Life events as risk factors for myocardial infarction: a pilot case-control study in Kolkata, India.

This case-control study was conducted in the Cardiology Department of Medical College, Kolkata, India, during 2000-2001, to explore the link between stressful life events and subsequent myocardial infarction (MI). One hundred consecutive confirmed MI patients were selected as a case group. One hundred age-, sex- and income-matched controls were selected from visitors other than relatives who attended these patients. The subjects were interviewed and asked to rate 61 life events with a number between 0 and 20. They also noted which of these they had experienced in the last one year. The main exposure variables included life events as per E.S. Paykel, smoking, alcohol consumption, chewing of tobacco, marital status, literacy, employment, and monthly per-capita income. The results showed that an MI patient was likely to experience 4.16 stressful life events, which were twice as much as the control group (2.24). The total stress score was the highest for serious personal illness followed by illness of family members and unemployment for the MI patients. For the controls, conflict between husband and wife, death of friends, and personal illness had the highest total stress score. The mean stress score for the MI patients was 35.5 compared to 17.35 among the controls. The MI subjects were more likely to have experienced stressful life events than the controls.

Mass DEC campaign for filariasis in a hyper endemic district of West Bengal.

As a part of the on-going National Filaria Control Programme, National Filaria Day was observed in Purulia district of West Bengal on 26th Nov 2000 with an extensively organized mass DEC consumption campaign preceded by IEC activities and followed by mopping up operations. In all 81.07% of the targeted population was covered, females (84.3%) being more available than males (78.3%). Percentage of coverage declines with increase in age. Municipalities and notified areas had less coverage as well as supervised consumption than in other areas. Consumption was highest in Balarampur block (88.6%). Supervised consumption among surveyed population was highest in Bandwan (56.0%). Overall patient compliance was very good, with side effects of vomiting, dizziness, headache and fever being 2.12% only among surveyed population. Disease prevalence among population covered showed 0.1% hand swelling, 0.6 to 0.8% leg swelling, while 1.1% of males had hydrocele.

Pro-xenopsin(s) in vesicles of mammalian brain, liver, stomach and intestine is apparently released into blood and cerebral spinal fluid.

Mammalian pro-xenopsins (proXP), proteins (such as alpha-coatomer) that yield XP-related peptides when digested by pepsin-related proteases, are ubiquitously distributed in rats, with highest concentrations in liver and gastrointestinal tissues. Here, the cellular and subcellular distributions of canine and rat proXP were determined in brain, liver, stomach and intestine. Elutriation and percoll density centrifugation of collagenase-dispersed cells demonstrated that proXP was primarily associated with hepatocytes in liver, chief and parietal cells in stomach and endocrine/exocrine cells in intestine. When fragmented cells were subjected to differential centrifugation, congruent with85% of proXP was associated with particulate fractions and only congruent with15% was cytosolic. Sucrose-gradient centrifugation of crude mitochondrial preparations (P2 pellets) for liver, stomach and intestine demonstrated that proXP was localized to vesicles (density, congruent with1.19; size, 80-400 micrometer), which contained material of variable electron density. In isotonic homogenates of brain, proXP migrated primarily with synaptosomes (density, congruent with1. 15) which contained vesicles (size, 50-100 micrometer). During HPLC-sizing and ion exchange chromatography, proXP gave at least three components, the major one being an anionic 140-kDa protein. ProXP-like activity was found in human and rat blood, human cerebral spinal fluid and in contents of the gastrointestinal lumen. These results are consistent with the idea that these vesicle-associated protein(s) could be released during endocrine and/or exocrine secretion and serve as precursors to XP-related peptides.

Agonist induced conformation alteration of neurotensin receptor and the mechanism behind Na+ inhibition of 125I-NT binding.

