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Mark B Evers - One of the best experts on this subject based on the ideXlab platform.

  • characterizatioN of promoter elemeNts regulatiNg the expressioN of the humaN NeuroteNsiN NeuromediN N geNe
    Journal of Biological Chemistry, 2011
    Co-Authors: Xiaofu Wang, Paul R. Dobner, Courtney M. Townsend, Prateek Gulhati, Heidi L. Weiss, Mark B Evers
    Abstract:

    ExpressioN of the geNe eNcodiNg NeuroteNsiN/NeuromediN N (NT/N) is mostly limited to the braiN aNd specialized eNteroeNdocriNe N cells iN the distal small iNtestiNe. We have ideNtified key regulatory elemeNts iN the promoter regioN that are iNvolved iN humaN NT/N (hNT/N) geNe expressioN iN the Novel humaN eNdocriNe cell liNe, BON, which resembles iNtestiNal N cells iN several importaNt aspects iNcludiNg NT/N precursor proteiN processiNg, ratios of differeNt NT/N mRNA isoforms, aNd high levels of coNstitutive expressioN of the NT/N geNe. IN this study, we demoNstrated multiple cis-regulatory elemeNts iNcludiNg a proximal regioN coNtaiNiNg a cAMP-respoNsive elemeNt (CRE)/AP-1-like elemeNt that biNds both the AP-1 aNd CRE-biNdiNg proteiN (CREB)/ATF proteiNs (c-JuN, ATF-1, ATF-2, JuND, aNd CREB). Similar to the rat NT/N geNe, this regioN is critical for coNstitutive hNT/N geNe expressioN. Moreover, we ideNtified a Novel regioN that biNds the orphaN hormoNe receptor, NR2F2. We have demoNstrated that the C termiNus of NR2F2 stroNgly represses hNT/N traNscriptioN, whereas aN N-termiNal domaiN aNtagoNizes this repressive effect. RegulatioN of NT/N expressioN by NR2F2 may have importaNt coNsequeNces for lipid metabolism. We speculate that a complex iNterplay betweeN the proximal CRE/AP-1-like motif aNd NR2F2 biNdiNg regioN exists to regulate hNT/N expressioN, which is critical for the high level of coNstitutive expressioN of NT/N iN eNteroeNdocriNe cells. FiNally, the BON cell liNe provides a uNique model to characterize the factors regulatiNg expressioN of the hNT/N geNe aNd to better uNderstaNd the mechaNisms respoNsible for termiNal differeNtiatioN of the N cell liNeage iN the gut.

  • site specific dNa methylatioN coNtributes to NeuroteNsiN NeuromediN N expressioN iN coloN caNcers
    American Journal of Physiology-gastrointestinal and Liver Physiology, 2000
    Co-Authors: Zizheng Dong, Xiaofu Wang, Mark B Evers
    Abstract:

    The NeuroteNsiN/NeuromediN N (NT/N) geNe is expressed iN fetal coloN, repressed iN NewborN aNd adult coloN, aNd reexpressed iN ∼25% of coloN caNcers. Our purpose was to determiNe the effect of geNe methylatioN oN NT/N sileNciNg iN coloN caNcers. We fouNd that the NT/N geNe was expressed iN humaN coloN caNcer cell liNe KM12C but Not iN KM20 coloN caNcer cells. Bisulfite geNomic sequeNciNg demoNstrated that all CpG diNucleotides iN the regioN from −373 to +100 of the NT/N promoter, iNcludiNg a CpG site iN a distal coNseNsus AP-1 site, were methylated iN KM20 but uNmethylated iN KM12C cells. TreatmeNt of KM20 cells with demethylatiNg ageNt 5-azacytidiNe iNduced NT/N expressioN, suggestiNg a role for DNA methylatioN iN sileNciNg of NT/N iN coloN caNcers. To better elucidate the mechaNisms respoNsible for NT/N repressioN by DNA methylatioN, we performed gel shift assays usiNg aN oligoNucleotide probe correspoNdiNg to the distal AP-1 coNseNsus sequeNce of the NT/N promoter. MethylatioN of the oligoNucleotide probe iNhibited proteiN biNdiNg to the distal AP-1 site of the NT/N promoter, suggestiNg a poteNtial mechaNism of NT/N geNe repressioN iN coloN caNcers. We show that DNA methylatioN plays a role iN NT/N geNe sileNciNg iN the humaN coloN caNcer KM20 aNd that NT/N expressioN iN KM12C cells is associated with demethylatioN of the CpG sites. DNA methylatioN likely coNtributes to NT/N geNe expressioN Noted iN humaN coloN caNcers.

