Internet-based behavioural activation to improve depressive symptoms and prevent child abuse in postnatal women (SmartMama): a protocol for a pragmatic randomized controlled trial.

BACKGROUND: Child abuse and postnatal depression are two public health problems that often co-occur, with rates of childhood maltreatment highest during the first year of life. Internet-based behavioural activation (iBA) therapy has demonstrated its efficacy for improving postnatal depression. No study has examined whether the iBA program is also effective at preventing child abuse. This study aims to investigate whether iBA improves depressive symptoms among mothers and prevents abusive behaviours towards children in postpartum mothers in a randomized controlled trial, stratifying on depressive mood status. The study also evaluates the implementation aspects of the program, including how users, medical providers, and managers perceive the program in terms of acceptability, appropriateness, feasibility, and harm done. METHODS: The study is a non-blinded, stratified randomized controlled trial. Based on cut-off scores validated on Japanese mothers, participants will be stratified to either a low Edinburgh Postnatal Depression Scale (EPDS) group, (EPDS 0-8 points) or a high EPDS group (EPDS ≥9 points). A total of 390 postnatal women, 20 years or older, who have given birth within 10 weeks and have regular internet-access will be recruited at two hospitals. Participants will be randomly assigned to either treatment, with treatment as usual (TAU) or through intervention groups. The TAU group receives 12 weekly iBA sessions with online assignments and feedback from trained therapists. Co-primary outcomes are maternal depressive symptoms (EPDS) and psychological aggression toward children (Conflict Tactic Scale 1) at the 24-week follow-up survey. Secondary outcomes include maternal depressive symptoms, parental stress, bonding relationship, quality of life, maternal health care use, and paediatric outcomes such as physical development, preventive care attendance, and health care use. The study will also investigate the implementation outcomes of the program. DISCUSSION: The study investigates the effectiveness of the iBA program for maternal depressive symptoms and psychological aggression toward children, as well as implementation outcomes, in a randomized-controlled trial. The iBA may be a potential strategy for improving maternal postnatal depression and preventing child abuse. TRIAL REGISTRATION: The study protocol (issue date: 2019-Mar-01, original version 2019005NI-00) was registered at the UMIN Clinical Trial Registry (UMIN-CTR: ID UMIN 000036864 ).

Protease-Activated Receptor-2 Plays a Critical Role in Vascular Inflammation and Atherosclerosis in Apolipoprotein E-Deficient Mice.

BACKGROUND: The coagulation system is closely linked with vascular inflammation, although the underlying mechanisms are still obscure. Recent studies show that protease-activated receptor (PAR)-2, a major receptor of activated factor X, is expressed in both vascular cells and leukocytes, suggesting that PAR-2 may contribute to the pathogenesis of inflammatory diseases. Here we investigated the role of PAR-2 in vascular inflammation and atherogenesis. METHODS: We generated apolipoprotein E-deficient ( ApoE-/-) mice lacking systemic PAR-2 expression ( PAR-2-/- ApoE-/-). ApoE-/- mice, which lack or express PAR-2 only in bone marrow (BM) cells, were also generated by BM transplantation. Atherosclerotic lesions were investigated after 20 weeks on a Western-type diet by histological analyses, quantitative reverse transcription polymerase chain reaction, and Western blotting. In vitro experiments using BM-derived macrophages were performed to confirm the proinflammatory roles of PAR-2. The association between plasma activated factor X level and the severity of coronary atherosclerosis was also examined in humans who underwent coronary intervention. RESULTS: PAR-2-/- ApoE-/- mice showed reduced atherosclerotic lesions in the aortic arch ( P<0.05) along with features of stabilized atherosclerotic plaques, such as less lipid deposition ( P<0.05), collagen loss ( P<0.01), macrophage accumulation ( P<0.05), and inflammatory molecule expression ( P<0.05) compared with ApoE-/- mice. Systemic PAR2 deletion in ApoE-/-mice significantly decreased the expression of inflammatory molecules in the aorta. The results of BM transplantation experiments demonstrated that PAR-2 in hematopoietic cells contributed to atherogenesis in ApoE-/- mice. PAR-2 deletion did not alter metabolic parameters. In vitro experiments demonstrated that activated factor X or a specific peptide agonist of PAR-2 significantly increased the expression of inflammatory molecules and lipid uptake in BM-derived macrophages from wild-type mice compared with those from PAR-2-deficient mice. Activation of nuclear factor-κB signaling was involved in PAR-2-associated vascular inflammation and macrophage activation. In humans who underwent coronary intervention, plasma activated factor X level independently correlated with the severity of coronary atherosclerosis as determined by Gensini score ( P<0.05) and plaque volume ( P<0.01). CONCLUSIONS: PAR-2 signaling activates macrophages and promotes vascular inflammation, increasing atherosclerosis in ApoE-/- mice. This signaling pathway may also participate in atherogenesis in humans.

