Patent Application: US-201314429733-A

Abstract:
methods and compositions for increasing a plant &# 39 ; s resistance to an insect pest such as the corn rootworm are provided . methods are provided for overexpression of crw2 , or variants thereof , in a host plant or plant cell to increase resistance to an insect pest in a plant such as maize . methods are also provided for identifying variants of crw2 that when incorporated into a plant via transgenic or traditional breeding means increase resistance to an insect pest in a plant such as maize . also provided are methods for increasing resistance by overexpressing crw1 and crw2 .

Description:
detailed descriptions of one or more embodiments are provided herein . it is to be understood , however , that the present invention may be embodied in various forms . therefore , specific details disclosed herein are not to be interpreted as limiting , but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner . where ever the phrase “ for example ,” “ such as ,” “ including ” and the like are used herein , the phrase “ and without limitation ” is understood to follow unless explicitly stated otherwise . similarly “ an example ,” “ exemplary ” and the like are understood to be non - limiting . the terms “ comprising ” and “ including ” and “ having ” and “ involving ” ( and similarly “ comprises ”, “ includes ,” “ has ,” and “ involves ”) and the like are used interchangeably and have the same meaning . specifically , each of the terms is defined consistent with the common united states patent law definition of “ comprising ” and is therefore interpreted to be an open term meaning “ at least the following ,” and is also interpreted not to exclude additional features , limitations , aspects , etc . thus , for example , “ a process involving steps a , b , and c ” means that the process includes at least steps a , b and c . where ever the terms “ a ” or “ an ” are used , “ one or more ” is understood , unless such interpretation is nonsensical in context . as used herein , the term “ effective amount ” is intended to encompass contexts such as a pharmaceutically effective amount or therapeutically effective amount . for example , in certain embodiments , the effective amount is capable of achieving a beneficial state , beneficial outcome , functional activity in a screening assay , or improvement of a clinical condition . the terms “ polypeptide ,” “ protein ” and “ peptide ” are used interchangeably and mean a polymer of amino acids not limited to any particular length . the term does not exclude modifications such as myristylation , sulfation , glycosylation , phosphorylation and addition or deletion of signal sequences . the term “ antibody ” as used herein includes monoclonal antibodies , polyclonal antibodies , multispecific antibodies ( e . g ., bispecific antibodies ), and antibody fragments , so long as they exhibit the desired biological activity , e . g ., specifically bind to prr . the term “ immunoglobulin ” ( ig ) is used interchangeably with “ antibody ” herein . monoclonal or polyclonal antibodies specifically reacting with prr may be made by methods known in the art , and are commercially available . see , e . g ., harlow and lane ( 1988 ) antibodies : a laboratory manual , cold spring harbor laboratories ; goding ( 1986 ) monoclonal antibodies : principles and practice , 2d ed ., academic press , new york ; and ausubel et al . ( 1999 ) current protocols in molecular biology , john wiley & amp ; sons , inc ., new york . the term “ isolated ” refers to a subject prr antagonist , e . g ., a thioether bridge modified prr antagonist peptide , that has been separated and / or recovered from a component of its natural environment , e . g ., a host cell culture environment . in certain embodiments , the isolated protein is substantially free from proteins or polypeptides or other contaminants that would interfere with its use ( therapeutic , diagnostic , prophylactic , research or otherwise ). the term “ polynucleotide ” as referred to herein means single - stranded or double - stranded nucleic acid polymers . in certain embodiments , the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide . the term “ polynucleotide ” specifically includes single and double stranded forms of dna . as will be also recognized by the skilled artisan , polynucleotides may be single - stranded ( coding or antisense ) or double - stranded , and may be dna ( genomic , cdna or