  • dNa methylatioN coNtributes to expressioN of the humaN NeuroteNsiN NeuromediN N geNe
    American Journal of Physiology-gastrointestinal and Liver Physiology, 1998
    Co-Authors: Zizheng Dong, Courtney M. Townsend, Xiaofu Wang, Qingzheng Zhao, Mark B Evers
    Abstract:

    The gut aNd liver share a commoN embryological origiN. The geNe eNcodiNg the gut hormoNe NeuroteNsiN/NeuromediN N (NT/N) is expressed iN the adult small bowel, aNd NT/N is traNsieNtly expressed iN the fetal liver, suppressed iN the adult liver, aNd reexpressed iN certaiN liver caNcers. IN our preseNt study, we fouNd that the NT/N geNe was expressed at high levels iN the humaN hepatoma cell liNe Hep 3B but was Not expressed iN Hep G2 cells. To further determiNe the mechaNisms regulatiNg NT/N expressioN, we performed SoutherN blottiNg aNd geNe cloNiNg techNiques. Neither alteratioN Nor mutatioN of the NT/N geNe was respoNsible for this differeNtial NT/N expressioN patterN. HumaN NT/N promoter coNstructs were traNsfected iNto either Hep 3B or Hep G2. Both cell liNes supported NT/N traNscriptioN, iNdicatiNg that the abseNce of NT/N expressioN iN Hep G2 cells was due to mechaNisms other thaN the abseNce of positive traNscriptioN factors. The role of DNA methylatioN was Next assessed. MethylatioN of NT/N promoter coNstructs iN vitro resulted iN a 67-fold reductioN iN promoter activity, whereas treatmeNt with the demethylatiNg ageNt 5-azacytidiNe iNduced NT/N expressioN iN Hep G2 cells, thus suggestiNg that DNA methylatioN plays a role iN the expressioN of the gut eNdocriNe geNe NT/N. DefiNiNg the mechaNisms regulatiNg NT/N expressioN iN these hepatic-derived cell liNes will provide Not oNly a better uNderstaNdiNg of cell-specific aNd developmeNtal regulatioN of a gut eNdocriNe geNe but also possible iNsight iNto liver cell liNeage patterNs aNd the derivatioN of certaiN hepatocellular caNcers.

  • src mediated activatioN of the humaN NeuroteNsiN NeuromediN N promoter
    Surgery, 1997
    Co-Authors: Nitesh A. Banker, Mark R. Hellmich, Hong Jin Kim, Courtney M. Townsend, Mark B Evers
    Abstract:

    Abstract BackgrouNd . ExpressioN of the geNe eNcodiNg the NeuroteNsiN/NeuromediN N (NT/N) is developmeNtally regulated iN the gut iN a distiNctive temporal aNd spatial fashioN. Src kiNase, a NoNreceptor tyrosiNe kiNase, has beeN implicated iN the growth aNd differeNtiatioN of various tissues; its role iN gut differeNtiatioN is Not kNowN. The purpose of this study was to determiNe whether the Src sigNaliNg pathway plays a role iN the activatioN of the humaN NT/N promoter. Methods . Caco-2 cells, a humaN coloN caNcer cell liNe that caN differeNtiate to a small bowel pheNotype, were traNsieNtly traNsfected with humaN NT/N promoter fragmeNts liNked to luciferase aNd various amouNts of Src expressioN plasmids or domiNaNt Negative Raf; luciferase aNd β-galactosidase activities were measured after 48 hours. Results . CotraNsfectioN of Src resulted iN aN approximate eightfold iNcrease of NT/N promoter activity; mutatioN of a proximal activatiNg proteiN-1/cyclic adeNosiNe moNophosphate respoNsive elemeNt site resulted iN a dramatic decrease of Src-mediated NT/N iNductioN. CotraNsfectioN with a domiNaNt Negative Raf plasmid partially blocked Src-mediated NT/N activatioN. CoNclusioNs . Src iNcreases NT/N promoter activity iN Caco-2 cells actiNg, iN part, through a proximal AP-1/CRE promoter elemeNt. IN additioN, Src regulatioN of the NT/N promoter appears to be mediated through a Raf-depeNdeNt pathway. We propose that Src may play a role iN tissue-specific geNe expressioN iN the gut.

  • boN cells display the iNtestiNal patterN of NeuroteNsiN NeuromediN N precursor processiNg
    Regulatory Peptides, 1994
    Co-Authors: Robert E. Carraway, Sankar P Mitra, Mark B Evers, Courtney M. Townsend
    Abstract:

    Abstract ANtisera towards the bioactive peptides, NeuroteNsiN (NT, 13 residues) aNd NeuromediN N (NMN, 6 residues), as well as towards three regioNs of their 147-residue caNiNe precursor were used to ideNtify aNd to quaNtitate precursor-derived peptides iN extracts of humaN BON cells. This cell-liNe, which was obtaiNed from a humaN paNcreatic carciNoid tumor, coNstituitively expresses NT/NMN mRNA aNd secretes NT. QuaNtitatioN of seveN precursor-derived peptides led us to coNclude that BON cells display the iNtestiNal patterN of NT/NMN precursor processiNg, which is primarily characterized by the productioN of a large molecular (125 amiNo acid) form of NMN. Four large molecular compoNeNts, ideNtified by immuNochemical aNalyses aNd WesterN blottiNg, displayed physico-chemical properties which, for the most part, were coNsisteNt with the structures predicted from the partially-kNowN humaN mRNA sequeNce. However, as showN previously for these peptides iN caNiNe gut, the empirically determiNed M r aNd p I values were slightly higher thaN those predicted solely from the amiNo acid coNteNt, perhaps due to the preseNce of additioNal substitueNts. These results suggest that BON cells may provide a good iN vitro model iN which to study the regulatioN of iNtestiNal NT/NMN precursor processiNg aNd the Nature of the eNzyme(s) iNvolved.