In the absence of Na+, 125I-Neurotensin (125I-NT) binding to the Neurotensin receptor (NTR) produces a stable noncovalent 125I-NT-NTR complex whose dissociation rate is extremely low even after the addition of 1 microM NT, 100 microM SR48692 (antagonist), 100 microM GPPNHP or 100 mM NaCl. Lowering the medium pH to 4.5 enhances the process (approximately 70% in 10 minutes). Labeling by photoactivatable 125I-Tyr3-Azo4-NT identifies a approximately 50 KD Mr band along with several other minor components. Interestingly, the labeling intensity is drastically reduced when binding is performed in the presence of Na+ or GPPNHP. However, a minor reduction is noticed when Na+ or GPPNHP is added to the medium after binding. The binding kinetics indicates that Na+ lowers the rate of 125I-NT association by acting as a noncompetitive inhibitor. On the contrary, Na+ favors the interaction of antagonist, SR48692 by lowering the value of Ki. GTPgamma35S binding to membranes in the presence of 30 mM NaCl suggests that Na+ inhibition of 125I-NT binding is due to the uncoupling of NTR associated G protein(s). In order to explain the entire phenomenon, a two-step, binding model has been proposed. In Step-1, interaction between NT and NTR produces a transient complex, which attains a stable state in the absence of NaCl via step-2, thereby altering the native NTR conformation. The presence of Na+ prevents step-2 by dissociating the transition complex.

Synergistic effects of neurotensin and beta-adrenergic agonist on 3,5-cyclic adenosine monophosphate accumulation and DNA synthesis in prostate cancer PC3 cells.

Since neurotensin is often co-stored with catecholamines and since it can excite the release of dopamine and norepinephrine, responses to this peptide might depend upon the activity of catecholaminergic systems. In this study, we used prostate cancer PC3 cells, which express neurotensin receptors and 12-adrenergic receptors, to demonstrate that neurotensin can potentiate the effects of isoproterenol on 3',5'-cyclic adenosine monophosphate (cAMP) formation and on inhibition of DNA synthesis. While neurotensin had only a slight effect on basal cAMP levels, it nearly doubled the response to isoproterenol even at maximal levels without altering potency. Neurotensin increased the rate of cAMP accumulation and the steady-state level achieved. Consistent with the known antimitogenic action of dibutyryl-cAMP in PC3 cells, isoproterenol was found to inhibit DNA synthesis concentration-dependently, measured using [3H]thymidine. Neurotensin enhanced DNA synthesis when given alone. However, it inhibited DNA synthesis when given with a threshold level of isoproterenol, which by itself had no significant effect. These results, demonstrating cross-talk in the neurotensin and beta-adrenergic signaling pathways, suggest that there may be other physiologic instances of similar interactions between neurotensin and catecholamines.

Neurotensin enhances agonist-induced cAMP accumulation in PC3 cells via Ca2+ -dependent adenylyl cyclase(s).

A human prostate cancer cell line (PC3) with abundant neurotensin (NT) receptors was used to demonstrate that NT potentiated 3',5'-cyclic adenosine monophate (cAMP) accumulation in response to a variety of stimuli, including both direct forskolin (F) and indirect (prostaglandin, (PGE2), isoproterenol (ISO) and cholera toxin (CTx)) activators of adenylyl cyclase. Several mechanisms were investigated and our results indicated an effect on the rate of cAMP formation and not on degradation or extrusion. For each stimulus, NT enhanced efficacy without altering EC50. The effect of NT did not involve stimulatory G-protein (Gs)-activation or interference with a tonic inhibitory G-protein (Gi)-mediated inhibition. A similar response was obtained when NT was added with the stimulus or given as a two minute pulse which was removed prior to addition of stimulus. The potentiating activity disappeared with a t1,2 of approximately 15 min. NT transiently elevated cellular [Ca2+]i and its effects on cAMP could be mimicked by [Ca2+]i-elevating agents (uridine triphosphate (UTP), thapsigargin and ionomycin). Buffering cellular [Ca2+]i with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) inhibited cAMP responses to ISO and F in presence and absence of NT. These data support the idea that NT potentiated cAMP formation in response to a variety of stimuli by facilitating the activation of Ca2+ -dependent adenylyl cyclases.

Neurotensin receptor expression in prostate cancer cell line and growth effect of NT at physiological concentrations.

