the uptake of glysar from ph 6 . 0 and 7 . 4 uptake buffers containing 0 . 025 , 0 . 1 , 0 . 4 , 1 . 6 , 6 . 4 , or 25 . 6 mm glysar was evaluated ( fig1 ). uptake of glysar was greatest ( p & lt ; 0 . 001 ) from the ph 6 . 0 buffers , at all concentrations . h + - dependent glysar uptake was saturable , consistent with an apparent k m for glysar of about 1 . 1 mm . these values are consistent with our preliminary trials that estimated a k m of 1 . 1 mm for glysar uptake by mdck cells using only ph 6 . 0 uptake buffer and indicate that h + - dependent glysar uptake is predominately , if not completely , a result of low affinity ( mm ) h + / peptide cotransporter activity ( pept1 ). as a comparative value , the reported k m of for glysar uptake by the pept1 - expressing caco - 2 cells also is 1 . 1 mm . it is of interest also to note that glysar uptake in the absence of a ph gradient ( ph 7 . 4 buffers ) also displayed linear ( p & lt ; 0 . 01 ) and quadratic ( p & lt ; 0 . 001 ) components , ( 1 ) reflects that the ph “ 7 . 4 ” buffer was in fact slightly acidic , ( 2 ) represents the activity of the putative basalateral peptide transporter running in “ reverse ”, or ( 3 ) indicates the presence of a non - characterized peptide transport system . as a result of this experiment , subsequent h + - dependent peptide transport trials were conducted using 100 μm glysar , a value well below the k m but one that will result in increased transport activity , and thus , sensitivity . characteristic hallmarks of low affinity h + / peptide cotransport activity , classically defined using membrane vesicles of several species , and more recently by functional expression studies using human , rat , and rabbit pept1 cdna , is the recognition of some , but not all , β - lactam antibiotics . in addition , pept1 recognition of cefadroxil is low ( the k l of cefadroxil inhibition of glysar uptake by pept1 is 3 mm ), whereas recognition of cefadroxil by pept2 is high ( the k i of cefadroxil inhibition of pept2 transport of glysar is 30 μm ). to determine whether mdck cpept1 activity shared these functional features , the uptake of 100 μm glysar in the absence and presence of ph 7 . 5 and ph 6 . 0 buffer , and , in ph 6 . 0 buffers , the presence of 1 mm additional glysar ( self - inhibitor control ), 3 mm penicillin - g , 30 μm cefadroxil , or 3 mm cefadroxil was compared ( fig1 ). h + - dependent glysar uptake was not inhibited by penicillin - g or 30 μm cefadroxil , but was inhibited about 76 % by 3 mm cefadroxil . as expected , the presence of 1 mm glysar self - inhibited 100 μm glysar uptake by 64 %. these results indicate that h +- dependent uptake of glysar by mdck cells is by pept1 activity . other hallmarks of pept1 function are the decreased ability of gly - containing peptides to inhibit glysar , in proportion to their length , and sensitivity to inhibition by carnosine ( β - ala - his ). to determine if cpept1 activity behaves as reported for other pept1 activities , the relative abilities of 1 mm gly ([ 3 h ]- gly free amino acid control ), glygly , [ gly ] 4 , or [ gly ] 5 to inhibit h + - dependent 100 μm glysar was determined ( fig1 ). gly ( 5 . 0 %) and [ gly ] 5 ( 7 . 3 %) did not influence uptake , whereas glygly inhibited and [ gly ] 4 tended to inhibit uptake by 63 and 23 %, respectively . this pattern of gly - containing peptides to inhibit glysar uptake in an inverse proportion to the number of glycyl residues in the canine mdck cell model is consistent with pept1 activities reported for other species . similarly , glysar uptake was inhibited 50 % by 1 mm carnosine ( data not shown but listed in table 2 below ). together with the molecular identification of pept1 mrna expression in mdck cells using full - length rabbit cdna and our canine rt - pcr product ( see example 1 data ), the above biochemical characterization data indicate that h + - dependent glysar uptake activity in mdck cells is consistent with the low - affinity , high - capacity of the pept1 transport protein . collectively , the above experiments resulted in the generation