Unique XCI evolution in Tokudaia: initial XCI of the neo-X chromosome in Tokudaia muenninki and function loss of XIST in Tokudaia osimensis.

X chromosome inactivation (XCI) is an essential mechanism to compensate gene dosage in mammals. Here, we show that XCI has evolved differently in two species of the genus Tokudaia. The Amami spiny rat, Tokudaia osimensis, has a single X chromosome in males and females (XO/XO). By contrast, the Okinawa spiny rat, Tokudaia muenninki, has XX/XY sex chromosomes like most mammals, although the X chromosome has acquired a neo-X region by fusion with an autosome. BAC clones containing the XIST gene, which produces the long non-coding RNA XIST required for XCI, were obtained by screening of T. osimensis and T. muenninki BAC libraries. Each clone was mapped to the homologous region of the X inactivation center in the X chromosome of the two species by BAC-FISH. XIST RNAs were expressed in T. muenninki females, whereas no expression was observed in T. osimensis. The sequence of the XIST RNA was compared with that of mouse, showing that the XIST gene is highly conserved in T. muenninki. XIST RNAs were localized to the ancestral X region (Xq), to the heterochromatic region (pericentromeric region), and partially to the neo-X region (Xp). The hybridization pattern correlated with LINE-1 accumulation in Xq but not in Xp. Dosage of genes located on the neo-X chromosome was not compensated, suggesting that the neo-X region is in an early state of XCI. By contrast, many mutations were observed in the XIST gene of T. osimensis, indicating its loss of function in the XO/XO species.

A case with concurrent duplication, triplication, and uniparental isodisomy at 1q42.12-qter supporting microhomology-mediated break-induced replication model for replicative rearrangements.

BACKGROUND: Complex genomic rearrangements (CGRs) consisting of interstitial triplications in conjunction with uniparental isodisomy (isoUPD) have rarely been reported in patients with multiple congenital anomalies (MCA)/intellectual disability (ID). One-ended DNA break repair coupled with microhomology-mediated break-induced replication (MMBIR) has been recently proposed as a possible mechanism giving rise to interstitial copy number gains and distal isoUPD, although only a few cases providing supportive evidence in human congenital diseases with MCA have been documented. CASE PRESENTATION: Here, we report on the chromosomal microarray (CMA)-based identification of the first known case with concurrent interstitial duplication at 1q42.12-q42.2 and triplication at 1q42.2-q43 followed by isoUPD for the remainder of chromosome 1q (at 1q43-qter). In distal 1q duplication/triplication overlapping with 1q42.12-q43, variable clinical features have been reported, and our 25-year-old patient with MCA/ID presented with some of these frequently described features. Further analyses including the precise mapping of breakpoint junctions within the CGR in a sequence level suggested that the CGR found in association with isoUPD in our case is a triplication with flanking duplications, characterized as a triplication with a particularly long duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) structure. Because microhomology was observed in both junctions between the triplicated region and the flanking duplicated regions, our case provides supportive evidence for recently proposed replication-based mechanisms, such as MMBIR, underlying the formation of CGRs + isoUPD implicated in chromosomal disorders. CONCLUSIONS: To the best of our knowledge, this is the first case of CGRs + isoUPD observed in 1q and having DUP-TRP/INV-DUP structure with a long proximal duplication, which supports MMBIR-based model for genomic rearrangements. Molecular cytogenetic analyses using CMA containing single-nucleotide polymorphism probes with further analyses of the breakpoint junctions are recommended in cases suspected of having complex chromosomal abnormalities based on discrepancies between clinical and conventional cytogenetic findings.

