Genomes and Genes
Summary: EXOPEPTIDASES that specifically act on dipeptides. EC 3.4.13.
- Nitanai Y, Satow Y, Adachi H, Tsujimoto M. Crystal structure of human renal dipeptidase involved in beta-lactam hydrolysis. J Mol Biol. 2002;321:177-84 pubmed..The barrel and active-site architectures utilizing catalytic metal ions exhibit unexpected similarities to those of the murine adenosine deaminase and the catalytic domain of the bacterial urease. ..
- Miltyk W, Karna E, Palka J. Prolidase-independent mechanism of camptothecin-induced inhibition of collagen biosynthesis in cultured human skin fibroblasts. J Biochem. 2007;141:287-92 pubmed..The data suggest that CPT induces inhibition of collagen biosynthesis in cultured human skin fibroblasts by stimulation of NF-kappaB signalling. ..
- Wilson C, Abbott C. Expression profiling of dipeptidyl peptidase 8 and 9 in breast and ovarian carcinoma cell lines. Int J Oncol. 2012;41:919-32 pubmed publisher..Overall, this study suggests a potential role for DP8 and DP9 in breast and ovarian cancer and further investigations in this area are required. ..
- Riedl E, Koeppel H, Brinkkoetter P, Sternik P, Steinbeisser H, Sauerhoefer S, et al. A CTG polymorphism in the CNDP1 gene determines the secretion of serum carnosinase in Cos-7 transfected cells. Diabetes. 2007;56:2410-3 pubmed
- Miltyk W, Surazynski A, Kasprzak K, Fivash M, Buzard G, Phang J. Inhibition of prolidase activity by nickel causes decreased growth of proline auxotrophic CHO cells. J Cell Biochem. 2005;94:1210-7 pubmed
- Surazynski A, Pałka J, Wołczyński S. Phosphorylation of prolidase increases the enzyme activity. Mol Cell Biochem. 2001;220:95-101 pubmed..These results provide evidence that prolidase is phosphotyrosine enzyme and suggest that the activity of prolidase may be up regulated by the enzyme phosphorylation. ..
- Verit F, Geyikli I, Yazgan P, Celik A. Correlations of serum prolidase activity between bone turnover markers and mineral density in postmenopausal osteoporosis. Arch Gynecol Obstet. 2006;274:133-7 pubmed..To conclude, in postmenopausal osteoporotic women with increased bone turnover, serum prolidase concentration was not correlated with the biomarkers of bone formation or bone resorption and with BMD. ..
- Yazbeck R, Howarth G, Abbott C. Dipeptidyl peptidase inhibitors, an emerging drug class for inflammatory disease?. Trends Pharmacol Sci. 2009;30:600-7 pubmed publisher..This review highlights recent insights into the applicability of DPP inhibitors as novel pharmacological agents for inflammatory disease. ..
- Lupi A, Rossi A, Campari E, Pecora F, Lund A, Elcioglu N, et al. Molecular characterisation of six patients with prolidase deficiency: identification of the first small duplication in the prolidase gene and of a mutation generating symptomatic and asymptomatic outcomes within the same family. J Med Genet. 2006;43:e58 pubmed..The E412K substitution is responsible for the first reported phenotypic variability within a family with severe and asymptomatic outcomes. ..
- Forlino A, Lupi A, Vaghi P, Icaro Cornaglia A, Calligaro A, Campari E, et al. Mutation analysis of five new patients affected by prolidase deficiency: the lack of enzyme activity causes necrosis-like cell death in cultured fibroblasts. Hum Genet. 2002;111:314-22 pubmed..Our results provide the first evidence that absence of prolidase activity causes the activation of a necrosis-like cellular death, which could be responsible for the typical skin lesions in PD. ..
- Okamoto T, Matsumura N, Mandai M, Oura T, Yamanishi Y, Horiuchi A, et al. Distinguishing primary from secondary mucinous ovarian tumors: an algorithm using the novel marker DPEP1. Mod Pathol. 2011;24:267-76 pubmed publisher..The algorithm using immunohistochemical and clinical factors to distinguish metastases of digestive organ cancers from primary mucinous ovarian cancers will be useful to establish a protocol for the diagnosis of ovarian metastasis. ..