B. Mark Evers - One of the best experts on this subject based on the ideXlab platform.

  • CharacterizatioN of Promoter ElemeNts RegulatiNg the ExpressioN of the HumaN NeuroteNsiN/NeuromediN N GeNe
    The Journal of biological chemistry, 2010
    Co-Authors: Xiaofu Wang, Paul R. Dobner, Courtney M. Townsend, Prateek Gulhati, Heidi L. Weiss, B. Mark Evers
    Abstract:

    ExpressioN of the geNe eNcodiNg NeuroteNsiN/NeuromediN N (NT/N) is mostly limited to the braiN aNd specialized eNteroeNdocriNe N cells iN the distal small iNtestiNe. We have ideNtified key regulatory elemeNts iN the promoter regioN that are iNvolved iN humaN NT/N (hNT/N) geNe expressioN iN the Novel humaN eNdocriNe cell liNe, BON, which resembles iNtestiNal N cells iN several importaNt aspects iNcludiNg NT/N precursor proteiN processiNg, ratios of differeNt NT/N mRNA isoforms, aNd high levels of coNstitutive expressioN of the NT/N geNe. IN this study, we demoNstrated multiple cis-regulatory elemeNts iNcludiNg a proximal regioN coNtaiNiNg a cAMP-respoNsive elemeNt (CRE)/AP-1-like elemeNt that biNds both the AP-1 aNd CRE-biNdiNg proteiN (CREB)/ATF proteiNs (c-JuN, ATF-1, ATF-2, JuND, aNd CREB). Similar to the rat NT/N geNe, this regioN is critical for coNstitutive hNT/N geNe expressioN. Moreover, we ideNtified a Novel regioN that biNds the orphaN hormoNe receptor, NR2F2. We have demoNstrated that the C termiNus of NR2F2 stroNgly represses hNT/N traNscriptioN, whereas aN N-termiNal domaiN aNtagoNizes this repressive effect. RegulatioN of NT/N expressioN by NR2F2 may have importaNt coNsequeNces for lipid metabolism. We speculate that a complex iNterplay betweeN the proximal CRE/AP-1-like motif aNd NR2F2 biNdiNg regioN exists to regulate hNT/N expressioN, which is critical for the high level of coNstitutive expressioN of NT/N iN eNteroeNdocriNe cells. FiNally, the BON cell liNe provides a uNique model to characterize the factors regulatiNg expressioN of the hNT/N geNe aNd to better uNderstaNd the mechaNisms respoNsible for termiNal differeNtiatioN of the N cell liNeage iN the gut.

  • Site-specific DNA methylatioN coNtributes to NeuroteNsiN/NeuromediN N expressioN iN coloN caNcers.
    American journal of physiology. Gastrointestinal and liver physiology, 2000
    Co-Authors: Zizheng Dong, Xiaofu Wang, B. Mark Evers
    Abstract:

    The NeuroteNsiN/NeuromediN N (NT/N) geNe is expressed iN fetal coloN, repressed iN NewborN aNd adult coloN, aNd reexpressed iN ∼25% of coloN caNcers. Our purpose was to determiNe the effect of geNe methylatioN oN NT/N sileNciNg iN coloN caNcers. We fouNd that the NT/N geNe was expressed iN humaN coloN caNcer cell liNe KM12C but Not iN KM20 coloN caNcer cells. Bisulfite geNomic sequeNciNg demoNstrated that all CpG diNucleotides iN the regioN from −373 to +100 of the NT/N promoter, iNcludiNg a CpG site iN a distal coNseNsus AP-1 site, were methylated iN KM20 but uNmethylated iN KM12C cells. TreatmeNt of KM20 cells with demethylatiNg ageNt 5-azacytidiNe iNduced NT/N expressioN, suggestiNg a role for DNA methylatioN iN sileNciNg of NT/N iN coloN caNcers. To better elucidate the mechaNisms respoNsible for NT/N repressioN by DNA methylatioN, we performed gel shift assays usiNg aN oligoNucleotide probe correspoNdiNg to the distal AP-1 coNseNsus sequeNce of the NT/N promoter. MethylatioN of the oligoNucleotide probe iNhibited proteiN biNdiNg to the distal AP-1 site of the NT/N promoter, suggestiNg a poteNtial mechaNism of NT/N geNe repressioN iN coloN caNcers. We show that DNA methylatioN plays a role iN NT/N geNe sileNciNg iN the humaN coloN caNcer KM20 aNd that NT/N expressioN iN KM12C cells is associated with demethylatioN of the CpG sites. DNA methylatioN likely coNtributes to NT/N geNe expressioN Noted iN humaN coloN caNcers.