Ancestral Y-linked genes were maintained by translocation to the X and Y chromosomes fused to an autosomal pair in the Okinawa spiny rat Tokudaia muenninki.

Two species of the genus Tokudaia lack the Y chromosome and SRY, but several Y-linked genes have been rescued by translocation or transposition to other chromosomes. Tokudaia muenninki is the only species in the genus that maintains the Y owing to sex chromosome-autosome fusions. According to previous studies, many SRY pseudocopies and other Y-linked genes have evolved by excess duplication in this species. Using RNA-seq and RT-PCR, we found that ZFY, EIF2S3Y, TSPY, UTY, DDX3Y, USP9Y, and RBMY, but not UBA1Y, had high deduced amino acid sequence similarity and similar expression patterns with other rodents, suggesting that these genes were functional. Based on FISH and quantitative real-time PCR, all of the genes except for UTY and DDX3Y were amplified on the X and Y chromosomes with approximately 10-66 copies in the male genome. In a comparative analysis of the 372.4-kb BAC sequence and Y-linked gene transcripts from T. muenninki with the mouse Y genomic sequence, we observed that multiple-copy genes in the ancestral Y genome were nonfunctional, indicating that the gene functions were assumed by amplified copies. We also found a LTR sequence at the distal end of a SRY duplication unit, suggesting that unequal sister chromatid exchange mediated by retrotransposable elements could have been involved in SRY amplification. Our results revealed that the Y-linked genes were rescued from degeneration via translocations to other sex chromosomal regions and amplification events in T. muenninki.

Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance.

Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9 (-/-) ) macrophages. Fat-fed Tlr9 (-/-) mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9 (-/-) mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography-determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance.

The cryptic Y-autosome translocation in the small Indian mongoose, Herpestes auropunctatus, revealed by molecular cytogenetic approaches.

In initial studies of the eutherian small Indian mongoose (Herpestes auropunctatus), the Y chromosome could not be identified in somatic cells. The male chromosome number is uniquely odd, 2n = 35, whereas that of females is 2n = 36. Previous reports indicated that this unique karyotype resulted from a translocation of the ancestral Y chromosome to an autosome. However, it has been difficult to identify the chromosomes that harbor the translocated Y chromosomal segment because it is an extremely small euchromatic region. Using a Southern blot analysis, we detected four conserved Y-linked genes, SRY, EIF2S3Y, KDM5D, and ZFY, in the male genome. We cloned homologues of these genes and determined their sequences, which showed high homology to genes in two carnivore species, cat and dog. To unambiguously identify the Y-bearing autosome, we performed immunostaining of pachytene spermatocytes using antibodies against SYCP3, γH2AX, and the centromere. We observed trivalent chromosomes, and the associations between the distal ends of the chromosomes were consistent with those of Y and X1 chromosomes. The centromere of the Y chromosome was located on the ancestral Y chromosomal segment. We mapped the complementary DNA (cDNA) clones of these genes to the male chromosomes using fluorescence in situ hybridization (FISH), and the linear localization of all genes was confirmed by two-colored FISH. These Y-linked genes were localized to the proximal region of the long arm of a single telomeric chromosome, and we successfully identified the chromosome harboring the ancestral Y chromosomal segment.

Initiation of recombination suppression and PAR formation during the early stages of neo-sex chromosome differentiation in the Okinawa spiny rat, Tokudaia muenninki.