- Pandya V, Ekka M, Dutta R, Kumaran S. Mass spectrometry assay for studying kinetic properties of dipeptidases: characterization of human and yeast dipeptidases. Anal Biochem. 2011;418:134-42 pubmed publisher..The ESI-MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo.
- Besio R, Monzani E, Gioia R, Nicolis S, Rossi A, Casella L, et al. Improved prolidase activity assay allowed enzyme kinetic characterization and faster prolidase deficiency diagnosis. Clin Chim Acta. 2011;412:1814-20 pubmed publisher..We optimized a faster spectrophotometric method to measure the activity when the enzyme is fully activated, this is crucial to allow a reliable evaluation of prolidase activity from different sources. ..
- Chakkera H, Hanson R, Kobes S, Millis M, Nelson R, Knowler W, et al. Association of variants in the carnosine peptidase 1 gene (CNDP1) with diabetic nephropathy in American Indians. Mol Genet Metab. 2011;103:185-90 pubmed publisher..04; 18.8ml/min per copy of the C allele; p=0.03; and -13.4ml/min per copy of the C allele; p=0.001, respectively). These findings provide nominal evidence supporting a role between CNDP1 variants and diabetic kidney disease. ..
- Surazynski A, Liu Y, Miltyk W, Phang J. Nitric oxide regulates prolidase activity by serine/threonine phosphorylation. J Cell Biochem. 2005;96:1086-94 pubmed
- Janssen B, Hohenadel D, Brinkkoetter P, Peters V, Rind N, Fischer C, et al. Carnosine as a protective factor in diabetic nephropathy: association with a leucine repeat of the carnosinase gene CNDP1. Diabetes. 2005;54:2320-7 pubmed..Diabetic patients with the CNDP1 Mannheim variant are less susceptible for nephropathy. Carnosine protects against the adverse effects of high glucose levels on renal cells. ..
- Freedman B, Hicks P, Sale M, Pierson E, Langefeld C, Rich S, et al. A leucine repeat in the carnosinase gene CNDP1 is associated with diabetic end-stage renal disease in European Americans. Nephrol Dial Transplant. 2007;22:1131-5 pubmed..This replicates the CNDP1 gene association with DN that was initially detected in European Caucasians and in Arabs, and further demonstrates that the CNDP1 gene and carnosine pathway appear to play a role in susceptibility to DN. ..
- Lankas G, Leiting B, Roy R, Eiermann G, Beconi M, Biftu T, et al. Dipeptidyl peptidase IV inhibition for the treatment of type 2 diabetes: potential importance of selectivity over dipeptidyl peptidases 8 and 9. Diabetes. 2005;54:2988-94 pubmed..These results suggest that assessment of selectivity of potential clinical candidates may be important to an optimal safety profile for this new class of antihyperglycemic agents. ..
- McIver C, Lloyd J, Hewett P, Hardingham J. Dipeptidase 1: a candidate tumor-specific molecular marker in colorectal carcinoma. Cancer Lett. 2004;209:67-74 pubmed..This is the first report of DPEP1 as a marker for disseminated colon tumor cells. ..
- Guszczyn T, Sobolewski K. Deregulation of collagen metabolism in human stomach cancer. Pathobiology. 2004;71:308-13 pubmed..The data presented suggest an enhancement of collagen turnover in stomach cancer. It may be suggested that the increased degradation of collagen by gelatinase in cancer tissue is balanced by an increased biosynthesis of this protein. ..
- Chen Y, Chien C, Goparaju C, Hsu J, Liang P, Chen X. Purification and characterization of human prolyl dipeptidase DPP8 in Sf9 insect cells. Protein Expr Purif. 2004;35:142-6 pubmedDPP8 is a new member of the prolyl dipeptidases, many of which have important biological functions in vivo. DPP8 catalyzes the cleavage at the carboxyl side of the proline residue at the penultimate position...