  • DNA methylatioN coNtributes to expressioN of the humaN NeuroteNsiN/NeuromediN N geNe.
    The American journal of physiology, 1998
    Co-Authors: Zizheng Dong, Courtney M. Townsend, Xiaofu Wang, Qingzheng Zhao, B. Mark Evers
    Abstract:

    The gut aNd liver share a commoN embryological origiN. The geNe eNcodiNg the gut hormoNe NeuroteNsiN/NeuromediN N (NT/N) is expressed iN the adult small bowel, aNd NT/N is traNsieNtly expressed iN the fetal liver, suppressed iN the adult liver, aNd reexpressed iN certaiN liver caNcers. IN our preseNt study, we fouNd that the NT/N geNe was expressed at high levels iN the humaN hepatoma cell liNe Hep 3B but was Not expressed iN Hep G2 cells. To further determiNe the mechaNisms regulatiNg NT/N expressioN, we performed SoutherN blottiNg aNd geNe cloNiNg techNiques. Neither alteratioN Nor mutatioN of the NT/N geNe was respoNsible for this differeNtial NT/N expressioN patterN. HumaN NT/N promoter coNstructs were traNsfected iNto either Hep 3B or Hep G2. Both cell liNes supported NT/N traNscriptioN, iNdicatiNg that the abseNce of NT/N expressioN iN Hep G2 cells was due to mechaNisms other thaN the abseNce of positive traNscriptioN factors. The role of DNA methylatioN was Next assessed. MethylatioN of NT/N promoter coNstructs iN vitro resulted iN a 67-fold reductioN iN promoter activity, whereas treatmeNt with the demethylatiNg ageNt 5-azacytidiNe iNduced NT/N expressioN iN Hep G2 cells, thus suggestiNg that DNA methylatioN plays a role iN the expressioN of the gut eNdocriNe geNe NT/N. DefiNiNg the mechaNisms regulatiNg NT/N expressioN iN these hepatic-derived cell liNes will provide Not oNly a better uNderstaNdiNg of cell-specific aNd developmeNtal regulatioN of a gut eNdocriNe geNe but also possible iNsight iNto liver cell liNeage patterNs aNd the derivatioN of certaiN hepatocellular caNcers.

  • Src-mediated activatioN of the humaN NeuroteNsiN/NeuromediN N promoter
    Surgery, 1997
    Co-Authors: Nitesh A. Banker, Mark R. Hellmich, Hong Jin Kim, Courtney M. Townsend, B. Mark Evers
    Abstract:

    Abstract BackgrouNd . ExpressioN of the geNe eNcodiNg the NeuroteNsiN/NeuromediN N (NT/N) is developmeNtally regulated iN the gut iN a distiNctive temporal aNd spatial fashioN. Src kiNase, a NoNreceptor tyrosiNe kiNase, has beeN implicated iN the growth aNd differeNtiatioN of various tissues; its role iN gut differeNtiatioN is Not kNowN. The purpose of this study was to determiNe whether the Src sigNaliNg pathway plays a role iN the activatioN of the humaN NT/N promoter. Methods . Caco-2 cells, a humaN coloN caNcer cell liNe that caN differeNtiate to a small bowel pheNotype, were traNsieNtly traNsfected with humaN NT/N promoter fragmeNts liNked to luciferase aNd various amouNts of Src expressioN plasmids or domiNaNt Negative Raf; luciferase aNd β-galactosidase activities were measured after 48 hours. Results . CotraNsfectioN of Src resulted iN aN approximate eightfold iNcrease of NT/N promoter activity; mutatioN of a proximal activatiNg proteiN-1/cyclic adeNosiNe moNophosphate respoNsive elemeNt site resulted iN a dramatic decrease of Src-mediated NT/N iNductioN. CotraNsfectioN with a domiNaNt Negative Raf plasmid partially blocked Src-mediated NT/N activatioN. CoNclusioNs . Src iNcreases NT/N promoter activity iN Caco-2 cells actiNg, iN part, through a proximal AP-1/CRE promoter elemeNt. IN additioN, Src regulatioN of the NT/N promoter appears to be mediated through a Raf-depeNdeNt pathway. We propose that Src may play a role iN tissue-specific geNe expressioN iN the gut.

  • BON cells display the iNtestiNal patterN of NeuroteNsiN/NeuromediN N precursor processiNg
    Regulatory peptides, 1994
    Co-Authors: Robert E. Carraway, Sankar P Mitra, B. Mark Evers, Courtney M. Townsend
    Abstract:

    Abstract ANtisera towards the bioactive peptides, NeuroteNsiN (NT, 13 residues) aNd NeuromediN N (NMN, 6 residues), as well as towards three regioNs of their 147-residue caNiNe precursor were used to ideNtify aNd to quaNtitate precursor-derived peptides iN extracts of humaN BON cells. This cell-liNe, which was obtaiNed from a humaN paNcreatic carciNoid tumor, coNstituitively expresses NT/NMN mRNA aNd secretes NT. QuaNtitatioN of seveN precursor-derived peptides led us to coNclude that BON cells display the iNtestiNal patterN of NT/NMN precursor processiNg, which is primarily characterized by the productioN of a large molecular (125 amiNo acid) form of NMN. Four large molecular compoNeNts, ideNtified by immuNochemical aNalyses aNd WesterN blottiNg, displayed physico-chemical properties which, for the most part, were coNsisteNt with the structures predicted from the partially-kNowN humaN mRNA sequeNce. However, as showN previously for these peptides iN caNiNe gut, the empirically determiNed M r aNd p I values were slightly higher thaN those predicted solely from the amiNo acid coNteNt, perhaps due to the preseNce of additioNal substitueNts. These results suggest that BON cells may provide a good iN vitro model iN which to study the regulatioN of iNtestiNal NT/NMN precursor processiNg aNd the Nature of the eNzyme(s) iNvolved.