of an experimental regimen for the culture and determination of h + - dependent peptide transport activity in mdck cells , with which to evaluate the relative substrate preferences of canine pept1 ( cpept1 ). accordingly , the following general regimen was used to perform a series of experiments that evaluated the relative abilities of candidate di -( primarily ) and tri - peptides to inhibit glysar uptake by endogenously expressed cpept1 in mdck cells : 1 . sixty thousand cells / well were plated into collagen - coated 24 - well trays and cultured at 37 ° c . in an atmosphere of 95 % air / 5 % co 2 in dmem / 10 % fcs that contained antibiotics for 1 day . 2 . the media was removed and cells were cultured in lhm / 10 % fcs / antibiotics for 1 day . 3 . the media was removed and cells cultured in lhm / 10 % fcs ( no antibiotics ) for 20 h . 4 . the media was removed and cells cultured for 30 min in air at 37 ° c . in depletion medium ( 25 mm hepes / tris ( ph 7 . 5 ), 140 mm nacl , 5 . 4 mm kcl , 1 . 8 mm cacl2 , 0 . 8 mm mgso4 , and 5 mm glucose , to normalize intracellular nutrient pools . 5 . transport was initiated by replacing depletion medium with uptake medium ( depletion medium adjusted to ph 6 . 0 or kept at ph 7 . 4 ) that contained 100 μm glysar ( at a specific activity of 5 μci / ml , with [ 3 h ]- glysar supplying 2 . 88 % of total glysar substrate ) and ( or ) 1 mm of inhibiting peptide . an inhibitory substrate concentration of 1 mm was selected because the literature indicates that typical k m values for pept1 ranges from 0 . 5 to 5 mm . therefore , by selecting an inhibitor concentration of 1 mm ( not expected to completely inhibit uptake ), our goal was to more finely delineate the relative abilities of candidate inhibitors than if the typical 5 mm inhibitor concentration ( expected to achieve close to 100 % inhibition of glysar uptake ) was used . candidate peptides were selected based on their containing trp , leu , met , and ( or ) arg , substrates . in total , 23 inhibitory peptides and 2 drug compounds were screened using this protocol . to determine the potential of trp and leu absorption as dipeptides by cpept1 , the ability of trpleu versus leutrp dipeptides to inhibit 100 μm glysar uptake was evaluated ( fig1 ). the presence of either trpleu or leutrp in the ph 6 . 0 uptake buffer abolished h + - dependent glysar uptake by 117 % or 114 %, respectively . in contrast , neither leu nor trp significantly influenced h + - dependent glysar uptake . these results indicate that a lesser concentration of inhibitor would be required to delineate the relative recognition of trpleu and leutrp by cpept1 . with regard to the mechanism of h + - independent glysar uptake observed throughout these experiments , it is of interest to note that trpleu and leutrp inhibited h + - independent glysar uptake by 36 % and 46 %, respectively . to further evaluate the potential of trp to be absorbed in the form of peptides by cpept1 , the ability of trptrp , trpgly , and trpglygly to inhibit glysar uptake was compared ( fig1 ). as observed for trpleu ( fig1 ), trptrp abolished h + - dependent glysar uptake and inhibited h + - independent uptake by about 22 %. trpgly abolished h + - dependent glysar uptake but did not influence h + - independent uptake . the tripeptide trpglygly also significantly inhibited glysar uptake , but to a lesser extent ( 73 %) than did trptrp or trpgly . to determine the relative potential of other amino acids ( met , arg , lys , phe , for example ) to be absorbed in the peptide - bound form , additional glysar competitive inhibition experiments were conducted using the above - described regimen and a variety candidate peptides at 1 mm . the results of these experiments are summarized in table 2 , which also includes those experiments described in fig1 , 14 , 15 , and 16 for comparative purposes . the inhibitors are listed within groupings in order of their relative ability to inhibit 100 μm of glysar uptake . in addition to the listed peptides , the constituent free amino acids were tested within the