Patent Application: US-93077797-A

Abstract:
methods of inhibiting leukocyte o 2 − production and attracting luekocytes using specific peptides are disclosed . theses peptides include the proline - arginine - rich antimicrobial peptide known as pr - 39 and truncated analogs thereof . these peptides can be used as medicaments that fight infection by attracting leukocytes to a wound site , yet restrict tissue damage at the wound site caused by excessive oxygen radicals produced by these leukocytes .

Description:
the following example illustrates the preferred practice of the invention . it is to be understood , however , that this example is provided by way of illustration only and nothing therein should be taken as a limitation upon the overall scope of the invention . peptide design and synthesis . theoretical predictions of peptide characteristics , relative to hydrophilicity , hydrophobicity , and antigenicity , were accomplished using a computer software program ( peptide companion , peptide international , louisville , ky .). the following peptides were synthesized : pr - 39 , pr - 26 , pr - 23 , pr - 19 , pr - 16 , pr - 15 , and pr - 14 ; the sequences of these peptides appear in the sequence listing as sequence id nos . 1 through 7 , respectively . peptides were synthesized by the solid - phase method using t - boc chemistry with an applied biosystems model 431 peptide synthesizer ( abi , foster city , calif .). amino acid derivatives having the l - configuration were used . peptide purification and characterization were conducted as described previously ( shi et al ., 1994b ; shi et al ., 1996 ). briefly , the peptides were purified on a reversed - phase high - performance liquid chromatography ( rp - hplc ) system ( beckman instruments , fullerton , calif .) with a vydac 218 tp c 18 column ( 0 . 46 × 25 cm ), analyzed by fast - atom mass spectrometry ( autospec - q ; vg analytical ltd ., manchester , united kingdom ), and visualized by acid - urea polyacrylamide gel electrophoresis ( au - page ). in the au - page analysis , peptides ( 10 μg ) were dissolved in 20 μl of sample buffer ( 3m urea with 5 % acetic acid ) and the gels were run at 150v for approximately 15 min . or until the dye front ( methyl green ) had migrated to the end of the gel . the gel was stained with 0 . 3 % amido black . antibacterial activity assays . synthetic peptides were evaluated for antibacterial activity by previously described gel - overlay and “ lawn - spotting ” assays ( shi et al ., 1994b ), by determination of the minimal inhibitory concentrations ( mics ) and the minimal bactericidal concentrations ( mbcs ) of pr - 26 and pr - 39 , and by determination of the postantibiotic effect ( pae ) of pr - 26 and pr - 39 and the bacterial susceptibility to neutrophil phagocytosis by pr - 26 and pr - 39 . ( i ) gel - overlay assay . peptides were subjected to au - page as described previously ( shi et al ., 1994b ). the acid - urea gels were overlaid with 10 6 bacteria ( e . coli , atcc 25922 ) in 3 % tryptcase soy broth ( tsb ) and 1 % agarose medium , and the overlaid gels then were incubated at 37 ° c . for 18 hr . bactericidal activity was indicated by clear zones on the agarose gels . ( ii ) lawn - sooting assay . lawns of bacteria ( e . coli , attc 25922 , or s . typhimurium , ksu isolate 7 ) were made on sheep - blood or brain - heart - infusion agar plates . after drying at room temperature for 10 min , 5 μl of the various peptides ( dissolved in 0 . 01 % acetic acid or phosphate buffered saline ( pbs ), ph 7 . 4 ) and a medium control were spotted on the surface of the bacterial lawn . plates were incubated at 37 ° c . for 18 hr . bactericidal activity was indicated by a clear zone on the bacterial lawn ( shi et al ., 1994b ). ( iii ) mics and mbcs . for peptides that showed antibacterial activity using the gel - overlay and lawn spotting assays , mics and mbcs were determined by the microdilution broth method ( nccls , 1990 ). briefly , 50 μl of twofold serial dilutions ( 128 to 0 . 25 μm ) of synthetic peptides were dispensed into wells of 96 - well tissue culture plates . bacteria ( e . coli , atcc 25922 ; e . coli , k88 ; s . typhimurium , fresh isolate from a dog ; salmonella choleraesuis atcc 6962 ; streptococcus suis , fresh isolate from porcine spleen ; and staphylococcus aureus ) from sheep - blood or brain - heart - infusion agar plates were standardized to 0 . 