Tohru Mochizuki - One of the best experts on this subject based on the ideXlab platform.

  • proNeuroteNsiN NeuromediN N secreted from small cell luNg carciNoma cell liNes as a poteNtial tumor marker
    Proteomics Clinical Applications, 2008
    Co-Authors: Shun-ichiro Ogura, Kumi Kaneko, Shoji Miyajima, Keiichi Ohshima, Ken Yamaguchi, Tohru Mochizuki
    Abstract:

    ProteiNs secreted from specific caNcer cells have a high poteNtial for use as tumor markers. We ideNtified secreted proteiNs produced by 15 differeNt carciNoma cell liNes growN iN serum-free medium usiNg MS/MS. ProNeuroteNsiN/NeuromediN N (proNT/NMN) was fouNd iN coNditioNed medium from four of seveN small cell luNg carciNoma cell liNes but Not from eight NoNsmall cell luNg carciNoma cell liNes. These results iNdicate proNT/NMN has poteNtial as a specific tumor marker of small cell luNg carciNoma.

  • ProNeuroteNsiN/NeuromediN N secreted from small cell luNg carciNoma cell liNes as a poteNtial tumor marker.
    Proteomics. Clinical applications, 2008
    Co-Authors: Shun-ichiro Ogura, Kumi Kaneko, Shoji Miyajima, Keiichi Ohshima, Ken Yamaguchi, Tohru Mochizuki
    Abstract:

    ProteiNs secreted from specific caNcer cells have a high poteNtial for use as tumor markers. We ideNtified secreted proteiNs produced by 15 differeNt carciNoma cell liNes growN iN serum-free medium usiNg MS/MS. ProNeuroteNsiN/NeuromediN N (proNT/NMN) was fouNd iN coNditioNed medium from four of seveN small cell luNg carciNoma cell liNes but Not from eight NoNsmall cell luNg carciNoma cell liNes. These results iNdicate proNT/NMN has poteNtial as a specific tumor marker of small cell luNg carciNoma.

Robert E. Carraway - One of the best experts on this subject based on the ideXlab platform.

  • BON cells display the iNtestiNal patterN of NeuroteNsiN/NeuromediN N precursor processiNg
    Regulatory peptides, 1994
    Co-Authors: Robert E. Carraway, Sankar P Mitra, B. Mark Evers, Courtney M. Townsend
    Abstract:

    Abstract ANtisera towards the bioactive peptides, NeuroteNsiN (NT, 13 residues) aNd NeuromediN N (NMN, 6 residues), as well as towards three regioNs of their 147-residue caNiNe precursor were used to ideNtify aNd to quaNtitate precursor-derived peptides iN extracts of humaN BON cells. This cell-liNe, which was obtaiNed from a humaN paNcreatic carciNoid tumor, coNstituitively expresses NT/NMN mRNA aNd secretes NT. QuaNtitatioN of seveN precursor-derived peptides led us to coNclude that BON cells display the iNtestiNal patterN of NT/NMN precursor processiNg, which is primarily characterized by the productioN of a large molecular (125 amiNo acid) form of NMN. Four large molecular compoNeNts, ideNtified by immuNochemical aNalyses aNd WesterN blottiNg, displayed physico-chemical properties which, for the most part, were coNsisteNt with the structures predicted from the partially-kNowN humaN mRNA sequeNce. However, as showN previously for these peptides iN caNiNe gut, the empirically determiNed M r aNd p I values were slightly higher thaN those predicted solely from the amiNo acid coNteNt, perhaps due to the preseNce of additioNal substitueNts. These results suggest that BON cells may provide a good iN vitro model iN which to study the regulatioN of iNtestiNal NT/NMN precursor processiNg aNd the Nature of the eNzyme(s) iNvolved.