appropriate experiment to evaluate whether the peptide - bound or free amino acid was responsible for any affect on glysar uptake . as expected , the presence of 1 mm constituent free amino acid did not influence glysar uptake . inhibition percentages of 50 % indicate that the inhibitor substrate was recognized at least as well as was glysar , given that the k m of glysar was determined to be about 1 mm ( fig1 ) and that the substrate was present at 1 mm . of the 19 treatment peptides evaluated , eleven abolished h + - dependent glysar uptake , with seven of these also displaying the ability to inhibit h + - independent glysar uptake . of the remaining eight peptides tested , four displayed greater than 80 % inhibition while four inhibited glysar uptake by 50 % or less . these results indicate that a wide variety of peptides of nutritionally important constituent amino acids are recognized by cpept1 . overall , the observation that cpept1 activity was sensitive to a number of substrates is typical of pept1 function . however , what was surprising was the large number of peptides that completely inhibited glysar uptake . to establish a more sensitive relative inhibitory order among peptides that inhibited glysar uptake by more than 80 %, and , therefore , a more accurate potential for recognition , fourteen peptides were re - screened for their ability to inhibit 100 μm glysar uptake using the same cell culture and transport regimen but using only 10 % of the previous inhibitor concentration ( 100 μm ). the data from an experiment to directly compare the ability of 100 μm trp - containing peptides are shown in fig1 . all trp - containing peptides inhibited h + - dependent glysar uptake . however , trpleu inhibited more ( 92 %) than did leutrp ( 58 %), trptrp ( 62 %), or trpgly ( 45 %). these values and the results of other experiments comparing the relative ability of leu -, met -, and arg - containing peptides are listed in table 3 . overall , four of the peptides inhibited glysar uptake by at least 80 %, six by more than 40 %, and four less than 40 %, thus establishing a relative ranking for recognition by cpept1 . among the five trp - containing peptides ( fig1 , table 3 ), trpleu demonstrated the greatest ability to inhibit glysar uptake . trpleu also demonstrated the greatest ability to inhibit glysar uptake ( 94 %) among the leu - containing peptides . among the met - containing substrates that were directly compared within the same experiment , the neutral peptides , metmet and metphe , inhibited more glysar uptake than did the anionic ( metglu ) or cationic ( metlys ) carboxyl residues . interestingly , as a group the arg peptides demonstrated the least inhibitory ability , seemingly in keeping with the apparent lesser recognition by pept1 of substrates with charged residues . however , it is of interest to note that 100 μm argleu demonstrated a much greater ability to inhibit glysar uptake than did leuarg ( 49 versus 8 . 9 %). to confirm the relative ranking of trpleu & gt ; trptrp inhibition of glysar ( tables 2 and 3 ), michaelis - menton constants for substrate inhibition ( k i ) of glysar uptake by trpleu and trptrp were generated by graphical analyses of ic 50 experiments ( fig1 ). in keeping with the results achieved in the 100 μm - inhibition studies , trpleu inhibited glysar uptake at lower concentrations than did trptrp ( k l = 0 . 2 versus 0 . 75 μm , respectively ). collectively , the results of cpept1 competitive inhibition trials using mdck cells indicate that trpleu is better recognized by cpept1 than any other tested peptide . the results also indicate that a number of trp -, leu , and met - containing peptides also are well recognized by cpept 1 . ultimately , in the intestinal environment , it is the combination of recognition by the transporter and relative resistance of the peptide to luminal and membrane - bound peptidases that will determine how much of a given peptide will be absorbed . in this regard , there is some evidence to suggest that gly - x peptides are more resistant than other peptides , especially