5 mcfarland in demineralized water using a radiometer / sensititre ( chelsea instrument ltd ., england ). the water / bacteria suspension ( 100 μl ) was immediately transferred to 10 ml of cation - adjusted muller - hinton broth and 50 μl of the bacterial suspension then were added to each well of the microtiter plate . plates were incubated for 20 hr . at 37 ° c . and mics were determined . ten microliters of bacterial suspensions in muller - hinton broth also were diluted to determine the actual bacterial concentration using standard colony forming unit ( cfu ) counting . after determination of the mics , 10 μl of each bacteria - peptide suspension were plated on sheep - blood or brain - heart - infusion agar plates and incubated for 24 hr . at 37 ° c . to determine the mbcs . mbc was considered that peptide concentration that inhibited 99 . 9 % of the original cfus ( nccls , 1990 ). ( iv ) pae . s . typhimurium was used to evaluate the pae of pr - 26 and pr - 39 . stationary phase bacteria were adjusted to 5 × 10 7 bacteria / ml in brain - heart - infusion agar and incubated with different concentrations of pr - 26 or pr - 39 at 37 ° c . for 2 hr . control tubes without pr - peptides were treated in an identical manner to the experimental tubes . pr - 26 and pr - 39 were removed by centrifugation ( 13 , 600 × g for 1 min .) and 100 μl of the bacteria were resuspended in 0 . 9 ml of pr - peptide - free brain - heart - infusion agar and incubated at 37 ° c . bacteria ( 20 μl ) were diluted in sterile saline immediately after removal of the pr - peptides and then at hourly intervals , and 20 μl aliquots were spread on nutrient agar plates . viable bacteria were counted . tests were repeated on three different days . pae was determined by calculating the difference in time required for the number of test and control bacteria to increase 1 log 10 above the number present immediately after removal of pr - peptides from the test cultures . the results were expressed as the mean ± standard deviation . a pae greater than 30 min . was considered significant ( mackenzie and gould , 1993 ). ( v ) susceptibility to neutrophil phagocytosis . porcine neutrophils were isolated from 6 to 8 week - old - crossbred pigs by density - gradient centrifugation and hypotonic lysis as previously described ( shi et al ., 1994a ). s . choleraesuis , atcc 6962 was used in this experiment . bacteria were incubated with pr - 26 or pr - 39 for 10 min . at 37 ° c . peptides were removed from the bacterial cultures by centrifugation at 13 , 600 × g for 1 min . bacteria were resuspended in 1 ml of pbs and 0 . 1 ml of bacteria was mixed with 2 × 10 6 porcine neutrophils . the final volume was adjusted to 0 . 5 ml , 15 % porcine serum . bacteria without neutrophils and neutrophils without bacteria were used as controls . tubes were incubated at 37 ° c . in a reciprocating water bath at 110 oscillations / min . aliquots of 50 μl from the experimental tubes were used to prepare slides using a cytospin 2 centrifuge ( shandon products ltd ., pittsburgh , pa .). slides were stained with leukostat solutions ( fisher , pittsburgh , pa .). phagocytosis was determined by light microscopy at a magnification of 1 . 000 . at least 200 neutrophils were examined . the degree of phagocytosis was calculated according to the following formula : phagocytic index =( percentage of neutrophils containing at least one bacteria )×( mean number of bacteria per positive cell ). tests were repeated on three different days . results were expressed as the mean ± standard deviation . influence of pr - 39 on neutrophil chemotaxis . chemotaxis of porcine neutrophils was measured by the procedure of salak et al . ( 1993 ). briefly , pr - 14 , pr - 15 , pr - 16 , pr - 26 , or pr - 39 ( 30 μl in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ) were placed in the bottom chamber of a modified boyden chamber ( neuro probe , cabin john , md .) and porcine neutrophils ( 50 μl at 5 × 10 6 cells / ml ) were placed in the top chamber . the chambers were incubated at 37 ° c . for 30 min . cells that migrated through the porous membrane ( pore size 5 μm ) were stained using leukostat solution and enumerated . five microscope fields were counted and the cells that migrated through the membrane were standardized to the medium control and referred to as the migration index . influence of pr - 26 and pr - 39 on intestinal epithelial cells . a nonradioactive assay based on the cellular conversion by viable cells of a tetrazolium salt into a blue formazan product was used to determine if pr - 26 or pr - 39 were toxic to intestinal epithelial cells . ninety - six - well microtiter plates were seeded with the rat intestinal epithelial cell line , iec6 , ( 5 × 10 4 cells / well ) in dmem containing 10 % fetal bovine serum , 1 % antibiotic / antimycotic , and 0 . 