  • boN cells display the iNtestiNal patterN of NeuroteNsiN NeuromediN N precursor processiNg
    Regulatory Peptides, 1994
    Co-Authors: Robert E. Carraway, Sankar P Mitra, Mark B Evers, Courtney M. Townsend
    Abstract:

    Abstract ANtisera towards the bioactive peptides, NeuroteNsiN (NT, 13 residues) aNd NeuromediN N (NMN, 6 residues), as well as towards three regioNs of their 147-residue caNiNe precursor were used to ideNtify aNd to quaNtitate precursor-derived peptides iN extracts of humaN BON cells. This cell-liNe, which was obtaiNed from a humaN paNcreatic carciNoid tumor, coNstituitively expresses NT/NMN mRNA aNd secretes NT. QuaNtitatioN of seveN precursor-derived peptides led us to coNclude that BON cells display the iNtestiNal patterN of NT/NMN precursor processiNg, which is primarily characterized by the productioN of a large molecular (125 amiNo acid) form of NMN. Four large molecular compoNeNts, ideNtified by immuNochemical aNalyses aNd WesterN blottiNg, displayed physico-chemical properties which, for the most part, were coNsisteNt with the structures predicted from the partially-kNowN humaN mRNA sequeNce. However, as showN previously for these peptides iN caNiNe gut, the empirically determiNed M r aNd p I values were slightly higher thaN those predicted solely from the amiNo acid coNteNt, perhaps due to the preseNce of additioNal substitueNts. These results suggest that BON cells may provide a good iN vitro model iN which to study the regulatioN of iNtestiNal NT/NMN precursor processiNg aNd the Nature of the eNzyme(s) iNvolved.

  • Tissue-specific processiNg of NeuroteNsiN/NeuromediN-N precursor iN cat
    Regulatory Peptides, 1993
    Co-Authors: Robert E. Carraway, Sankar Mitra, Terrence J. Joyce
    Abstract:

    Abstract ANtisera were raised towards the putative N-termiNal sequeNce of the caNiNe precursor to NeuroteNsiN (NT) aNd NeuromediN N (NMN), residues 24–35 iN the pre-prohormoNe, as well as its C-termiNal TAIL, residues 164–170. These were used iN coNjuNctioN with previously developed immuNoassays towards NT aNd NMN to characterize the precursor aNd to study its processiNg iN feliNe braiN, heart, adreNal aNd iNtestiNe. The precursor was ideNtified as a 18 kDa proteiN that reacted with both the N- aNd C-termiNal aNtisera aNd yielded NT 3–13 aNd NT 4–13 upoN treatmeNt with pepsiN. It represeNted 5–10% of the NT activity iN ileum. ProcessiNg was fouNd to be tissue-specific, yieldiNg primarily NT, NMN aNd TAIL iN most tissues but also giviNg rise to N-termiNally exteNded forms of these peptides, which were particularly evideNt iN extracts of adreNal aNd iNtestiNe. Two of the four molecular forms detected usiNg the TAIL immuNoassay were teNatively ideNtified as NT-TAIL aNd Gly-Ser-Tyr-Tyr-Tyr based upoN chromatographic evideNce. WesterN blottiNg iNdicated that ileal extracts coNtaiNed large molecular forms of NT (17 kDa) aNd NMN (16 kDa), whereas braiN extracts coNtaiNed primarily the N-termiNal of the precursor without NT aNd NMN attached (14.3 kDa). It was coNcluded that the structure of the feliNe precursor iN its N- aNd C-termiNal regioNs, aNd the positioNiNg of the sigNal peptide cleavage site were as predicted from cDNA work iN the dog. ON the other haNd, processiNg of the precursor was complex, occurriNg iN a tissue-specific maNNer.

  • tissue specific processiNg of NeuroteNsiN NeuromediN N precursor iN cat
    Regulatory Peptides, 1993
    Co-Authors: Robert E. Carraway, Sankar P Mitra, Terrence J. Joyce
    Abstract:

    Abstract ANtisera were raised towards the putative N-termiNal sequeNce of the caNiNe precursor to NeuroteNsiN (NT) aNd NeuromediN N (NMN), residues 24–35 iN the pre-prohormoNe, as well as its C-termiNal TAIL, residues 164–170. These were used iN coNjuNctioN with previously developed immuNoassays towards NT aNd NMN to characterize the precursor aNd to study its processiNg iN feliNe braiN, heart, adreNal aNd iNtestiNe. The precursor was ideNtified as a 18 kDa proteiN that reacted with both the N- aNd C-termiNal aNtisera aNd yielded NT 3–13 aNd NT 4–13 upoN treatmeNt with pepsiN. It represeNted 5–10% of the NT activity iN ileum. ProcessiNg was fouNd to be tissue-specific, yieldiNg primarily NT, NMN aNd TAIL iN most tissues but also giviNg rise to N-termiNally exteNded forms of these peptides, which were particularly evideNt iN extracts of adreNal aNd iNtestiNe. Two of the four molecular forms detected usiNg the TAIL immuNoassay were teNatively ideNtified as NT-TAIL aNd Gly-Ser-Tyr-Tyr-Tyr based upoN chromatographic evideNce. WesterN blottiNg iNdicated that ileal extracts coNtaiNed large molecular forms of NT (17 kDa) aNd NMN (16 kDa), whereas braiN extracts coNtaiNed primarily the N-termiNal of the precursor without NT aNd NMN attached (14.3 kDa). It was coNcluded that the structure of the feliNe precursor iN its N- aNd C-termiNal regioNs, aNd the positioNiNg of the sigNal peptide cleavage site were as predicted from cDNA work iN the dog. ON the other haNd, processiNg of the precursor was complex, occurriNg iN a tissue-specific maNNer.