- Kayadibi H, Gultepe M, Yasar B, Ince A, Ozcan O, Ipcioglu O, et al. Diagnostic value of serum prolidase enzyme activity to predict the liver histological lesions in non-alcoholic fatty liver disease: a surrogate marker to distinguish steatohepatitis from simple steatosis. Dig Dis Sci. 2009;54:1764-71 pubmed publisher..It could be helpful for distinguishing steatohepatitis from simple steatosis and reducing the need for liver biopsy in the majority of patients with NAFLD. ..
- Cakmak A, Zeyrek D, Atas A, Celik H, Aksoy N, Erel O. Serum prolidase activity and oxidative status in patients with bronchial asthma. J Clin Lab Anal. 2009;23:132-8 pubmed publisher..Moreover, the high level of TOS indicates that these patients were exposed to severe oxidative stress with an increased TAC response. ..
- Surazynski A, Miltyk W, Prokop I, Palka J. Prolidase-dependent regulation of TGF ? (corrected) and TGF ? receptor expressions in human skin fibroblasts. Eur J Pharmacol. 2010;649:115-9 pubmed publisher..It seems, that products of prolidase activity, Pro or HyPro may act as an interface between mTOR and phospho-mTOR in regulation of numerous TGF ? receptor-dependent functions. ..
- Theriot C, Du X, Tove S, Grunden A. Improving the catalytic activity of hyperthermophilic Pyrococcus prolidases for detoxification of organophosphorus nerve agents over a broad range of temperatures. Appl Microbiol Biotechnol. 2010;87:1715-26 pubmed publisher..coli strain JD1 (lambdaDE3) (auxotrophic for proline [DeltaproA] and having deletions in pepQ and pepP dipeptidases with specificity for proline-containing dipeptides) was constructed for screening mutant P...
- Zhang G, Schetter A, He P, Funamizu N, Gaedcke J, Ghadimi B, et al. DPEP1 inhibits tumor cell invasiveness, enhances chemosensitivity and predicts clinical outcome in pancreatic ductal adenocarcinoma. PLoS ONE. 2012;7:e31507 pubmed publisher..In view of these findings, we propose that DPEP1 may be a candidate target in PDAC for designing improved treatments. ..
- Lupi A, Rossi A, Vaghi P, Gallanti A, Cetta G, Forlino A. N-benzyloxycarbonyl-L-proline: an in vitro and in vivo inhibitor of prolidase. Biochim Biophys Acta. 2005;1744:157-63 pubmed..Our results demonstrated that Cbz-Pro is a potent inhibitor of prolidase in cultured fibroblasts and it can be used in vivo to better characterize the prolidase enzyme and further investigate PD physiopathology. ..
- Peters V, Kebbewar M, Jansen E, Jakobs C, Riedl E, Koeppel H, et al. Relevance of allosteric conformations and homocarnosine concentration on carnosinase activity. Amino Acids. 2010;38:1607-15 pubmed publisher..Homocarnosine treatment can be expected to reduce CN1 activity toward carnosine, resulting in higher carnosine levels. ..
- Theriot C, Tove S, Grunden A. Characterization of two proline dipeptidases (prolidases) from the hyperthermophilic archaeon Pyrococcus horikoshii. Appl Microbiol Biotechnol. 2010;86:177-88 pubmed publisher..recombinantly in the Escherichia coli strain BL21 (lambdaDE3), and both were shown to function as proline dipeptidases. Biochemical characterization of these prolidases shows they have higher catalytic activities over a broader pH ..
- Wanic K, Placha G, Dunn J, Smiles A, Warram J, Krolewski A. Exclusion of polymorphisms in carnosinase genes (CNDP1 and CNDP2) as a cause of diabetic nephropathy in type 1 diabetes: results of large case-control and follow-up studies. Diabetes. 2008;57:2547-51 pubmed publisher..Our large, comprehensive study did not find an association between the D18S880 microsatellite or any other polymorphisms in the CNDP2-CNDP1 genomic region and susceptibility for diabetic nephropathy in type 1 diabetes. ..
- Lupi A, Tenni R, Rossi A, Cetta G, Forlino A. Human prolidase and prolidase deficiency: an overview on the characterization of the enzyme involved in proline recycling and on the effects of its mutations. Amino Acids. 2008;35:739-52 pubmed publisher..The pathophysiology of PD is still poorly understood; we offer here a review of the molecular mechanisms so far hypothesized. ..