synthetic ) or rna molecules . standard techniques for cloning , dna isolation , amplification and purification , for enzymatic reactions involving dna ligase , dna polymerase , restriction endonucleases and the like , and various separation techniques are those known and commonly employed by those skilled in the art . the term “ operably linked ” means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions . for example , a transcription control sequence “ operably linked ” to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences . the term “ vector ” is used to refer to any molecule ( e . g ., nucleic acid , plasmid , or virus ) used to transfer a polynucleotide sequence to a host cell . the term “ expression vector ” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control expression of inserted nucleic acid sequences . expression includes , but is not limited to , processes such as transcription , translation , and rna splicing , if introns are present . calculations of sequence similarity or sequence identity between sequences ( the terms are used interchangeably herein ) are performed as follows . to determine the percent identity of two amino acid sequences , or of two nucleic acid sequences , the sequences are aligned for optimal comparison purposes ( e . g ., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non - homologous sequences can be disregarded for comparison purposes ). in certain embodiments , the length of a reference sequence aligned for comparison purposes is at least 30 %, preferably at least 40 %, more preferably at least 50 %, 60 %, and even more preferably at least 70 %, 80 %, 90 %, 100 % of the length of the reference sequence . the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared . when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence , then the molecules are identical at that position . the percent identity between the two sequences is a function of the number of identical positions shared by the sequences , taking into account the number of gaps , and the length of each gap , which need to be introduced for optimal alignment of the two sequences . the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm . in a preferred embodiment , the percent identity between two amino acid sequences is determined using the needleman and wunsch , ( 1970 , j . mol . biol . 48 : 444 - 453 ) algorithm which has been incorporated into the gap program in the gcg software package , using either a blossum 62 matrix or a pam250 matrix , and a gap weight of 16 , 14 , 12 , 10 , 8 , 6 , or 4 and a length weight of 1 , 2 , 3 , 4 , 5 , or 6 . in yet another preferred embodiment , the percent identity between two nucleotide sequences is determined using the gap program in the gcg software package , using a nwsgapdna . cmp matrix and a gap weight of 40 , 50 , 60 , 70 , or 80 and a length weight of 1 , 2 , 3 , 4 , 5 , or 6 . a particularly preferred set of parameters ( and the one that should be used unless otherwise specified ) are a blossum 62 scoring matrix with a gap penalty of 12 , a gap extend penalty of 4 , and a frameshift gap penalty of 5 . the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of e . meyers and w . miller ( 1989 , cabios , 4 : 11 - 17 ) which has been incorporated into the align program ( version 2 . 0 ), using a pam120 weight residue table , a gap length penalty of 12 and a gap penalty of 4 . as used herein , “ treating ” or “ treatment ” refers to an approach for obtaining beneficial or desired results , including and preferably clinical results . treatment can involve optionally either the amelioration of symptoms of the disease or condition , or the delaying of the progression of the disease or condition . as used herein , unless the context makes clear otherwise , “ prevention ,” and similar words such as “ prevented ,” “ preventing ” etc ., indicates an approach for preventing , inhibiting , or reducing the likelihood of , the onset or recurrence of a disease or condition . it also refers to preventing , inhibiting , or reducing the likelihood of , the occurrence or recurrence of the symptoms of a disease or condition , or optionally an approach for delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition . as used herein , “ prevention ” and similar words also includes reducing the intensity , effect , symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition . the present invention is based in part on the finding that blocking the ( pro ) renin receptor ( prr ) reduces angiotensin ii ( ang ii ) generation and also prevents ang ii - independent signal activation . accordingly , one aspect of the invention relates to prr antagonists . prr antagonists include , e . g ., small molecule compounds , antisense polynucleotides , polypeptides , including prr - binding peptides , and anti - prr antibodies . a prr antagonist inhibits or blocks the ligand - receptor interaction of prorenin to prr . in one embodiment , a prr antagonist blocks prorenin from binding prr . in another embodiment , a prr antagonist competes with prorenin for binding to prr . in one embodiment , a polynucleotide variant of a prr antagonist is provided . a polynucleotide variant may have substantial identity to a prr antagonist polynucleotide sequence described herein . for example , a polynucleotide may be a polynucleotide comprising at least 70 % sequence identity , preferably at least 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % or higher , sequence identity compared to a reference polynucleotide sequence , using the methods described herein , ( e . g ., blast analysis using standard parameters ). in another embodiment , a polypeptide variant of a prr antagonist is provided . a peptide variant may have substantial identity to a prr antagonist peptide sequence described herein . for example , a peptide variant may be a peptide comprising at least 70 % sequence identity , preferably at least 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 % or higher , sequence identity compared to a prr antagonist peptide described herein . in one embodiment of the invention , the prr antagonist is a peptide that inhibits the binding of prorenin to prr . examples of functional prr antagonist peptides include the peptides disclosed herein , pr10 , pr20 , pr30 , and pr40 , with sequences ifdniisqgvlkedvf ( seq id no : 1 ), lptdtttfkriflkrmpsi ( seq id no : 2 ), lptdtttfkriflkrmpsire ( seq id no : 16 ), and lptrtatferiplkkmpsvre lptrtatferiplkkmpsvre ( seq id no : 17 ), respectively . administration of either pr10 or pr20 intracerebroventricularly ( icv ) was shown to significantly reduce prorenin ( pr ) induced pressor response as described further in the examples below . one embodiment of the invention provides a polynucleotide sequence encoding a prr antagonist peptide . in one embodiment , the polynucleotide is codon optimized for expression in the host cell , e . g ., lactococcus lactis . the polynucleotide sequence encoding the prr antagonist peptide may be part of a construct . accordingly , the polynucleotide sequence encoding the prr antagonist peptide may be contained in a vector , such as an expression vector for expression and production by a host cell , e . g ., l . lactis . prr antagonist peptides include modified peptides , e . g ., peptides comprising a thioether bridge and / or amino acids that are not standard or naturally occurring in humans , e . g ., amino acids found in polypeptides of microbial origin . examples of non - standard amino acids include , but are not limited to , dehydroalanine ( dha ), 2 - aminobutyric acid ( abu ), and dehydrobutyrine ( referred to herein interchangeably as “ dht ” or “ dhb ”). in one embodiment , thioether - bridge modified peptides are designed based on the core amino acid sequences of pr10 , pr20 , pr30 , and pr40 in order to avoid peptide degradation by peptidase in vivo . the introduction of one or more thioether bridges makes the resulting peptides more stable and , therefore , strong prr antagonists . the nisbtc encoding plasmid ( ptu - btc ) and substrate - peptide - encoding plasmids ( ppr103 , ppr105 , ppr107 , ppr201 , ppr202 ) were constructed and introduced into the lactic acid producing bacterium l . lactis to produce the thioether - bridge containing peptides pr103 ( seq id no : 3 ), pr105 ( seq id no : 4 ), pr107 ( seq id no : 5 ), pr201 ( seq id no : 6 ) and pr202 ( seq id no : 7 ) respectively . one alternative embodiment is pr203 ( seq id no : 8 ) as provided in fig1 . in one embodiment , a prr antagonist peptide comprises common amino acid substitutions or modifications . for example , a prr antagonist peptide derived from the core amino acid sequence of pr20 comprises amino acid residues 3 , 4 , 6 , 7 , and 18 of the amino acid sequence set forth in seq id no : 2 . in a related embodiment , the prr antagonist peptide derived from pr20 further comprises amino acid residues 8 , 10 , and 11 of seq