BACKGROUND: Neurotensin (NT), a neuroendocrine peptide, exerts trophic effects in vivo and stimulates growth of some tumor cells in vitro. Androgen-sensitive prostate cells derived from lymph node carcinoma of the prostate (LNCaP) secrete NT and exhibit growth responses to NT. This study examines NT secretion, NT receptor and NT-growth responses in androgen-independent prostatic carcinoma (PC3) cells derived from prostate adenocarcinoma metastatic to bone. METHODS: Binding of 125I-NT to PC3 membranes was studied by filtration. NT was measured by RIA. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for NT and NT receptor mRNA. Growth was measured as 3H-thymidine incorporation into DNA. RESULTS: Scatchard analyses gave two binding components (Kd1 = 40 pM and Kd2 = 300 pM) in equal amounts (15-30 x 10(3) sites/cell). The bioactive region of NT was essential and the specific, non-peptide NT antagonist, SR48692, inhibited (IC50 = 3 nM). GTP analogs, sodium ion and SH-directed alkylating agents also inhibited. Glutaraldehyde crosslinking labeled two substances (M(r) of 23 and 46 kDa). RT-PCR indicated robust expression of authentic NT receptor but little for NT precursor. NT was stable in PC3 cultures but it was not found in cells or conditioned media. Incubated with PC3 cells, NT exhibited a mitogenic effect with bell-shaped dose-response and maximum at 100 pM NT. CONCLUSIONS: PC3 cells expressed genuine NT receptors and generated growth responses to physiologic levels of NT which were blocked by SR48692. If NT contributes to the survival of prostate tumor cells upon androgen deprivation therapy, NT antagonists might be useful agents in further treatment.

High affinity binding of 125I-neurotensin to dispersed cells from chicken liver and brain.

Dispersed cells from chicken brain and liver were found to possess cell surface binding sites for 125I-neurotensin (125I-NT). Scatchard analyses indicated the presence of high affinity (K4, 25-80 pM) and low affinity (Kd, 250-450 pM) components in adult tissues. Binding capacity was reduced 25-40% by incubation with pertussis toxin. Ontogenetic studies indicated that NT receptor capacity increased approximately 20-fold from the embryonic stage to adult. Cross-linking of 125I-NT to intact cells labeled one major band (52 kDa, > or = 90%) and two minor bands (40 and 90 kDa, < or = 10%) which could represent distinct NT-receptors or one receptor partly degraded or cross-linked to G-protein(s). The binding of 125I-NT to dispersed cells was enhanced by reduction with dithoithreitol and suppressed by alkylation with N-ethyl-maleimide (NEM), maleimidocaproic acid (MCA) and p-chloromercuribenzenesulfonate (PCMBS). Since MCA and PCMBS do not permeate cells, this suggests that the sulfhydryl group(s) critical to binding are located within the NT receptor itself. Preincubation of cells with NT prior to treatment with NEM diminished its inhibitory effect, suggesting that the critical SH-group(s) were within the NT binding pocket or were protected by an allosteric effect. These results suggest that one or more of the nine cysteine residues in the NT receptor is involved in the NT binding reaction.

Chicken liver contains a large quantity of a G-protein-linked neurotensin receptor.

Using 125I-labeled neurotensin (NT), chicken liver was found to contain high affinity, G-protein-linked receptors directed specifically towards the bioactive C-terminal portion of NT. Binding was proportional to membrane and optimal at pH 7.5. The apparent Kd (approximately 91 pM) for this single class of binding sites was similar to Kds reported for the high-affinity components of NT binding to mammalian brain and intestinal membranes. However, the binding capacity (Bmax, approximately 2.3 pmol/mg) was 10-100 times higher than values reported for these mammalian tissues. Binding was inhibited by GTP analogues and by treatment with pertussis toxin but not by cholera toxin. Treatments with alkaline solutions, shown to inactivate G-proteins, decreased subsequent binding at pH 7.5. Whereas low concentrations of Mg2+ (optimum, approximately 0.5 mM) enhanced NT binding, concentrations of 5 mM and above were inhibitory. Cross-linking of 125I-labeled NT to liver membranes using glutaraldehyde specifically labeled two substances of approximately 52 and approximately 90 kDa, which could represent different binding proteins or complexes. These data demonstrate the presence in chicken liver of large amounts of high-affinity NT receptor(s) coupled to pertussis toxin-sensitive G-protein(s).

BON cells display the intestinal pattern of neurotensin/neuromedin N precursor processing.

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, constitutively 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 pI 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.

Binding and biologic activity of neurotensin in guinea pig ileum.