- Ajami K, Pitman M, Wilson C, Park J, Menz R, Starr A, et al. Stromal cell-derived factors 1alpha and 1beta, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8. FEBS Lett. 2008;582:819-25 pubmed publisher..This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes. ..
- Maher M, Ghosh M, Grunden A, Menon A, Adams M, Freeman H, et al. Structure of the prolidase from Pyrococcus furiosus. Biochemistry. 2004;43:2771-83 pubmed publisher..The final R and R(free) values are 0.24 and 0.28, respectively...
- Duong H, Zhang Q, Le A, Kelly A, Kamdar R, Messadi D. Elevated prolidase activity in keloids: correlation with type I collagen turnover. Br J Dermatol. 2006;154:820-8 pubmed..The combination of elevated prolidase activity and associated higher collagen synthesis to degradation ratio in keloids suggests a possible metabolic process for the excessive accumulation of type I collagen in keloids. ..
- Cechowska Pasko M, Pałka J, Wojtukiewicz M. Enhanced prolidase activity and decreased collagen content in breast cancer tissue. Int J Exp Pathol. 2006;87:289-96 pubmed..On the basis of the data, it seems that compounds endowed with gelatinolytic and prolidase inhibitory activities may be considered as a potential drug candidates for breast cancer therapy. ..
- Wołczyński S, Surazynski A, Swiatecka J, Pałka J. Estrogenic and antiestrogenic effects of raloxifene on collagen metabolism in breast cancer MCF-7 cells. Gynecol Endocrinol. 2001;15:225-33 pubmed..Our data suggest that the effects of raloxifene on collagen synthesis, prolidase and metalloproteinase activities in breast cancer may explain its role in the prevention of breast cancer development. ..
- Chen X. Biochemical properties of recombinant prolyl dipeptidases DPP-IV and DPP8. Adv Exp Med Biol. 2006;575:27-32 pubmed
- Pitman M, Menz R, Abbott C. Hydrophilic residues surrounding the S1 and S2 pockets contribute to dimerisation and catalysis in human dipeptidyl peptidase 8 (DP8). Biol Chem. 2010;391:959-72 pubmed publisher..This study provides insights into how the DP8 substrate pocket and dimer interface differ from DPIV and FAP which could be utilised for designing more selective DP8 inhibitors. ..
- Karna E, Pałka J, Wołczyński S. Doxycycline-induced inhibition of prolidase activity in human skin fibroblasts and its involvement in impaired collagen biosynthesis. Eur J Pharmacol. 2001;430:25-31 pubmed..The data presented here raise the possibility that the doxycycline-induced decrease in collagen biosynthesis is mostly due to the inhibition of prolidase activity. ..
- Lupi A, Della Torre S, Campari E, Tenni R, Cetta G, Rossi A, et al. Human recombinant prolidase from eukaryotic and prokaryotic sources. Expression, purification, characterization and long-term stability studies. FEBS J. 2006;273:5466-78 pubmed..Generation of the prolidase in Escherichia coli, because of its high yield, stability, and similarity to native prolidase, appears to be the best approach for future structural studies and enzyme replacement therapy. ..
- Savas M, Yeni E, Celik H, Ciftci H, Utangac M, Oncel H, et al. The association of serum prolidase activity and erectile dysfunction. J Androl. 2010;31:146-54 pubmed publisher..78. Findings of this study have shown that serum prolidase activity is significantly associated with the presence and severity of vasculogenic ED, and elevated serum prolidase activity might be an independent predictor of ED. ..
- Mooyaart A, van Valkengoed I, Shaw P, Peters V, Baelde H, Rabelink T, et al. Lower frequency of the 5/5 homozygous CNDP1 genotype in South Asian Surinamese. Diabetes Res Clin Pract. 2009;85:272-8 pubmed publisher..Our study provides an indication that South Asian Surinamese are genetically at risk for developing diabetic nephropathy. ..