by blood and renal peptidases . if so , then glyleu may be a better candidate substrate than trpleu to supply leu . similarly , tripeptides , as a group , are thought to be relatively resistant to hydrolysis . thus , more trpglygly may prove to be absorbed in larger amounts by the intestine than trpleu . an important result of this set of experiments was the establishment of a sensitive experimental regimen / model to evaluate potential affecters of peptide transport capacity . accordingly , this experimental model of mdck cells grown in lhm affords an opportunity to evaluate the effects of various peptide and drug substrates , and hormones and ( or ) growth factors , on the expression of pept1 . thus , the culture of mdck cells in lhm versus dmem results in an increase of h + - dependent glysar uptake ( k m = 1 . 1 mm ) that is consistent with mammalian pept1 - like activity . using this stimulated model , the ability of twenty - three di - and tripeptides at 1 mm , and fourteen at 100 μm , extracellular concentrations were screened for their ability to inhibit 100 μm glysar uptake , as an indicator of recognition by pept1 . of the trp - and ( or ) leu - containing peptides evaluated , trpleu ( k i = 0 . 2 μm ) and leutrp ( k l = 0 . 75 μm ) demonstrated the greatest ability to inhibit glysar uptake , with trpleu demonstrating a relatively higher affinity ( lower k l ) for pept1 . of the met - containing peptides evaluated , four ( metmet , metphe , leumet , metleu ) appear particularly well recognized by pept1 . in contrast , as a group , arg - containing peptides displayed the least inhibition of pept1 activity . overall , these results indicate that epept1 is capable of recognizing a variety of di - and tripeptides , including , for example , those that contain leucine and tryptophan . experimental model to determine whether the h + / peptide transport capacity expressed by mdck cells is sensitive to substrate regulation examples 1 and 2 above demonstrated that madin - darby canine kidney ( mdck ) cells express pept1 mrna and characterized h + - dependent biochemical properties . therefore , mdck cells were chosen as the experimental model to determine whether the h + / peptide transport capacity expressed by mdck cells is sensitive to substrate regulation . research from example 2 demonstrated that mdck cells grown in lactalbumin hydrolysate medium ( lhm ) had elevated levels of peptide uptake capacity . accordingly , to avoid potential confounding effects of the peptide - containing lhm and individual treatment peptides , dmem ( contains no peptides ) and not lhm was selected as the appropriate medium to test the influence of extracellular peptides on canine pept1 functional capacity of mdck cells . glyphe was selected as a substrate because it has been reported to increase brush border membrane content of pept1 , ( shiraga t , miyamoto k , tanaka h , yamamoto h , taketani y , morita k , tamai i , tsuji a , takada e . cellular and molecular mechanisms of dietary regulation on rat intestinal h +/ peptide transporter pept1 . gastroenterology 1999 ; 116 : 354 - 362 ), whereas phe and gly were tested as constituent free amino acid treatment controls . carnosine was selected because of its high content in meat - based diets . all cells were plated ( 60 , 000 / 2 cm 2 well ) and cultured ( 95 % air / 5 % co 2 , 37 ° c .) for 24 h in dulbecco &# 39 ; s modified eagle media / 10 % fetal calf serum ( fcs )/ 1 % antibiotic / antimicrobial solution ( abam ) ( dmem media ). following these initial common culture conditions , cells then were cultured in dmem , or dmem that contained 10 mm of camosine , glyphe , phe , or gly . media were changed every 24 h . media treatments ( n = 8 ) were as follows : the measurement of [ 3 h ] glysarcosine uptake was performed by using a 24 - well cluster tray method ( kilberg m s . measurement of amino acid transport by hepatocytes in suspension and monolayer culture . methods enzym 1989 ; 173 : 564 - 575 . matthews j c , aslanian a , mcdonald k k , yang w , malandro m s , novak d a , kilberg m s . an expression system for mammalian amino acid transport using a stably maintained episomal vector . anal biochem 1997 ; 254 : 208 - 214 ), and used in examples 1 and 2 . cells were cultured for 30 min in air at 37 ° c . in depletion medium ( 25 mm hepes / tris ( ph 7 . 