id no : 2 . in another embodiment , a prr antagonist peptide comprises amino acid residues 3 , 4 , 6 , 7 , and 18 of one of seq id nos : 18 - 23 . in a related embodiment , the prr antagonist peptide further comprises amino acid residues 8 , 10 , 11 , and 14 of one of seq id nos : 18 - 23 . in one embodiment , the prr antagonist is a peptide comprising an amino acid sequence having at least 50 % identity to an amino acid sequence set forth in one of seq id nos : 4 - 8 and 18 - 23 . in another embodiment , the prr antagonist is a peptide comprising an amino acid sequence having at least 60 % identity to an amino acid sequence set forth in one of seq id nos : 4 - 8 and 18 - 23 . in yet another embodiment , the prr antagonist is a peptide comprising an amino acid sequence having at least 70 % identity to an amino acid sequence set forth in one of seq id nos : 4 - 8 and 18 - 23 . in one embodiment , the prr antagonist is a peptide comprising an amino acid sequence having at least 80 % identity to an amino acid sequence set forth in one of seq id nos : 4 - 8 and 18 - 23 . in one embodiment , the prr antagonist is a peptide comprising an amino acid sequence having at least 90 % identity to an amino acid sequence set forth in one of seq id nos : 4 - 8 and 18 - 23 . in another embodiment , the prr antagonist is a peptide comprising the amino acid sequence set forth in one of seq id nos : 4 - 8 and 18 - 23 . one aspect of the invention provides a more efficient hypertension treatment because of the dual beneficial effect of prr blockade that both reduces ang ii generation and prevents ang ii - independent signal activation . the result of prr deletion mimics the effect of prr antagonism in hypertension . deletion of prr attenuates blood pressure in chronic hypertension and prevents development of hypertension . as described in the examples , intracerebroventricular ( icv ) administration of either pr10 or pr20 significantly reduced the prorenin ( icv ) induced pressor response in c57bi / 6j mice . accordingly , the significant reduction in the prorenin - induced pressor response indicated a functional activity of pr10 and pr20 in blocking the effects of prorenin / prr activation . in one embodiment , the prr antagonist is administered to a patient for the treatment or prevention of a disease or disorder including , e . g ., hypertension , diabetes , diabetic retinopathy , nephropathy , cardiac hypertrophy , vascular and kidney fibrosis . in certain embodiments , the disease or disorder is chronic hypertension or ras - dependent hypertension . in one embodiment , the hypertension is neurogenic hypertension . in another embodiment , the prr antagonist is used in the preparation of a medicament for the treatment or prevention of a disease or disorder . in one embodiment , the prr antagonist is a prr - binding peptide . in a preferred embodiment , the prr antagonist is a modified peptide . in one embodiment , the modified peptide prr antagonist comprises one or more thioether bridges . for example , the modified peptide prr antagonist may comprise one , two , three , or more thioether bridges . pharmaceutical compositions can be made using the prr antagonists described herein . an effective amount of a therapeutic composition is the minimum dose that produces a measurable effect in a subject , e . g ., produces a statistically significant reduction in blood pressure . pharmaceutical compositions are readily prepared by one of ordinary skill in the art . a prr antagonist of the present invention can be lyophilized for storage or formulated into various solutions known in the art for solubility and stability and consistent with safe administration into animals , including humans . administration of the prr antagonists described herein , in pure form or in an appropriate pharmaceutical composition , can be carried out via any of the accepted modes of administration of agents for serving similar utilities . the pharmaceutical compositions can be prepared by combining a prr antagonist or antagonist - containing composition with an appropriate physiologically acceptable carrier , diluent or excipient , and may be formulated into preparations in solid , semi solid , liquid or gaseous forms , such as tablets , capsules , powders , granules , ointments , solutions , suppositories , injections , inhalants , gels , microspheres , and aerosols . in addition , other pharmaceutically active ingredients ( including other anti - hypertensive agents ) and / or suitable excipients such as salts , buffers and stabilizers may , but need not , be present within the composition . “ carriers ” as used herein include pharmaceutically or physiologically acceptable carriers , excipients , or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed . often the physiologically acceptable carrier is an aqueous ph buffered solution . examples of physiologically acceptable carriers include buffers such as phosphate , citrate , and other organic acids ; antioxidants including ascorbic acid ; low molecular weight ( less than about 10 residues ) polypeptide ; proteins , such as serum albumin , gelatin , or immunoglobulins ; hydrophilic polymers such as polyvinylpyrrolidone ; amino acids such as glycine , glutamine , asparagine , arginine or lysine ; monosaccharides , disaccharides , and other carbohydrates including glucose , mannose , or dextrins ; chelating agents such as edta ; sugar alcohols such as mannitol or sorbitol ; salt - forming counterions such as sodium ; and / or nonionic surfactants such as polysorbate 20 ( tween ™) polyethylene glycol ( peg ), and poloxamers ( pluronics ™), and the like . administration may be achieved by a variety of different routes , including oral , parenteral , nasal , intravenous , intradermal , subcutaneous or topical . in one embodiment , the prr antagonist is administered intracerebroventricularly ( icv ). in another embodiment , the prr antagonist is administered orally . preferred modes of administration depend upon the nature of the condition to be treated or prevented . an amount that , following administration , reduces , inhibits , or prevents hypertension is considered effective . in particular embodiments , a thioether bridge - modified peptide is designed to be delivered intravenously or orally . the following examples are intended to illustrate but not limit the disclosure . while the disclosure provides certain specific embodiments , the invention is not limited to those embodiments . a person of ordinary skill will appreciate from the description herein that modifications can be made to the described embodiments and therefore that specification is broader in scope than the described embodiments . all examples are therefore non - limiting . neuron - specific prr knockout mice ( nefh - prr ) and wildtype ( wt ) littermates ( n = 5 / group ) were each implanted with a telemetric probe for blood pressure ( bp ) recording and an intracerebroventricular ( icv ) cannula for infusion of mouse prorenin ( 100 ng / ul ), mouse renin ( 100 ng / ul ), or an ang ii type 1 receptor ( at1r ) blocker ( losartan , 10 ug / ul ) at 0 . 3 ul / minute for 10 minutes . mouse prorenin infusion increased the bp ( mmhg ) in wt mice ( δmap : 41 ± 5 ); however , the prorenin induced pressor response was abolished in nefh - prr mice ( δmap : 5 ± 1 ). infusion of mouse renin similarly increased bp in nefh - prr ( δmap : 27 ± 2 ) and wt ( δmap : 31 ± 5 ) mice . the pressor response induced by prorenin or renin was completely blocked by the infusion of losartan . the data suggest that icv prorenin , via prr , mediates ang ii - dependent pressor response in wt mice . to determine whether prr contributes to the development of brain ras - dependent hypertension , nefh - prr and wt littermates ( n = 8 / group ) were treated with 50 mg of deoxycorticosterone acetate ( doca ) subcutaneously , plus 0 . 9 % nacl drinking water for 21 days . the baseline bp was similar between nefh - prr ( 101 ± 2 ) and wt ( 101 ± 3 ) mice . bp was increased in wt mice ( 132 ± 6 ) by doca - salt treatment , while nefh - prr mice remained normotensive ( 108 ± 3 ). in summary , prorenin via prr mediates angii / at1r - dependent pressor response in the brain . neuronspecific prr deletion attenuates the development of doca - salt hypertension likely due to the lack of ang ii / at1r activation . blocking prr improves baroreflex sensitivity and autonomic function and is linked to the attenuation of doca - salt hypertension elevated expression and activity of ras components in the brain cv control regions support the concept that the brain ras is involved in the pathogenesis of hypertension , including doca - salt hypertension . prr participates in ang ii generation , triggering both ang ii - dependent and - independent activation of signaling pathways , and plays a significant role in regulating cv function . accordingly , decreasing prr expression in cv regulatory