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38 degrees C. NT contracted the longitudinal muscle (ED50, approximately 0.3 nM), the 2-3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 microM), > or = 50% of the response was blocked and the residual effect gave an ED50 of approximately 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 microM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED50, 0.3 nM) and one substance P (ED50, 1.4 nM). Using membrane preparations and 125I-labeled NT, specific, high affinity receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (Kds: approximately 0.1 nM and approximately 2 nM). Sodium ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg++ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.

Enhanced expression of neurotensin/neuromedin N mRNA and products of NT/NMN precursor processing in neonatal rats.

Intestinal levels of immunoreactive neurotensin (iNT) and neuromedin N (iNMN), as well as mRNA for the NT/NMN precursor, were elevated during the suckling period in rats. While transient expression of NT/NMN was observed at 1-5 days of age in the proximal small intestine and colon, NT/NMN levels in the ileum increased to peak at 10-20 days of age and then decreased to adult levels. The levels of these peptides were not elevated in the central nervous system and pituitary over this time period. Chromatographic analyses of jejunoileal extracts indicated that large molecular forms of iNT and iNMN were present, constituting approximately 1.3% of total iNT and approximately 56% of total iNMN, respectively. Treatment of the large forms with pepsin, which is known to generate the fully immunoreactive peptides, NT(3-13), NT(4-13), and NMN, increased immunoreactivity tenfold (iNT) and 1.2-fold (iNMN). Thus, large forms actually constituted approximately 13% (iNT) and approximately 60% (iNMN). Based upon its physicochemical properties, large molecular iNMN was tentatively identified as a 125 residue peptide with NMN at its C-terminus [i.e., rat prepro-NT/NMN(23-147)]. The properties of large molecular iNT were most similar to those predicted for the entire precursor [i.e., rat prepro-NT/NMN(23-169)]. These results indicate a) that enhanced expression of NT/NMN occurs in a tissue-specific manner in rats during the suckling period; b) that the pattern of precursor processing in intestine yields primarily NT and a large molecular form of NMN.

A common precursor to neurotensin and LANT6 and its differential processing in chicken tissues.

Antisera towards neurotensin (NT) and the structurally related peptide, LANT6, were used to characterize immunoreactive peptides and proteins in extracts of chicken tissues. A 17 kDa protein was identified by Western blotting as a potential precursor to NT and LANT6. However, the posttranslational processing of this common precursor appeared to be tissue specific, giving rise to disproportionate amounts of NT and LANT6, along with varying expression of a large molecular LANT6 (M(r), 15 kDa). The intestinal cells containing immunoreactive NT, LANT6, and large molecular LANT6 behaved similarly during fractionation by size and density. These activities also banded together in particles resembling vesicles during centrifugation of isotonic homogenates of tissue. These results suggest that chicken NT and LANT6 are biosynthesized as parts of the same precursor, the processing of which can give rise to a variety of products stored within secretory vesicles.

Importance of thiol group(s) in the binding of 125I-labeled neurotensin to membranes from porcine brain.

In a radioreceptor assay employing 125I-labeled neurotensin (125I-NT) and membranes from porcine brain, the reducing agent, dithiothreitol (DTT), was found to enhance binding (ED50, approximately 10 microM), whereas alkylating agents such as N-ethyl-maleimide (NEM) suppressed binding (ED50, approximately 0.7 mM). The enhanced binding appeared to be due to an effect on disulfide group(s) within the NT-receptor or associated protein(s), since the stability of 125I-NT in the presence of membranes was not altered by 2 mM DTT. Scatchard analysis indicated that treatment with 2 mM DTT increased the total number of binding sites approximately 1.6-fold without much effect on the apparent KdS for the high- and low-affinity states. In a similar manner, the effect of 1 mM NEM was shown to result primarily from a decrease (approximately 60%) in the number of binding sites with little change in the KdS. The additional receptors gained by exposure to DTT appeared not to be sensitive to NEM unless pretreated with DTT, suggesting that reduction of disulfide bond(s) converted latent receptors into active receptors. Interestingly, nine Cys residues have been found to be present in the recently cloned rat NT receptor. In other studies, preincubation of membranes with NT prior to treatment with NEM diminished the inhibitory effect of NEM on agonist binding, suggesting that the critical sulfhydryl group(s) were located at the NT binding site or were protected by an allosteric effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of GTP analogs and metal ions on the binding of neurotensin to porcine brain membranes.