5 ), 140 mm nacl , 5 . 4 mm kcl , 1 . 8 mm cacl2 , 0 . 8 mm mgso 4 , and 5 mm glucose ), to normalize intracellular nutrient pools before transport . the transport assays are initiated by replacing depletion medium with uptake medium ( depletion medium adjusted to ph 6 . 0 ) that contained 100 μm glysar ( 5 μci / ml , with [ 3 h ]- glysar supplying 2 . 88 % of total glysar ). after a 30 minute incubation period , transport was terminated with four rinses of 4 ° c . depletion medium ( ph 7 . 5 ). two hundred and twenty μl of 10 % trichloroacetic acid was added to each well , and the radioactivity of the supernatant quantified by liquid scintillation counting . the cells of each well are solubilized in 0 . 2 n naoh / 0 . 2 % sds and the protein quantified by using the modified lowry assay , using bovine serum as a standard . id . peptide uptake will be reported as pmol * mg − 1 protein * 30 min − 1 . uptake measurements were taken after 24 , 48 , and 72 hours of culture in treatment media . the previous research characterizing h + - dependent peptide transport by mdck cells ( example 2 above ) clearly showed that transport velocity is dependent on protein content . therefore , to make a valid comparison of various treatment parameters on glysar uptake , the protein content of compared treatment groups must not differ . accordingly , the influence of culture media on mdck cellular protein was evaluated ( fig1 ). all media treatments supported cellular growth from 1 to 3 d and no difference in protein content among treatments was observed . similarly , no difference in uptake velocity ( capacity ) was observed among treatment groups , for any culture period ( fig2 ). the results from trial 1 suggest that either canine pept1 is not sensitive to substrate regulation or that the substrates and ( or ) stimulation time were inadequate to influence h + - dependent peptide uptake in mdck cells . again , dmem was selected as the basal medium to allow the effect of individual peptides on peptide transport activity to be evaluated . to evaluate the latter two possibilities , a second trial was conducted that included a culture period of 9 d . glysar was added as another potential affecter of h + - dependent peptide transport capacity because 10 mm glysar it is reported capable of stimulating increased pept1 activity ( adibi s . the oligopeptide transporter pept1 in human intestine : biology and function . gastroenterology 1997 ; 113 : 332 - 340 ) in caco - 2 cells . glypro was added as a treatment because of its high content in muscle tissue , thus is likely to be abundant in meat - based diets . the mdck cell line was maintained as described previously in the methods section of trial 1 . following initial and common culture conditions , cells were cultured in dmem , or dmem that contained 10 mm glysar , glypro , glyphe , or carnosine . media were changed every 24 h . media treatments ( n = 8 ) were as follows : the measurement of [ 3 h ] glysarcosine uptake was performed by using the 24 - well cluster tray method as previously described in the methods section of trial 1 . peptide uptake will be reported as pmol * mg − 1 protein * 30 min − 1 . uptake measurements were taken after 4 , 12 , 24 , 36 , 72 , 120 , 168 , and 216 hours of culture in treatment media . protein content in all treatment groups increased linearly from 4 to 216 h ( 9 d ) of culture , for all treatment groups ( fig2 ). however , within a culture period , protein contents of treatment groups did not differ . over the 216 - h culture period , protein increased about 4 . 