nuclei will reduce ang ii generation and / or decrease ang ii - independent signals , resulting in vasodilation and reduction of bp . a conditional prr knockout mouse model with deletion of prr specifically in neurons was generated and characterized . the mouse prr exon 2 gene was deleted by breeding prr floxed mice with mice that express cre recombinase under the control of the neuron - specific neurofilament - h ( nefh ) promoter ( nefh - cre mice from jackson laboratory , maine ) ( fig5 a ). these prr knockout mice ( nefh - prr ) appear to be vital and exhibit global prr ablation in the brain regions that are involved in central regulation of bp , such as the subfornical organ ( sfo ) and paraventricular nucleus ( pvn ) ( fig5 b ), as well as the rostral ventral lateral medulla ( rvlm ), solitary nucleus ( nts ), and non - cv regulatory nuclei . prr is critical for early development . to test whether neurons are functionally intact in neuron - specific prr knockout mice , carbachol ( a cholinergic agonist ), ang ii , renin , and prorenin were icv infused to both nefh - prr and wild type ( wt ) mice . the pressor response to icv infusion of carbachol and ang ii was similar between nefh - prr and wild type ( wt ) mice suggesting that the nefh - prr mice harbor functional acetylcholine receptor and at1r ( fig6 a , b ). interestingly , the pressor response to icv prorenin was significantly reduced in nefh - prr compared to wt mice ( fig6 d ) indicating that the pressor response to prorenin requires prr . in addition , the prorenin - induced pressor effect is primarily the direct action of prorenin but not from its conversion to renin , since the pressor response to icv renin is similar between nefh - prr and wt mice ( fig6 c ). these data demonstrate that prorenin , via binding to prr , regulates bp in the cns . more importantly , although the baseline bp and hr were not different between nefh - prr and wt mice , the brain - targeted prr deletion attenuated the development of doca - salt hypertension ( fig7 ), indicating a critical role of brain prr in the development of hypertension . however , the mechanism by which prr affects bp remains unclear . accordingly , without wishing to be bound by theory , it is hypothesized that brain - targeted prr deletion reduces ang ii - dependent and ang ii - independent signaling pathways , leading to reduced sympathetic activity , improved baroreflex sensitivity , and ultimately reduced bp and improved cv function . telemetry bp recording , baroreflex and autonomic function analysis , and molecular biology techniques may be utilized to determine the role and mechanisms of prr in the development of doca - salt hypertension . the control mice for nefh - prr mice experiments are wild type littermates ( wt ) that are heterozygous for nefh - cre to exclude the possible effects of cre - mediated toxicity on phenotypes . delineating the autonomic mechanisms and cv consequences of brain - targeted prr deletion in the development of doca - salt hypertension the nefh - prr and wt mice are implanted with telemetric transmitters and receive doca - salt , high salt only , or sham treatment as described above . spontaneous baroreflex sensitivity ( sbrs ), cardiac , and vasomotor sympathetic tone are assessed as described previously ( see , e . g ., li w , et al . brain - targeted ( pro ) renin receptor knockdown attenuates angiotensin ii - dependent hypertension . hypertension . 2012 , 59 : 1188 - 1194 ; which is hereby incorporated by reference in its entirety ). the sbrs is calculated at four different time points ( 0 , 7 , 14 , and 21 days ) without additional animals needed using the sequence method ( hemolab software ). in addition , baroreflex reflex sensitivity ( brs ) is assessed using a pharmacological method consisting of infusion of sodium nitroprusside ( 5 μg / min , iv ) and phenylephrine ( 50 ng / min iv ) to decrease and increase bp respectively after 21 days of doca - salt treatment . autonomic function is assessed using intraperitoneal injection of propranolol ( β - blocker , 4 mg / kg ), methyl - atropine ( muscarinic receptor blocker , 1 mg / kg ), and chlorisondamine ( ganglionic blocker , 5 mg / kg ) as described previously . changes in hr or bp are calculated after administration of the antagonists . at the end of the protocol , echocardiography is performed as previously described , and mice are sacrificed after echocardiography . the hearts are collected