1 bovine insulin . and incubated at 37 ° c . for three days to achieve confluency . cells then were incubated with different concentrations of pr - 26 or pr - 39 in medium without antibiotic / antimycotic for three days . well contents then were aspirated and monolayers were washed with medium . medium ( 100 μl ) and 15 μl of dye solution ( promega , madison , wis .) were added to each well and plates were incubated for 4 hr . at 37 ° c . solubilization buffer ( 100 μl , promega , madison , wis .) was added to each well and plates were incubated overnight to allow solubilization of the formazan crystals . absorbance then was read at 570 nm using a microplate reader . effect of pr - 26 on survival of mice challenged with s . typhimurium . three separate s . typhimurium challenge studies were conducted with mice . in the first experiment , 45 a / j mice ( jackson labs , bar harbor , me .) were orally challenged with s . typhimurium . feed was withdrawn 4 hrs . prior to an oral inoculation . twenty min . prior to the inoculation with s . typhimurium mice were given a 30 μl dose of a 10 % sodium bicarbonate solution in pbs . s . typhimurium , strain ksu007 , was started in brain heart infusion broth overnight and then grown for 6 hrs . in luria broth base ( gibco , gaithersburg , md .). after centrifugation , bacteria were suspended in a 1 % gelatin solution ( final concentration 1 . 24 × 10 10 s . typhimurium / ml ), and then 25 μl were given orally . immediately following oral inoculation with s . typhimurium , mice were given 0 , 100 , or 250 μg of pr - 26 in pbs . a second dose of pr - 26 or pbs was given 4 hrs . later . in the second experiment , 45 balb / c ( salmonella susceptible ) mice were given i . p . injections of s . typhimurium ( 2 × 10 4 / ml ) that had been grown as previously described , but resuspended in pbs . mice then received either 0 , 50 , or 100 μg of pr - 26 in 100 μl pbs i . p . immediately after the s . typhimurium . in the third experiment , the efficacy of timing of delivery of pr - 26 was evaluated . the protocol was the same as in the second experiment , except that only the 50 μg / mouse dose of pr - 26 was used and the pr - 26 was delivered immediately ( time 0 ), or 24 , 48 , or 72 hrs . after the s . typhimurium injection . a control group received pbs at time 0 . fifteen mice were used in all treatments except the 72 - hour treatment , which had 16 . ( i ) o 2 − production assays . whole - cell o 2 − production by porcine peripheral blood neutrophils was determined by the superoxide dismutase - inhibitable reduction of ferricytochrome c as previously described ( shi et al ., 1994a ). cell - free o 2 − production was measured as previously described ( leto et al ., 1991 ) using 96 - well plates and a molecular devices thermomax microplate reader ( menlo park , calif .). reactions ( 100 μl ) contained 10 6 cell equivalents of human neutrophil cytosol and 5 × 10 5 cell equivalents of deoxycholate - solubilized membranes prepared from human peripheral blood neutrophils . reaction mixtures contained 50 mm potassium phosphate ( ph 7 ), 0 . 2 mm acetylated cytochrome c , 4 mm mgcl 2 , 1 mm egta , 10 μm fad , 1 μm gtp - γs , and 200 μm nadph . the reactions were initiated by addition of 40 μm arachidonic acid . control reactions contained 5 μg of superoxide dismutase . superoxide anion generation was calculated based on superoxide dismutase - inhibitable changes in cytochrome c absorbance observed at 551 nm . the reactions were followed for 20 min . after addition of arachidonic acid , with absorbance readings taken at 1 - min . intervals . maximum rates of superoxide generation were calculated from a linear least squares fit of 10 consecutive 1 - min . data points . determinations were based on reactions performed in duplicate . ( ii ) binding assays . neutrophils were disrupted by sonication , and unbroken cells , debris , and granules were removed by centrifugation ( 13 , 000 × g for 60 min .). cytosol fractions were prepared by centrifugation ( 100 , 000 g for 45 min .) of the supematants to remove residual membranes . cytosolic proteins ( 15 μg in reducing buffer ) were separated on a 7 . 