5 times , from about 40 to 220 μg / well . in contrast to trial 1 results , media treatment did influence glysar uptake capacity ( fig2 ). in addition , a treatment × time effect was observed that represents differences in the time of culture required for glysar and carnosine treatment stimulation of glysar uptake capacity . specifically , glysar containing dmem culture treatment resulted in an increase in glysar uptake capacity of about 30 % over dmem control media by 24 h of culture time . this level of increase was maintained through 216 h . in contrast , culture in carnosine - containing media did not result in a significant ( 23 %) increase of glysar uptake capacity over that by dmem - cultured cells until 72 h of culture . this stimulation then steadily increased to 291 % over 216 h of culture . the nature of stimulated uptake between the two peptide substrates also differed . that is , the magnitude of carnosine - stimulated glysar uptake was essentially constant from 72 to 216 h , whereas that for glysar culture decreased during this period . collectively , these data indicate that h + - dependent peptide transport in cultured mdck cells can be stimulated by at least two of pept1 substrates , glysar and carnosine . the data from trial 2 indicate that h + - dependent glysar uptake capacity by fed mdck cells can be upregulated by the inclusion of 10 mm glysar for at least 24 h and 10 mm carnosine for at least 72 h . it is of equal interest to understand if h + - dependent glysar uptake capacity is sensitive to nutrient deprivation and ( or ) stimulation by glucocorticoids . a preliminary study indicates that fasting increases the expression of pept1 in rat small intestine epithelia . thamotharan m , bawani s , zhou x , adibi s . functional and molecular expression of intestinal oligopeptide transporter ( pept1 ) after a brief fast . metabolism 1999 ; 48 : 681 - 684 . to initiate investigation of potential influence of fasting and glucocorticoids on mdck cells expression of glysar uptake capacity , the h + - dependent uptake of glysar was evaluated over a 72 period of nutrient deprived or fed and cultured with dexamethasone ( dex ) and compared to that by cells cultured in dmem or dmem that contained insulin ( negative control ) ( trial 3a ). the “ nutrient deprived ” treatment actually contained 5 mm glucose and appropriate salts to ensure adequate basal metabolic conditions . although recruitment of pept1 protein and activity appears sensitive to insulin - stimulated recruitment from cytosolic vesicles in caco - 2 cells ( thamotharan m , bawani s , zhou x , adibi s . hormonal regulation of oligopeptide transporter pept1 after a brief fast . am j physiol 1999 ; 276 : c821 - 826 , mdck cells are reported to be insensitive to insulin , likely as an inability to express the insulin receptor . hofmann c , crettaz m , bruns p , hessel p , hadawi g . cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors . j cell biol 1985 ; 27 : 401 - 414 . in contrast to the lack of insulin sensitivity , igf - i is known to stimulate dna synthesis and cell proliferation in mdck cells . sukegawa i , hizuka n , takano k , asakawa k , shizume k . characterization of igf - 1 receptors on mdck cell line . endocrinol japan 1987 ; 34 ( 3 ): 339 - 346 . mouzon s h , kahn r . insulin - like growth factor - mediated phosphorylation and proto - ontogeny induction in mdck cells . mol endocrinol 1991 ; 5 : 51 - 60 . the understanding that mdck cells are apparently insensitive to insulin stimulation yet are sensitive to igf - i stimulation appears to be a paradox given that the supraphysiologic levels of both substrates employed in the perspective studies and the known ability of insulin to cross react with the igf - i receptor . accordingly , another trial ( trial 3b ) was conducted to evaluate the influence of increasing igf - i concentrations on h + - dependent glysar uptake by mdck of the same plating stock . mdck cells were maintained as described in trial 1 , except that cells were cultured for only 1 d before transport trials were performed . following initial and common culture conditions , cells were cultured in a “ nutrient depleted ” buffer ( hepes / tris ( ph 7 . 5 ), 140 mm nacl , 5 . 4 mm kcl , 1 . 8 mm cacl 2 , 0 . 