for collagen deposition and cardiomyocyte diameter analysis to determine the degree of hypertrophy in the heart . plasma and urine are collected for norepinephrine ( ne ) measurement using a catcombi elisa kit ( ibl international , hamburg , germany ). prr deletion will improve sbrs and reduce sympathetic activity due to reduced ang ii generation and its stimulation of at1 receptors in the brain , and thus reverse cardiac hypertrophy and fibrosis following reduction of hypertension . determining the effects of prr deletion on ace / angii / at1r and ace2 / ang1 - 7 / masr axes in doca - salt hypertension the reduction of doca - salt hypertension by prr deletion in the brain can result from a decrease of ang ii generation , thus leading to a lesser stimulation of at1 receptors , or an inactivation of ang ii independent signals . despite several reports showing prr - mediated ang ii formation , the direct effects of brain prr deletion on ang ii formation during hypertension remained unknown . an ang ii measurement assay has been established , and using the assay , an increase in brain ang ii level was found despite a decrease in kidney ang ii level in the doca - salt hypertensive mice ( fig8 ). the nefh - prr and wt mice receive doca - salt , high salt , or sham treatment as described above . at the end of 21 days of treatment , mice are sacrificed ; plasma and brain tissues are harvested for ang ii , ang 1 - 7 measurement utilizing an elisa kit ( phoenix pharmaceutical inc .). fig8 shows the ability to measure the ang ii levels in wt mice receiving either doca - salt or sham treatment . ace , ace2 , at1r , and masr mrna and protein levels may be determined using real time pcr , immunofluorescent staining , and western blotting as described . the radioligand receptor binding assay for at1r is performed to determine the levels of functional receptors as described . the ace and ace2 activity are measured to evaluate the function of the enzymes using fluorogenic peptide vi . data is expressed as mean ± sem . data is analyzed by one - way or two - way , repeated measures anova followed by student &# 39 ; s modified t - test with bonferroni correction for multiple comparisons between means using the modified error mean square term from the anova . c57bi / 6j mice ( n = 5 / group ) were each implanted with a telemetric probe for bp recording and an intracerebroventricular ( icv ) cannula for infusion of mouse prorenin ( 100 ng / ul ), pr10 , or pr20 ( 45 ng / u1 ) at 0 . 3 ul / minute for 10 minutes . mouse prorenin infusion increased the bp ( mmhg ) in wt mice ( δmap : 31 . 3 ± 1 . 1 ); however , the prorenin - induced pressor response was abolished in either pr10 ( δmap : 16 . 9 ± 4 . 4 vs . prorenin ) or pr20 ( δmap : 12 . 7 ± 0 . 5 vs . prorenin ) infused mice . both of the peptides exhibited a 40 - 50 % reduction in pressor response induced by prorenin . to avoid peptide degradation by peptidase in vivo , thioether - bridge modified peptides were designed according to the core amino acid sequence of pr10 and pr20 . the nisbtc - encoding plasmid ( ptu - btc ) and substrate - peptide - encoding plasmids ( ppr103 , ppr105 , ppr107 , ppr201 , ppr202 ) are constructed and introduced into the lactic acid producing bacterium lactococcus lactis to produce the thioether - bridged peptides pr103 , pr105 , pr107 , pr201 and pr202 respectively . computational modeling was used to design modified pr10 and pr20 peptides comprising thioether - bridges . the thioether - bridges were designed according to the three - dimensional ( 3d ) structure of human renin ( provided on the ncbi &# 39 ; s website at the following address : ncbi . nlm . nih . gov / structure / mmdb / mmdbsrv . cgi ? uid = 65019 ). computational modeling was used to confirm the similarity of the thioether - bridge modified peptides to the original human renin peptide . examples of the computational 3d structures are shown in fig1 , 12 , and 14 . the genomic dna from strain nctc 6681 lactococcus lactis subsp . lactis , which contains the nisbtc gene , purchased from american type culture collection ( atcc ), was used to prepare the vector . primers for fusion nisbtc gene pcr were designed : pnis forward : 5 ′- ttgagtcttaaacatacttgaatgacc - 3 ′ ( seq id no : 9 ), reverse : 5 ′- gaactttttatcattttgagt gcctccttata - 3 ′ ( seq id no : 10 ); pnisbtc forward : 5 ′- ggcactcaaaatgataaaaagttcatttaaagctc - 3 ′ ( seq id no : 11 ), reverse : 5 ′- cttctcatttcctcttccctcc - 3 ′ ( seq id no : 12 ). pfusion forward : 5 ′- ctagtcttataactatactgacaatag - 3 ′ ( seq id no : 13 ), reverse : 5 ′- tcatttcctcttccctcctttc - 3 ′ ( seq id no : 14 ). the pcr product will be inserted into nice pnz9530 lactococcus lactis nisrnisk vector and amplified in nz9000 l . lactis ( purchased from boca scientific ). the nisa leader peptide ( mstkdfnldlhhhhhhdsgaspritsislctpgck tgalm , seq id no : 15 ) was designed , which comprises of a his tag for purification and a thrombin cleavage site for generating target peptide without extra amino acid residues . the polynucleotide sequences coding for peptides pr10 and pr20 were optimized to l . lactis for higher expression efficiency using optimizer software ( available at the following web address : genomes . urv . es / optimizer /). the dna sequences coding for peptides were synthesized with scai and xbal restriction enzyme sites for cloning into the nice pnz8150 expression vector to form ppr103 , ppr105 , ppr107 , ppr201 , and ppr202 constructs . expression of prr antagonist peptides was achieved by co - transfection of ptu - btc with ppr103 , ppr105 , ppr107 , ppr201 , or ppr202 in the nz9000 l . lactis . the prr antagonist peptides are expressed in the cell culture medium . peptides with a 6 × his tag are purified through nichel nitrilotriacetic ( ni - nta ) resin . the purified thioether - bridge containing peptides are tested for antagonist activity to prr in animal models of hypertension and other diseases . in order to determine which residues of the pr20 antagonist peptide are involved in binding prr , an alanine replacement assay was performed . a series of peptides comprising a single alanine substitution at each of the 19 amino acid positions were generated . each of the 19 alanine substituted peptides and the pr20 peptide were labeled with fitc . using fluorescent microscopy , each peptide was examined for binding to prr in mouse brain sections . as shown in fig2 , the amino acids at positions 3 , 4 , 6 , 7 , 18 are essential for pr20 binding to prr . amino acids at positions 8 , 10 , 11 were identified as being important , but not critical , for pr20 binding to prr . two additional prr antagonist peptides , pr30 ( seq id no : 16 ) and pr40 ( seq id no : 17 ), were derived from the core amino acid sequence of pr20 . amino acid residues 3 , 4 , 6 , 7 , and 18 ( fig2 and 22 ; underlined , bold text ) of pr30 and pr40 correspond to the positions that are essential for binding to prr . amino acid residues 8 , 10 , 11 , and 14 were determined to be involved in prr binding , but not essential ( fig2 and 22 , bold , italicized text ). the polynucleotides encoding the peptides were codon optimized for expression in l . lactis using optimizer software , and computational modeling was used to design modified pr30 and pr40 peptides comprising thioether - bridges . as described above , the thioether - bridges were designed according to the three - dimensional ( 3d ) structure of human renin . computational modeling was used to confirm the similarity of the thioether - bridge modified peptides to the original human renin peptide . as shown in fig2 , two modified peptides derived from pr30 comprising a single thioether bridge were designed , pr301 ( seq id no : 18 ) and pr302 ( seq id no : 19 ), and one modified peptide was designed with two thioether bridges , pr303 ( seq id no : 20 ). similarly , two modified peptides comprising a single thioether bridge were derived from pr40 , pr401 ( seq id no : 21 ) and pr402 ( seq id no : 22 ), and one modified peptide with two thioether bridges was also designed , pr 403 ( seq id no : 23 ), as shown in fig2 . while certain novel features of this invention shown and described below are pointed out in the claims , the invention is not intended to be limited to the details specified , since a person of ordinary skill in the relevant art will understand that various omissions , modifications , substitutions and changes in the forms and details of the invention illustrated and in its operation may be made without departing in any way from the spirit of the present invention . no feature of the invention is critical or essential unless it is expressly stated as being “ critical ” or “ essential .” all patents , patent applications , and references cited herein are incorporated in their entireties by reference . fisher j p , fadel p j . exp physiol . 2010 ; 95 : 572 - 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