  • IsolatioN aNd quaNtitatioN of several New peptides from the caNiNe NeuroteNsiN/NeuromediN N precursor
    Peptides, 1992
    Co-Authors: Robert E. Carraway, Sankar P Mitra, Rebecca Salmonsen
    Abstract:

    Abstract UsiNg aNtisera towards the bioactive peptides, NeuroteNsiN aNd NeuromediN N, as well as towards the N -termiNal aNd C -termiNal regioNs of their shared 170-residue precursor, peptides represeNtiNg various portioNs of the precursor were isolated from extracts of caNiNe ileum. IN total, seveN peptides were isolated, two of which had Not beeN previously ideNtified. ONe was the C -termiNal tail of the precursor (Gly-Ser-Tyr-Tyr-Tyr) aNd the other was the tail peptide exteNded N -termiNally to iNclude NeuroteNsiN (Glp-Leu-Tyr-Glu-AsN-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Lys-Arg-Gly-Ser-Tyr-Tyr-Tyr). By compariNg the measured coNceNtratioNs for each of the ideNtified peptides, it was established that processiNg at the three Lys-Arg cleavage sites withiN the precursor did Not occur to the same exteNt. Cleavage at the N -termiNus of NeuromediN N was ∼10% complete, that betweeN NeuroteNsiN aNd the tail was ∼90% complete, aNd that betweeN NeuromediN N aNd NeuroteNsiN was ∼95% complete. Three immuNoreactive proteiNs were also ideNtified by immuNochemical aNd chromatographic aNalyses: N -termiNally exteNded NeuromediN N (125 residues), N -termiNally exteNded NeuroteNsiN (140 residues), aNd the eNtire 147-residue precursor. It was coNcluded that NeuroteNsiN, tail aNd large molecular NeuromediN N were the primary products of processiNg for this precursor iN caNiNe ileum, while miNor products iNcluded NeuromediN N, NeuroteNsiN tail, aNd large molecular NeuroteNsiN.

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  • differeNtial processiNg of pro NeuroteNsiN NeuromediN N aNd relatioNship to pro hormoNe coNvertases
    Peptides, 2006
    Co-Authors: Patrick Kitabgi
    Abstract:

    NeuroteNsiN (NT) is syNthesized as part of a larger precursor that also coNtaiNs NeuromediN N (NN), a six amiNo acid NeuroteNsiN-like peptide. NT aNd NN are located iN the C-termiNal regioN of the precursor (pro-NT/NN) where they are flaNked aNd separated by three Lys-Arg sequeNces. A fourth dibasic sequeNce is preseNt iN the middle of the precursor. Dibasics are the coNseNsus sites recogNized aNd cleaved by eNdoproteases that beloNg to the receNtly ideNtified family of pro-proteiN coNvertases (PCs). IN tissues that express pro-NT/NN, the three C-termiNal Lys-Arg sites are differeNtially processed, whereas the middle dibasic is poorly cleaved. Pro-NT/NN processiNg gives rise maiNly to NT aNd NN iN the braiN, to NT aNd a large peptide eNdiNg with the NN sequeNce at its C-termiNus (large NN) iN the gut aNd to NT, large NN aNd a large peptide eNdiNg with the NT sequeNce (large NT) iN the adreNals. ReceNt evideNce iNdicates that PC1, PC2 aNd PC5-A are the pro-hormoNe coNvertases respoNsible for the processiNg patterNs observed iN the gut, braiN aNd adreNals, respectively. As NT, NN, large NT aNd large NN are all eNdowed with biological activity, the evideNce reviewed here supports the idea that post-traNslatioNal processiNg of pro-NT/NN iN tissues may geNerate biological diversity.

  • INactivatioN of NeuroteNsiN aNd NeuromediN N by ZN metallopeptidases.
    Peptides, 2006
    Co-Authors: Patrick Kitabgi
    Abstract:

    The two related peptides NeuroteNsiN (NT) aNd NeuromediN N (NN) are efficieNtly iNactivated by peptidases iN vitro. Whereas NT is primarily degraded by a combiNatioN of three ZN metallo-eNdopeptidases, Namely eNdopeptidases 24.11, 24.15 aNd 24.16, iN all systems examiNed, NN is esseNtially iNactivated by the ZN metallo-exopeptidase amiNopeptidase M. IN this paper we review the work that has led to the ideNtificatioN of the NT- aNd NN-degradiNg eNzymes aNd to the purificatioN aNd cloNiNg of EP 24.16, a previously uNideNtified peptidase. We provide a brief descriptioN of the three NT-iNactivatiNg eNdopeptidases aNd of their specific aNd mixed iNhibitors, some of them developed iN the course of studyiNg NT degradatioN. FiNally, we review iN vivo data obtaiNed with these iNhibitors that stroNgly support a physiological role for EP 24.11, 24.15 aNd 24.16 iN the termiNatioN of NT-geNerated sigNals aNd for amiNopeptidase iN termiNatiNg NN actioN. KNowledge of the NT aNd NN iNactivatioN mechaNisms offers the perspective to develop metabolically stable aNalogs of these peptides with poteNtial therapeutic value.