5 % sds page gel , transferred to pvdf membranes , and probed with biotinylated pr - 39 ( 0 . 3 μm ) for 2 hr . binding between pr - 39 and cytosolic proteins were visualized by a chemiluminescent streptavidin alkaline phosphatase system using an epson scanner ( torrance , calif .) and the nih image program . pr - 39 was labeled with biotin hydrazide at the carboxy - terminus ( pierce chemical co ., rockford , ill .) and purified by gel filtration chromatography . competitive ligand - blot assays were performed by incubating recombinant p47 phox ( 0 . 02 μm ) with or without various concentrations ( 0 . 006 , 0 . 06 , and 0 . 6 μm ) of pr - 39 for 1 hr . at 37 ° c . and then with biotinylated pr - 39 ( 0 . 06 μm ) for an additional 1 hr . at 37 ° c . the solutions were dialyzed against ddh 2 o for 1 hr . at 4 ° c . before blotting to nitrocellulose ( 5 μl ) and detection with a streptavidin alkaline - phosphatase detection system as described above . methods for production of the glutathione s - transferase ( gst )- p47 phox - sh3 ( residues 151 - 284 ) fusion protein have been previously described ( leto et al ., 1994 ). for solution binding assays , lysates of recombinant p22 phox protein ( residues 127 - 195 ) from baculovirus - infected cells ( 2 . 5 × 10 4 cell equivalents per assay ) were mixed with peptides and incubated for 1 hr . at 20 ° c . with 100 μl of gst - p47 phox bound to glutathione - sepharose beads ( 15 % suspension ). bound proteins were washed three times in 15 volumes of ice - cold 100 mm kcl / 3 mm nacl / 3 . 5 mm mgcl 2 / 0 . 15 mm phenylmethanesulfonyl fluoride / 10 mm pipes ( ph 7 . 5 ), eluted with 1 % sds , and analyzed by sds - page followed by immunoblotting with mouse anti - p22 phox antibody ( mab 449 ; a . verhoeven , central laboratories of the netherlands red cross ). controls included blotting of recombinant p22 phox lysate ( 5 × 10 3 cell equivalents ) alone ( p22 ) and bound complexes detected without competing pr peptides (−). peptide design and synthesis . pr - 39 and six analogs were synthesized and named pr - 39 ( whole molecule ), pr - 26 ( nh 2 - terminal segment 1 to 26 ), pr - 23 ( control segment 4 to 26 ), pr - 19 ( nh 2 - terminal segment 1 to 19 ), pr - 16 ( central segment 11 to 26 ). pr - 15 ( cooh - terminal segment 25 to 39 ), and pr - 14 ( nh 2 - terminal segment 1 to 14 ). peptide sequences are illustrated in fig1 . rationale for design of the peptides was based on the graphic protein hydrophilicity scale ( fig2 ), since the hydrophilicity profile indicates locations of important interaction sites such as antibody and receptor binding sites ( hopp , 1985 ). pr - 26 was designed to mimic the hydrophilicity profile of pr - 39 which has a hydrophilic nh 2 - terminus and a hydrophobic cooh - terminus . as most endogenous antibacterial peptides are cationic molecules , pr - 14 , the highest positively charged segment ( 43 % vs . 25 % of whole molecule ), was designed to test if cationicity itself was enough for antibacterial activity . the central domain , pr - 16 , having the average positive charge intensity ( 25 %) was synthesized as the control for pr - 14 . pr - 15 , the cooh - terminus of pr - 39 was designed to determine if it was one of the functional domains of pr - 39 . pr - 23 was designed to evaluated the importance of the first three arginine residues of pr - 26 . pr - 19 was designed to assess the contribution of the cooh - terminus to the activity of pr - 26 . calculated molecular weights and experimental determinations of the mass of synthetic peptides suggested that these peptides possessed the designed sequences ( 1813 , 1777 , 1939 , 3230 , and 4720 calculated molecular weight vs 1812 , 1777 , 1941 , 3230 , and 4720 experimental determination of mass for pr - 14 , pr - 15 , pr - 16 , pr - 26 , and pr - 39 , respectively ). synthetic peptides (& gt ; 95 % purity in rp - hplc ) were subjected to au - page to further determine the purity and charge intensity . fig3 shows the results of this analysis for pr - 14 , pr - 15 , pr - 16 , pr - 26 , and pr - 39 ( data not shown for pr - 19 and pr - 23 ). as expected , pr - 14 migrated in the front since it has the highest positive charge intensity ; pr - 15 , having the lowest positive charge