8 mm mgso 4 ) that contained 5 mm glucose as an energy source , but that lacked amino acid or vitamin sources . in contrast , cells cultured in dmem , or dmem that contained 5 nm dex , 500 nm dex , 5 nm insulin , or 500 nm insulin , were adequately nourished . media treatments ( n = 4 ) were as follows : the measurement of [ 3 h ] glysarcosine uptake was performed by using the 24 - well cluster tray method as previously described in the methods section of trial 1 . peptide uptake is reported as pmol * mg − 1 protein * 30 min − 1 . uptake measurements were taken after 30 min and 4 h of culture in treatment media . trial 3b was conducted in the same manner as described for trail 3a , except that cells were cultured in dmem or dmem that contained 1 nm igf - 1 , 5 nm igf - 1 , 25 nm igf - 1 , or 100 nm igf - 1 . uptake measurements were taken after 30 min and 4 h of culture time . media treatments ( n = 4 ) were as follows : protein content of the treatments within trails 3a or 3b did not differ . after 4 h of culture , however , the capacity for h + - dependent peptide uptake was reduced 35 % in cells deprived of nutrients but adequate in energy ( fig2 ). in contrast , dexamethasone had no effect on glysar uptake . as expected , and consistent with the concept that mdck cells are insulin - insensitive , the presence of insulin for 4 h had no effect on glysar uptake capacity . similarly , culture of cells with increasing amounts of igf - i elicited no significant stimulation of h + - dependent glysar uptake ( fig2 ). quantitatively , however , 1 to 25 nm of igf - i tended to increase glysar uptake capacity by 10 to 15 %. given the noted restrictions of trail 3 , and the low number of observations ( n = 4 ) results from trial 3a and 3b suggest that h + - dependent uptake of glysar by mdck is sensitive to nutrient deprivation and , perhaps , igf - i . clone12 ( 5 th round ; seq id no : 11 ) primer pair is gsp3 - 4 ; gsp3 - 1r using regular rt - pcr clone37 beginning ( 6 th round ; seq id no : 12 ) primer pair is gsp3 - 9 ; auap using 3 ′ race protocol gccatcgccatacccttctagtatgggccccc aataattaccgagtggtaaaggatggcc ttaaccagaagccagaaaa aggagaaaatggaatcagatttataaatagtctt aatgagagcctcaacatcaccatgggcgacaaagtttatgtgaatgtc accagtcacaatgccagcgagtatcagttctttt ctttgggcacaaaaaacattacaataagttcaacacaacagatctcac aaaattgtacaaaagttctccaatcatccaaccttgaa tttggtagtgcatatacctatgtaatcggaacgcagagcactggc tgccctgaatgcatatgtttgaagatattcaccc aacacagttaacatggctctgcagatcccgcagtacttcctcatcacc tgcggcgaggtggttttctctgtcacaggact ggagttctcatattctcaggccccctccaacatgaagtcggtgcttcagg cgggatggctgctgacagtggcttgttggcaa catcattgtgctcattgtggcaggagcaggccagttcagtgaacagtg ggctgaatacatcctatttgcggcattgctt ctggttgtctgtgtaatatttgccat catggcccggttttacacttacgtcaatc cagcagagattg catcttcttcatcgtggtcaatgagttgt gaaagattttcctactatggaatgagagca ctcctgattctgtacttcagacgg ttcatcgggtgggacgataatctgtccacgg rcatctaccacacgtttgtggctctgt gctacctgacgccgatcctcggc gcactgatcgcagactcctggctgggaaag ttcaagacaatcgtgtcactctccattgtctacacaattggacaggcggtc actgcagtaagctcaattaatgacctc acagactataacaaagatggaactcc tgacaatctgtccgtgcatgtggcactgt ccatgattggcctggccctgatagctct gggaactggaggaataaagccctgtg tgtctgcatttgtggagaccagtttg aagagggccaggaaaaacaaagaaacag attcttttccatcttttantggccatt aatgctggaagcttgatttccactattg tcactcccatgctcagagttcacgaatgt ggaatttacagtcagaaagcttgtta cccactggcatttggggttcctgctgct ctcatggccgtatctctgattgtatttgtca ttggcagtggaatgtacaagaagtttcag ccccagggtaatgtcatgggtaa agttgtcaagtgcattggttttgccctc aaaaataggtttaggcaccggagt aagcagtttcccaagagggagcactggct ggactgggctaaagagaaatacgatgag cggctcatctctcaaattaagatggtcac aaaagtgatgttcttgtacatccc actcccaatgttctgggccctgtttgacc agcagggctccaggtggacactgcaagc aacagctatgagtgggaaaattg gacttcttgaagttcagccagatcagat gcagactgtgaatgccatcttgattgtc gtcatggtccccatcatggatgccgt ggtgtaccctctgattgcaaaatgtggc ttcaatttcacctccttgaagaggatg acagttggaatgttcctggcttccatgg ccttcgtgatggcggcgattgttcagct ggaaattgataaaactcttccagtctt ccccaaacaaaatgaagtccaaatcaa agtactgaatataggaaatggtgccatg aatgtatcttttcctggagcggtggtg acagttagccaaatgagtcaatcagat ggatttatgacttttgatgtagacaaac tgacaagtataaacatttcttccact ggatcaccagtcattccagtgacttataact ttgagcagggccatcgccatacccftctag tatgggcccccaataattaccgag