  • DiffereNtial processiNg of pro-NeuroteNsiN/NeuromediN N aNd relatioNship to pro-hormoNe coNvertases.
    Peptides, 2006
    Co-Authors: Patrick Kitabgi
    Abstract:

    NeuroteNsiN (NT) is syNthesized as part of a larger precursor that also coNtaiNs NeuromediN N (NN), a six amiNo acid NeuroteNsiN-like peptide. NT aNd NN are located iN the C-termiNal regioN of the precursor (pro-NT/NN) where they are flaNked aNd separated by three Lys-Arg sequeNces. A fourth dibasic sequeNce is preseNt iN the middle of the precursor. Dibasics are the coNseNsus sites recogNized aNd cleaved by eNdoproteases that beloNg to the receNtly ideNtified family of pro-proteiN coNvertases (PCs). IN tissues that express pro-NT/NN, the three C-termiNal Lys-Arg sites are differeNtially processed, whereas the middle dibasic is poorly cleaved. Pro-NT/NN processiNg gives rise maiNly to NT aNd NN iN the braiN, to NT aNd a large peptide eNdiNg with the NN sequeNce at its C-termiNus (large NN) iN the gut aNd to NT, large NN aNd a large peptide eNdiNg with the NT sequeNce (large NT) iN the adreNals. ReceNt evideNce iNdicates that PC1, PC2 aNd PC5-A are the pro-hormoNe coNvertases respoNsible for the processiNg patterNs observed iN the gut, braiN aNd adreNals, respectively. As NT, NN, large NT aNd large NN are all eNdowed with biological activity, the evideNce reviewed here supports the idea that post-traNslatioNal processiNg of pro-NT/NN iN tissues may geNerate biological diversity.

  • ProhormoNe coNvertases differeNtially process pro-NeuroteNsiN/NeuromediN N iN tissues aNd cell liNes
    Journal of Molecular Medicine, 2006
    Co-Authors: Patrick Kitabgi
    Abstract:

    NeuroteNsiN (NT) is syNthesized as part of a larger precursor that also coNtaiNs NeuromediN N (NN), a six-amiNo acid NeuroteNsiN-like peptide. NT aNd NN are located iN the C-termiNal regioN of the precursor (pro-NT/NN) where they are flaNked aNd separated by three Lys-Arg sequeNces. A fourth dibasic sequeNce is preseNt iN the middle of the precursor. Dibasics are the coNseNsus sites recogNized aNd cleaved by specialized eNdoproteases that beloNg to the family of proproteiN coNvertases (PCs). IN tissues that express pro-NT/NN, the three C-termiNal Lys-Arg sites are differeNtially processed, whereas the middle dibasic is poorly cleaved. ProcessiNg gives rise maiNly to NT aNd NN iN the braiN, to NT aNd a large peptide with a C-termiNal NN moiety (large NN) iN the gut, aNd to NT, large NN, aNd a large peptide with a C-termiNal NT moiety (large NT) iN the adreNals. ReceNt evideNce iNdicates that PC1, PC2, aNd PC5-A are the prohormoNe coNvertases respoNsible for the processiNg patterNs observed iN the gut, braiN, aNd adreNals, respectively. As NT, NN, large NT, aNd large NN are all eNdowed with biological activity, the evideNce reviewed iN this paper supports the idea that posttraNslatioNal processiNg of pro-NT/NN iN tissues may geNerate biological diversity of pathophysiological relevaNce.

  • prohormoNe coNvertases differeNtially process pro NeuroteNsiN NeuromediN N iN tissues aNd cell liNes
    Journal of Molecular Medicine, 2006
    Co-Authors: Patrick Kitabgi
    Abstract:

    NeuroteNsiN (NT) is syNthesized as part of a larger precursor that also coNtaiNs NeuromediN N (NN), a six-amiNo acid NeuroteNsiN-like peptide. NT aNd NN are located iN the C-termiNal regioN of the precursor (pro-NT/NN) where they are flaNked aNd separated by three Lys-Arg sequeNces. A fourth dibasic sequeNce is preseNt iN the middle of the precursor. Dibasics are the coNseNsus sites recogNized aNd cleaved by specialized eNdoproteases that beloNg to the family of proproteiN coNvertases (PCs). IN tissues that express pro-NT/NN, the three C-termiNal Lys-Arg sites are differeNtially processed, whereas the middle dibasic is poorly cleaved. ProcessiNg gives rise maiNly to NT aNd NN iN the braiN, to NT aNd a large peptide with a C-termiNal NN moiety (large NN) iN the gut, aNd to NT, large NN, aNd a large peptide with a C-termiNal NT moiety (large NT) iN the adreNals. ReceNt evideNce iNdicates that PC1, PC2, aNd PC5-A are the prohormoNe coNvertases respoNsible for the processiNg patterNs observed iN the gut, braiN, aNd adreNals, respectively. As NT, NN, large NT, aNd large NN are all eNdowed with biological activity, the evideNce reviewed iN this paper supports the idea that posttraNslatioNal processiNg of pro-NT/NN iN tissues may geNerate biological diversity of pathophysiological relevaNce.