- Vistoli G, Orioli M, Pedretti A, Regazzoni L, Canevotti R, Negrisoli G, et al. Design, synthesis, and evaluation of carnosine derivatives as selective and efficient sequestering agents of cytotoxic reactive carbonyl species. ChemMedChem. 2009;4:967-75 pubmed publisher..This study describes a set of aryl derivatives that are characterized by high stability in human plasma and a quenching activity toward 4-hydroxy-trans-2-nonenal (HNE), chosen as a model of RCS, up to threefold greater than D-carnosine. ..
- Liu G, Nakayama K, Awata S, Tang S, Kitaoka N, Manabe M, et al. Prolidase isoenzymes in the rat: their organ distribution, developmental change and specific inhibitors. Pediatr Res. 2007;62:54-9 pubmed..Similarly, age-related alterations of prolidase isoenzymes suggest that intestinal PD II also participates in absorption of proline and other amino acids early in life. ..
- Surazynski A, Miltyk W, Palka J, Phang J. Prolidase-dependent regulation of collagen biosynthesis. Amino Acids. 2008;35:731-8 pubmed publisher..e. echistatin) beta(1)-integrin ligands or by nitric oxide donors (i.e. DETA/NO) affect prolidase at post-transcriptional level. All those factors may represent novel approach to pharmacotherapy of connective tissue disorders. ..
- Bjelke J, Christensen J, Nielsen P, Branner S, Kanstrup A, Wagtmann N, et al. Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV. Biochem J. 2006;396:391-9 pubmed
- Poon P, Szeto C, Kwan B, Chow K, Li P. Relationship between carnosinase gene CNDP1 leucine repeat polymorphism and the clinical outcome of Chinese PD patients. Clin Nephrol. 2010;74:343-5 pubmed..3). the CTGs polymorphism of the CNDP1 gene does not affect survival of Chinese PD subjects. The role of carnosine and CNDP1 gene polymorphism in the pathogenesis of cardiovascular disease requires further study. ..
- Sauerhöfer S, Yuan G, Braun G, Deinzer M, Neumaier M, Gretz N, et al. L-carnosine, a substrate of carnosinase-1, influences glucose metabolism. Diabetes. 2007;56:2425-32 pubmed..Instead, a significant correlation of L-carnosine levels with beta-cell mass was observed. hCN1-dependent susceptibility to diabetic nephropathy may at least in part be mediated by altered glucose metabolism in type 2 diabetic patients. ..
- McDonough C, Hicks P, Lu L, Langefeld C, Freedman B, Bowden D. The influence of carnosinase gene polymorphisms on diabetic nephropathy risk in African-Americans. Hum Genet. 2009;126:265-75 pubmed publisher..Protection from diabetic nephropathy afforded by 5L-5L homozygosity in CNDP1 may be masked by the effects of additional risk haplotypes in CNDP1 and CNDP2. ..
- Lowther W, Matthews B. Metalloaminopeptidases: common functional themes in disparate structural surroundings. Chem Rev. 2002;102:4581-608 pubmed
- Arioz D, Camuzcuoglu H, Toy H, Kurt S, Celik H, Aksoy N. Serum prolidase activity and oxidative status in patients with stage I endometrial cancer. Int J Gynecol Cancer. 2009;19:1244-7 pubmed publisher..001). Increased serum prolidase activity and oxidative stress may be associated with endometrial cancer, and increased serum prolidase activity may be related to local invasion of endometrial cancer. ..
- Camuzcuoglu H, Arioz D, Toy H, Kurt S, Celik H, Aksoy N. Assessment of preoperative serum prolidase activity in epithelial ovarian cancer. Eur J Obstet Gynecol Reprod Biol. 2009;147:97-100 pubmed publisher..The aim of this study was to evaluate preoperative serum prolidase activity, total oxidant status (TOS), and total antioxidant capacity (TAC) in patients with newly diagnosed epithelial ovarian cancer (EOC)...
- Mooyaart A, Zutinic A, Bakker S, Grootendorst D, Kleefstra N, van Valkengoed I, et al. Association between CNDP1 genotype and diabetic nephropathy is sex specific. Diabetes. 2010;59:1555-9 pubmed publisher..23). This study suggests that the association between the CNDP1 gene and diabetic nephropathy is sex specific and independent of susceptibility for type 2 diabetes. ..