intensity , migrated far behind pr - 14 even though its mass is slightly less than pr - 14 . because pr - 16 and pr - 26 are smaller molecules than pr - 39 , they migrated faster than the parent molecule . native pr - 39 and synthetic pr - 39 behaved identically in au - page and rp - hplc analyses ( data not shown ). ( i ) gel - overlay assay . in the gel - overlay bactericidal assay , only pr - 26 and pr - 39 were found to have antibacterial activity against e . coli . fig4 shows the results of this assay for pr - 14 , pr - 15 , pr - 16 , pr - 26 , and pr - 39 ( data not shown for pr - 19 and pr - 23 ). ( ii ) lawn - spotting assay . in the “ lawn - spotting ” antibacterial assay , pr - 14 , pr - 15 , pr - 16 , pr - 19 , pr - 23 , pr - 26 , and pr - 39 , and the combination of pr - 14 , pr - 15 , and pr - 16 , were tested . fig5 shows the results of this assay for pr - 14 , pr - 15 , pr - 16 , pr - 26 , and pr - 39 ( data not shown for pr - 19 , pr - 23 , and the combination of pr - 14 , pr - 15 , and pr - 16 ). only pr - 26 and pr - 39 had antibacterial activity . all of the other segments and their mixtures showed no antibacterial activity even at 1 mg / ml . these results suggest that : 1 ) the very cationic nh 2 - terminus of pr - 39 is not sufficient for antibacterial activity ; 2 ) the cooh - terminal segment 27 to 39 does not contribute to the antibacterial activity of pr - 39 ; 3 ) pr - 26 , the nh 2 - terminal segment 1 to 26 , contains the antibacterial domain of pr - 39 ; 4 ) the nh 2 - terminal segment 1 to 3 is essential for antibacterial activity ; ( 5 ) the cooh - terminal segment 20 to 26 is essential for antibacterial activity ; and 6 ) certain secondary structure conformation is required for the antibacterial activity of pr - 26 and pr - 39 since segment mixtures did not have any antibacterial activity . the “ lawn - spotting ” assay also showed that pr - 26 had greater antibacterial activity against e . coli and s . typhimurium than pr - 39 ( fig5 ). ( iii ) mics and mbcs . the mics of pr - 26 and pr - 39 for e . coli , 25922 ; e . coli , k88 ; s . typhimurium ; s . choleraesuis ; s . suis ; and s . aureus are shown in fig6 and table 1 . for the enteric , gram - negative bacteria , the mics of pr - 26 and pr - 39 ranged from 1 to 4 μm ; pr - 26 had a lower mic than pr - 39 . similarly , the mbcs of pr - 26 were the same or lower than the mbcs of pr - 39 and ranged from 2 to 8 μm for the gram - negative bacteria ( table 1 ). these findings suggest that pr - 26 may be an effective antibiotic against enterdc gram - negative bacteria such as e . coli or salmonella . ( iv ) pae . the relationship between peptide concentration and the duration of pae of pr - 26 and pr - 39 is shown in fig7 . suboptimal peptide concentrations ( 0 . 1 mic ) only caused a slight growth delay in both pr - 26 - and pr - 39 - treated bacteria . however , at 1 and 8 mics , paes against s . typhimurium were significantly increased . these findings agree with other antibacterial data and show clearly that pr - 26 limits the growth of enteric bacteria . ( v ) susceptibility to neutrophil phagocytosis . bacteria treated with pr - 26 were more susceptible to neutrophil phagocytosis ( fig8 ; pr - 26 was different from control , p & lt ; 0 . 05 ). a 10 min . exposure of s . choleraesuis to 2 mics of pr - 26 significantly increased the capability of porcine neutrophils to phagocytose the bacteria . treatment of s . choleraesuis with pr - 39 at 8 mics did not increase the phagocytic index of porcine neutrophils . neutrophils phagocytosed both single and filamentous bacteria . these data show that , in addition to the direct antibacterial activity of pr - 26 , this antibacterial peptide predisposes enteric bacteria to elimination by phagocytic cells . this property suggests that pr - 26 works synergistically with the host &# 39 ; s immune system to limit enteric bacterial growth . influence of pr - 39 on neutrophil chemotaxis . phagocytic cells migrate from the blood to areas of inflammation in response to chemotactic agents . fig9 shows that pr - 14 , pr - 15 , pr - 16 , pr - 26 , and pr - 39 are chemotactic agents for neutrophils ( pr - 14 , pr - 15 , pr - 16 , and pr - 26 are used at 1 μm , and pr - 39 was used at 0 . 05 μm ; the chemoattractant c5a , a positive control , was used at 1 × 10 − 8 m ; starred entries are different from the control , p & lt ; 0 . 