tggtaaaggatggccttaaccagaa gccagaaaaaggagaaaatggaatcaga tttataaatagtcttaatgagag cctcaacatcaccatgggcgacaaagttt atgtgaatgtcaccagtcacaatgcc agcgagtatcagttcttttcttt gggcacaaaaaacattacaataagttcaacacaa cagatctcacaaaattgtacaaaagttct ccaatcatccaaccttgaatttgg tagtgcatatacctatgtaatcggaacgca gagcactggctgccctgaattgcatat gtttgaagatatttcacccaacacag ttaacatggctctgcagatcccgcagtac ttcctcatcacctgcggcgaggtggttttct ctgtcacaggactggagttctcatattct caggccccctccaacatgaagtc ggtgcttcaggcgggatggctgctgacagtggct tgttggcaacatcattgtgtctcattgtggcaggagcaggccagtt cagtgaaacagtgggctgaatacatcctatttgcggcattgcttct ggttgtctgtgtaatatttgccatcatggcccggtttt acacttacgtcaatccagcagagattg after analyzing the protein sequence and performing alignment with other species , the underlined , italicized was removed for submission to genbank . catcttcttcatcgtggtcaatgagttctgtgaaa gattttcctactatggaatgagagca ctcctgattctgtacttcagacgg ttcatcgggtgggacgataatctgtcca cggccatctaccacacgtttgtggctct gtgctacctgacgccgatcctcggc gcactgatcgcagactcctggctgggaa agttcaagacaatcgtgtcactct ccattgtctacacaattggacaggcggtc actgcagtaagctcaattaatga cctcacagactataacaaagatgg aactcctgacaatctgtccgtgcatgtggcactgt ccatgattggcctggccctgatag ctctgggaaactggaggaataaag ccctgtgtgtctgcatttggtggagaccagtttg aagagggccaggaaaaacaaag aaacagattcttttccatcttttattt ggccattaatgctggaagcttgatttccacctattg tcactcccatgctcagagttcacgaat gtggaatttacagtcagaaagcttgtt acccactggcatttggggttcctgctgct ctcatggccgtatctctgattgtattt gtcattggcagtggaatgtacaagaag tttcagccccagggtaatgtcatgggtaa agttgtcaagtgcattggttttgccct caaaaataggtttaggcaccggagtaa gcagtttcccaagagggagcactggct ggactgggctaaagagaaatacgatga gcggctcatctctcaaattaagatggt cacaaaagtgatgttcttgtacatccc actcccaatgttctgggccctgtttga ccagcagggctccaggtggacactgc aagcaacagctatgagtgggaaaattg gacttcttgaagttcagccagatc agatgcagactgtgaatgccatcttgattgtcgtcatggtccccatcatggatgccgt ggtgtaccctctgattgcaaaatgt ggcttcaatttcacctccttgaaga ggatgacagttggaatgttcctggcttccatgg ccttcgtgatggcggcgattgttca gctggaaattgataaaactcttcc agtcttccccaaacaaaatgaagtccaaatcaa agtactgaatataggaaatggtgcc atgaatgtatcttttcctggagcgg tggtgacagttagccaaatgagtcaatcagat ggatttatgacttttgatgtagaca aactgacaagtataaacatttcttcc actggatcaccagtcattccagtgacttataact ttgagcagggccatcgccatacccttct agtatgggcccccaataattaccgagt ggtaaaggatggccttaaccagaag ccagaaaaaggagaaaatggaatcaga tttataaatagtcttaatgagagcctc aacatcaccatgggcgacaaagtttat gtgaatgtcaccagtcacaatgccag cgagtatcagttcttttctttgggcac aaaaaacattacaataagttcaacacaac agatctcacaaaattgtacaaaagttct ccaatcatccaaccttgaatttggtagt 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acctatgtaatcggaacgcagag cactggctgccctgaattgcatatgtttgaagatat ttcacccaacacagttaacat ggctctgcagatcccgcagtactt cctcatcacctgcggcgaggtggttttctctgtcaca ggactggagttatcatattctca ggccccctccaacatgaagtcggt gcttcaggcgggatggctgctgacagtggctgtt ggcaacatcattgtgctcattgtg gcaggagcaggccagttcagtgaa cagtgggctgaatacatcctatttgcggcattg cttctggttgtctgtgtaatattt gccatcatggcccggttttacactt acgtcaatccagcagagattgaagctcagtttgacg acgatgagaaaaagaacctggaaaa gatgaatgtatattccacggtaactccggtctcacagacacagatg mgmsksygcfgyplsiffivvnef cerfsyygmrallilyfrrfigwddnls taiyhtfvalcyltpilgaliads wlgkfktivslsivytigqavtavssindl tdynkdgtpdnlsvhvalsmig lalialgtggikpcvsafggdqfeegqek qrnrffsifylainagslisti vtpmlrvhecgiysqkacyplafgvpaalma vslivfvigsgmykkfqpqgn vmgkvvkcigfalknrfrhrskqfpkreh wldwakekyderlisqikmv tkvmflyiplpmfwalfdqqgsrwtlqata msgkigllevqpdqmqtvnai livvmvpimdavvypliakcgfnftslkrm tvgmflasmafvmaaivql eidktlpvfpkqnevqik vlnigngamnvsfp gavvtvsqmsqsdgfmifdvdklts inisstgspvipvtynfeqghrhtllv wapnnyrvvkdglnqkpekgeng irfinslneslnitmgdkvyvnvtshn aseyqffslgtknitisstqqis qnctkvlqssnlefgsaytyvigtqstgcpe lhmfedispntvnmalqipqyfli tcgevvfsvtglefsysqapsnmksvlq agwlltvavgniivlivagagqf seqwaeyilfaalllvvcvifaimarfyt yvnpaeieaqfdddekknlekmnvystvtpvsqtqm all publications , patents and patent documents are incorporated by reference herein , as though individually incorporated by reference . the invention has been described with reference to various specific and preferred embodiments and techniques . however , it should be understood that many variations and modifications may be made while remaining within the scope of the invention .