- Altindag O, Erel O, Aksoy N, Selek S, Celik H, Karaoglanoglu M. Increased oxidative stress and its relation with collagen metabolism in knee osteoarthritis. Rheumatol Int. 2007;27:339-44 pubmed..Decreased collagen metabolism may be related with oxidative stress, which has a role in the ethiopathogenesis and/or in the progression of the disease. ..
- Surazynski A, Donald S, Cooper S, Whiteside M, Salnikow K, Liu Y, et al. Extracellular matrix and HIF-1 signaling: the role of prolidase. Int J Cancer. 2008;122:1435-40 pubmed..These findings show that metabolism of imidodipeptides by prolidase plays a previously unrecognized role in angiogenic signaling. ..
- Ahluwalia T, Lindholm E, Groop L. Common variants in CNDP1 and CNDP2, and risk of nephropathy in type 2 diabetes. Diabetologia. 2011;54:2295-302 pubmed publisher..98, 95% CI 2.43-3.67, p < 0.0001). These data suggest that common variants in CNDP1 and CNDP2 play a role in susceptibility to kidney disease in patients with type 2 diabetes. ..
- Palka J, Surazynski A, Karna E, Orlowski K, Puchalski Z, Pruszynski K, et al. Prolidase activity disregulation in chronic pancreatitis and pancreatic cancer. Hepatogastroenterology. 2002;49:1699-703 pubmed
- Besio R, Alleva S, Forlino A, Lupi A, Meneghini C, Minicozzi V, et al. Identifying the structure of the active sites of human recombinant prolidase. Eur Biophys J. 2010;39:935-45 pubmed publisher..In both dinuclear units a histidine residue is bound to a Zn ion. ..
- Yu D, Wang X, McCaughan G, Gorrell M. Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis. FEBS J. 2006;273:2447-60 pubmed..This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell-extracellular matrix interactions, and thus may regulate tissue remodeling. ..
- Teufel M, Saudek V, Ledig J, Bernhardt A, Boularand S, Carreau A, et al. Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase. J Biol Chem. 2003;278:6521-31 pubmed..5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ). ..
- Karna E, Szoka L, Palka J. Thrombin-dependent modulation of ?1-integrin-mediated signaling up-regulates prolidase and HIF-1? through p-FAK in colorectal cancer cells. Mol Cell Biochem. 2012;361:235-41 pubmed publisher..It was accompanied by increase in the expression of MAP kinases, ERK1 and ERK2. It suggests that integrin-dependent signaling through p-FAK is up-regulated in DLD-1 cells and it may represent potential target for anti-cancer therapy. ..
- Du X, Tove S, Kast Hutcheson K, Grunden A. Characterization of the dinuclear metal center of Pyrococcus furiosus prolidase by analysis of targeted mutants. FEBS Lett. 2005;579:6140-6 pubmed publisherProlidases are dipeptidases specific for cleavage of Xaa-Pro dipeptides...
- Schoen P, Everts H, de Boer T, van Oeveren W. Serum carnosinase activity in plasma and serum: validation of a method and values in cardiopulmonary bypass surgery. Clin Chem. 2003;49:1930-2 pubmed
- Stremenova J, Mares V, Lisa V, Hilser M, Krepela E, Vanickova Z, et al. Expression of dipeptidyl peptidase-IV activity and/or structure homologs in human meningiomas. Int J Oncol. 2010;36:351-8 pubmed..It shows that DPP8 and DPP9 prevail over canonical DPP-IV/CD26 and FAPalpha in all examined patients. In addition, the study suggests an increase of DPP-IV-like enzymatic activity in these tumors of WHO grade II. ..
- Alkhalaf A, Bakker S, Bilo H, Gans R, Navis G, Postmus D, et al. A polymorphism in the gene encoding carnosinase (CNDP1) as a predictor of mortality and progression from nephropathy to end-stage renal disease in type 1 diabetes mellitus. Diabetologia. 2010;53:2562-8 pubmed publisher..CNDP1 polymorphism predicts progression to ESRD in patients with DN, but only late after baseline measurements. ..