Using 125I-labeled neurotensin (NT), porcine brain membranes were found to contain two types of high-affinity receptors, one class (approximately 1/3 of total) with an apparent Kd of 0.12 nM and another with an apparent Kd of 1.4 nM. Nonhydrolyzable analogs of GTP inhibited NT binding in a dose-dependent manner. In the presence of 60 microM guanosine 5'-(3-thio) 5'-(beta, gamma-imino) triphosphate. NT binding was decreased by 35% with an associated decrease in the number of binding sites and little change in the Kd. Cross-linking of 125I-labeled NT to brain membranes using disuccinimidyl suberate was found to specifically label two substances of approximately 120 kDa and approximately 160 kDa, which could represent different binding proteins or complexes. For a series of NT analogs, there was close agreement between the IC50 in the binding assay and the ED50 in a bioassay based on ability to contract the guinea pig ileum. In addition, metal ions inhibited NT binding and the contractile action of NT with the same order of potency (Hg++ > Zn++ > Cu++ > Mn++ > Mg++ > Li++). There was a linear relationship between the standard reduction potential for these ions and the logarithm of the IC50 in the binding assay. The results suggest that porcine brain contains high-affinity, G-protein-linked receptors for NT, the functioning of which depends upon group(s), perhaps sulfhydryl(s), which can interact strongly with certain heavy metal ions.

Tissue-specific processing of neurotensin/neuromedin-N precursor in cat.

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 NT3-13 and NT4-13 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.

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 approximately 10% complete, that between neurotensin and the tail was approximately 90% complete, and that between neuromedin N and neurotensin was approximately 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.

Pepsin-mediated processing of synthetic precursor-like sequence yields neurotensin-like peptide.

A 15 amino acid synthetic peptide, which spanned the dibasic cleavage site C-terminal to neurotensin (NT), in its 170-residue canine precursor, was synthesized by solid-phase methods. Using this substrate in combination with a radioimmunoassay specific for the C-terminal region of NT, a simple assay was developed to monitor protease-mediated cleavage of the Leu8-Lys9 bond in the substrate. Hog pepsin and the related enzymes, rhizopus pepsin, bovine cathepsin D, and mouse renin, were found to be effective in this assay, pepsin cleaving only this bond to liberate the NT-like sequence. The pH dependence of the reaction indicated that pepsin, cathepsin D, and renin exhibited significant activity at pH's characteristic for secretory vesicles (pH 5.5-6.5). In addition, pepsin and cathepsin D were shown to process the native precursor at pH's as high as 5.5. These results, although not proof, are consistent with the idea that endoproteases with pepsin-like specificity may be involved in the processing of the NT precursor in neural/endocrine cells.

Purification of large neuromedin N (NMN) from canine intestine and its identification as NMN-125.

A large molecular form of neuromedin N (NMN), a neurotensin (NT)-related peptide, was purified to homogeneity from canine ileal mucosa. The amino acid sequence of its N-terminal 20 residues was found to be SDSEEEMKALEADLLTNMHT, which is identical to residues 24-43 of the cDNA-predicted NT/NMN precursor. Prior work had indicated that the NMN moiety was located at the C-terminus of large NMN. Having now defined both the N-terminus and C-terminus of large NMN, we note that this molecule must be comprised of 125 amino acids (including residues 24-148 of prepro NT/NMN) and we suggest that it be named NMN-125. This information also defines the signal peptide cleavage site as the cysteine-serine bond within the precursor. The amino acid composition of the isolated peptide and its molar extinction coefficient for absorbance at 280 nm were similar to those for the 24-148 segment of prepro NT/NMN. However, the empirically determined molecular weight (17 kDa) and the isoelectric point (pI = 5.4) were slightly higher than those predicted solely from the peptide's amino acid content (14.43 kDa and pI = 4.65, respectively). In total, these results indicate that the major NMN-related peptide found in canine ileum is 125 residues in length, extending from the putative signal peptide to the C-terminus of NMN, and that it might contain non-amino acid substituents.