05 ). fig1 shows a dose response of pr - 39 for neutrophil chemotaxis . the ability of pr - 39 to function as a chemotactic agent increases the probability that sufficient phagocytic cells are present at an inflammatory site to limit an infection . influence of pr - 26 and pr - 39 on intestinal epithelial cells . fig1 shows the cytotoxic activity of pr - 26 and pr - 39 on rat small - intestine epithelial cells ( iec - 6 ). pr - 26 was not cytotoxic to iec - 6 cells even at concentrations ( 20 μm ) much greater than the mic for this peptide . however , iec - 6 cells were sensitive to pr - 39 as cytotoxicity occurred at 0 . 5 μm , which is lower than the mic for this peptide . these data show that pr - 26 does not damage cells of the small intestine and should , therefore , be a safe oral antibiotic . effect of pr - 26 on survival of mice challenged with s . typhimurium . in the first experiment , mice that received pr - 26 were resistant to salmonella ( 0 and 1 death for 250 and 100 μg / mouse , respectively ), but the control group had a 27 % mortality rate ( fig1 ; both pr - 26 treatments were significantly different from controls , p & lt ; 0 . 05 ). based on these data , pr - 26 was effective in increasing the survival rates in this salmonella - resistant strain of mice . there was no evidence of toxicity caused by pr - 26 . in the second experiment , both 50 and 100 μg pr - 26 were sufficient to increase survival to more than 50 % at 14 days post - infection ( fig1 ; both pr - 26 treatments were different from the pbs control at days 6 through 14 , p & lt ; 0 . 05 , but not different from each other . in the third experiment , administration of pr - 26 was most effective when given at time 0 ; however , the 24 - hour post - infection treatment slightly improved the survival compared to the pbs treatment by day 14 ( fig1 ; treatments at 0 , 24 , and 72 hrs . were different from pbs control at day 15 , p & lt ; 0 . 05 for treatments at 0 and 24 hrs ., p & lt ; 0 . 10 for treatment at 72 hrs .). the delivery of pr - 26 at 48 hrs . post - infection was not different than the pbs control treatment . the rapidity of uptake of s . typhimurium and delivery to the liver and spleen during an i . p . infection may be the cause of the reduced effectiveness of pr - 26 by 48 hrs . post - infection . regulation of neutrophil o 2 − production by pr - 26 and pr - 39 . ( i ) o 2 − production assays . when pr - 39 was incubated with neutrophils for at least 45 min . before simulation with phorbol myristate acetate ( pma ), a significant reduction in 02 generation was observed ( fig1 ; neutrophils ( 1 × 10 6 ) were incubated without ( control ) or with pr - 39 ( 5 μm ) for the indicated times before stimulation with pma ; values are means ± sem , n = 2 ; starred entries are different from control , p & lt ; 0 . 05 ). this effect was even more apparent with longer pre - incubation periods , approaching as much as 70 % oxidase inhibition . pr - 26 also significantly reduced o 2 − generation ( fig1 ; neutrophils were incubated with pr - 39 or derivatives for 150 min . before stimulation with pma ; results are reported as % relative to control ( no peptides ); however , the statistical analysis was based on nmoles of o 2 − produced ; values are means , n = 3 ; starred entries are different from control , p & lt ; 0 . 05 ; this experiment was conducted twice with similar results ). however , the shorter peptides , pr - 14 , pr - 15 , pr - 16 , pr - 19 , and pr - 23 , did not significantly reduce o 2 − generation by intact neutrophils . pr - 39 ( 20 μm ) added to neutrophils at the same time as pma activities did not affect generation of o 2 − , while longer incubation periods with this peptide did not affect neutrophil viability , as judged by trypan blue dye exclusion . to investigate the inhibition of neutrophil o 2 − generation by pr - 39 and its fragments in more detail , a cell - free assay system of o 2 − generation was used . pr - 26 and pr - 39 were very potent inhibitors of o 2 − generation in the cell - free assay ; concentrations that inhibited 50 % ( ic 50 ) of o 2 − generation were approximately 1 and 2 μm , respectively ( fig1 ; data are the average of duplicate reactions and are representative of 3 to 4 independent experiments ; control ( 100 %) activities ranged from 0 . 9 to 2 . 4 nmol o 2 − min / 5 × 10 5 cell equivalents of membrane ). at concentrations greater than 5 μm , both proline - rich peptides completely inhibited the generation of o 2 − . pr - 15 and pr - 16 did not affect o 2 − generation ; however , pr - 14 did reduce o 2 − generation at concentrations greater than 25 μm . pr - 19 and pr - 23 had some oxidase inhibitory activity ; ic 50 &# 39 ; s of pr - 19 and pr - 23 were approximately 5 μm and 25 μm , respectively ( fig1 ). these findings suggest that both peptides contain the oxidase inhibitory domain , although the first three arginine residues of pr - 26 contribute greatly to oxidase inhibitory activity . ( ii ) binding assays . the findings that pr - 26 and pr - 39 inhibited both cell - free and whole cell o 2 − production by neutrophils and that inhibition required preincubation of cells with peptides for at least 45 min . prior to pma stimulation suggested that these peptides act through some intracellular target , such as the nadph oxidase components themselves . to determine if pr - 39 bound to specific neutrophil cytosol components , human and porcine cytosolic proteins were separated by sds - page , transferred to a pvdf membrane , and probed with biotinylated pr - 39 . using this ligand - blot binding assay , it was found that pr - 39 bound to a 47 kda protein in both human and porcine cytosol preparations ( fig1 ; cytosolic protein ( 50 μg ) from human and porcine polymorphonuclear ( hpmn and ppmn ) leukocytes was subjected to sds - page , transferred to a pvdf membrane , and then probed with biotinylated pr - 39 ; molecular masses of standards in kda are shown on the left of the blots ). competitive binding assays were conducted to determine if pr - 39 bound to recombinant p47 phox . these solution binding experiments with pure recombinant p47 phox and pr - 39 showed that increasing concentrations of nonbiotinylated pr - 39 inhibited specific binding of biotinylated pr - 39 to recombinant p47 phox ( fig1 ; competitive binding analysis was conducted by incubating various concentrations of pr - 39 with recombinant p47 phox and then with biotinylated pr - 39 ; after dialysis , solutions were dot - blotted onto pvdf membranes and probed for bound biotinylated pr - 39 ). binding was inhibited 90 % at equimolar concentrations of labeled and unlabeled peptide ( 0 . 06 μm ) and was completely blocked by a tenfold excess of unlabeled peptide . this finding shows that pr - 39 binds to p47 phox , that binding is specific , and implies that pr - 39 decreases o 2 − generation by interferring with this cytosolic component of the nadph oxidase complex . at least two proline - rich sequences , the cooh - terminal region ( residues 358 - 371 ) of p47 phox and the cytoplasmic region ( residues 149 - 162 ) of p22 phox , are thought to bind to sh3 domains of p67 phox and p47 phox , respectively , and are essential for the activation of nadph oxidase in vivo ( leto et al ., 1994 ; sumimoto et al ., 1994 ; finan et al ., 1994 ; mcphail , 1994 ; de mendex et al ., 1996 ). data obtained by probing neutrophil cytosolic proteins with biotinylated pr - 39 suggested that pr - 39 did not bind to p67 phox ( fig1 ). because pr - 39 bound directly to p47 phox , it was reasoned that pr - 39 could interfere with assembly of nadph oxidase by blocking its interaction with p22 phox . to examine this hypothesis , solution binding assays were conducted between gst - p47 phox sh3 domain fusion protein and a recombinant p22 phox cytoplasmic domain ( residues 127 - 195 ) in the presence or absence of pr - 39 or its derived fragments . pr - 26 and pr - 39 effectively blocked the interaction between gst - p47 phox and recombinant p22 phox at concentrations close to their ic 50 &# 39 ; s observed in the cell - free oxidase assay ( fig2 ; pr - 26 or pr - 39 were mixed with recombinant p22 phox and incubated for 60 min . with gst - p47 phox - sh3 fusion proteins ( 0 . 5 μg ) bound to glutathione - sepharose beads and analyzed as described : after sds - page , sepharosebound proteins were analyzed by immunoblotting with anti - p22 phox antibody ; controls included blotting of recombinant p22 phox lysate ( 5 × 10 3 cell equivalents ) alone ( p22 ) and bound complexes detected without competing pr - peptides (−); concentrations of competing peptides defined above each blot are indicated below the blots ). using this binding assay , analysis of the inhibition by pr - 14 , pr - 15 , pr - 16 , pr - 19 , and pr - 23 revealed the importance of the cooh - terminal region of pr - 26 in sh3 binding ( fig2 ; inhibition by pr - 19 or pr - 23 , conducted as in fig2 ) ( fig2 ; comparison of inhibitory activities of smaller peptides at 25 μm ). these data not only supported the hypothesis that pr - 39 blocks the interaction between p47 phox and p22 phox , but also indicated that the main structural motif involved in the interaction between pr - 39 and sh3 domains of p47 phox was in the central segment ( pr - 16 ) of pr - 39 . this 16 - residue segment contains structural elements compatible with consensus features of both classes of sh3 peptide ligands ( feng et al ., 1994 ; lim et al ., 1994 ). two sequences within pr - 16 , rpppffp ( sequence id no . 8 ) and pprlppri ( sequence id no . 9 ), conform to consensus sequences for either class i (+) and class ii (−) binding orientations , x 1 px 2 ppx 3 p and x 3 ′ ppx 2 ′ ppx 1 ′ , respectively ( upper - case denotes critical contact residues , p denotes proline , and x 1 or x 1 ′ favor arginine residues ). since pr - 23 inhibits better than pr - 19 , a class ii binding orientation appears likely . however , because pr - 19 had greater inhibitory activity in the cell - free assay but was less potent in blocking the interaction of p47 phox sh3 domains with p22 phox when compared to pr - 23 , the polybasic motif of the amino - terminus of pr - 26 also represents a separate important component of oxidase inhibition . this observation is supported by studies showing that several other cationic peptides effectively inhibit nadph oxidase , including peptides derived from p47 phox ( joseph et al ., 1994 ). thus , critical regions at both ends of pr - 26 have been defined based on dramatic losses of function seen with the deleted forms , pr - 19 and pr - 23 . the polybasic motif at the amino - terminus of pr - 26 and pr - 39 , in addition to their overall amphiphatic character , may have a role in promoting internalization of these inhibitory peptides , since similar structural properties are thought to enable internalization of various synthetic peptides designed for intracellular targets ( derossi et al ., 1994 ; mann et al ., 1994 ; fawell et al ., 1994 ; vellette et al ., 1994 ; taffs et al ., 1992 ). pr - 39 uptake may involve an active endocytic process or direct membrane lipid interactions , since this peptide binds and induces conductance changes in pure lipid bilayers ( cabiaux et al ., 1992 ). pr - 39 is the first naturally occurring down regulator of phagocyte nadph oxidase identified that interferes with assembly of this enzyme by binding to p47 phox . little is known about the mechanisms governing respiratory burst kinetics , although models based on a continuous cycling of cytosolic components are consistent with the direct inhibitory effects of pr - 39 on oxidase component interactions . the paradoxical finding of a neutrophil peptide possessing both antibacterial and oxidase inhibitory activities is intriguing , since a switch from oxygenependent to oxygen - independent bactericidal mechanisms by an accumulation of this peptide within inflammatory sites could serve several important functions . pr - 39 , which has a well - documented role as an antibacterial peptide , might have several roles in tissue repair by directly inhibiting nadph oxidase activity and limiting related proinflammatory responses , while also affecting gene expression patterns that promote wound healing ( gallo et al ., 1994 ). furthermore , since reactive oxygen intermediates have also been shown to function as second messengers that can regulate gene expression ( schreck et al ., 1991 ), pr - 39 may indirectly influence gene expression patterns related to oxidative stress . these opposing activities of pr - 39 illustrate a fine balance required in host defense mechanisms : antibacterial activity is necessary to control microbial pathogens and oxidase inhibitory activity is important for restricting tissue damage caused by excessive oxygen radicals generated by nadph oxidase . in the case of pr - 39 , one peptide possesses both activities . these findings suggest a mechanism for interaction between oxidative and nonoxidative antimicrobial systems of neutrophils and may serve as a basis for design of drugs effective against production of oxidants in chronic or acute inflammatory disease states . abo , a ., boyhan , a ., west , i ., thrasher , a . j . & amp ; segal , a . w . 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