BACKGROUND: Sex chromosomes of extant eutherian species are too ancient to reveal the process that initiated sex-chromosome differentiation. By contrast, the neo-sex chromosomes generated by sex-autosome fusions of recent origin in Tokudaia muenninki are expected to be evolutionarily 'young', and therefore provide a good model in which to elucidate the early phases of eutherian sex chromosome evolution. Here we describe the genomic evolution of T. muenninki in neo-sex chromosome differentiation. RESULTS: FISH mapping of a T. muenninki male, using 50 BAC clones as probes, revealed no chromosomal rearrangements between the neo-sex chromosomes. Substitution-direction analysis disclosed that sequence evolution toward GC-richness, which positively correlates with recombination activity, occurred in the peritelomeric regions, but not middle regions of the neo-sex chromosomes. In contrast, the sequence evolution toward AT-richness was observed in those pericentromeric regions. Furthermore, we showed genetic differentiation between the pericentromeric regions as well as an accelerated rate of evolution in the neo-Y region through the detection of male-specific substitutions by gene sequencing in multiple males and females, and each neo-sex-derived BAC sequencing. CONCLUSIONS: Our results suggest that recombination has been suppressed in the pericentromeric region of neo-sex chromosomes without chromosome rearrangement, whereas high levels of recombination activity is limited in the peritelomeric region of almost undifferentiated neo-sex chromosomes. We conclude that PAR might have been formed on the peritelomeric region of sex chromosomes as an independent event from spread of recombination suppression during the early stages of sex chromosome differentiation.

Mutations in the testis-specific enhancer of SOX9 in the SRY independent sex-determining mechanism in the genus Tokudaia.

SRY (sex-determining region Y) is widely conserved in eutherian mammals as a sex-determining gene located on the Y chromosome. SRY proteins bind to the testis-specific enhancer of SOX9 (TES) with SF1 to upregulate SOX9 expression in undifferentiated gonads of XY embryos of humans and mice. The core region within TES, named TESCO, is an important enhancer for mammalian sex determination. We show that TESCO of the genus Tokudaia lost enhancer activity caused by mutations in its SRY and SF1 binding sites. Two species of Tokudaia do not have the Y chromosome or SRY, and one species has multiple SRYs located on the neo-Y chromosome consisting of the Y fused with an autosome. The sequence of Tokudaia TESCO exhibited more than 83% identity with mouse TESCO, however, nucleotide substitution(s) were found in two out of three SRY binding sites and in five out of six SF1 binding sites. TESCO of all species showed low enhancer activity in cells co-transfected with SRY and SF1, and SOX9 and SF1 in reporter gene assays. Mutated TESCO, in which nucleotide substitutions found in SRY and SF1 binding sites were replaced with mouse sequence, recovered the activity. Furthermore, SRYs of the SRY-positive species could not activate the mutated TESCO or mouse TESCO, suggesting that SRYs lost function as a sex-determining gene any more. Our results indicate that the SRY dependent sex-determining mechanism was lost in a common ancestor of the genus Tokudaia caused by nucleotide substitutions in SRY and SF1 binding sites after emergence of a new sex-determining gene. We present the first evidence for an intermediate stage of the switchover from SRY to a new sex-determining gene in the evolution of mammalian sex-determining mechanism.

The Y chromosome of the Okinawa spiny rat, Tokudaia muenninki, was rescued through fusion with an autosome.

The genus Tokudaia comprises three species, two of which have lost their Y chromosome and have an XO/XO sex chromosome constitution. Although Tokudaia muenninki (Okinawa spiny rat) retains the Y chromosome, both sex chromosomes are unusually large. We conducted a molecular cytogenetic analysis to characterize the sex chromosomes of T. muenninki. Using cross-species fluorescence in situ hybridization (Zoo-FISH), we found that both short arms of the T. muenninki sex chromosomes were painted by probes from mouse chromosomes 11 and 16. Comparative genomic hybridization analysis was unable to detect sex-specific regions in the sex chromosomes because both sex probes highlighted the large heterochromatic blocks on the Y chromosome as well as five autosomal pairs. We then performed comparative FISH mapping using 29 mouse complementary DNA (cDNA) clones of the 22 X-linked genes and the seven genes linked to mouse chromosome 11 (whose homologue had fused to the sex chromosomes), and FISH mapping using two T. muenninki cDNA clones of the Y-linked genes. This analysis revealed that the ancestral gene order on the long arm of the X chromosome and the centromeric region of the short arm of the Y chromosome were conserved. Whereas six of the mouse chromosome 11 genes were also mapped to Xp and Yp, in addition, one gene, CBX2, was also mapped to Xp, Yp, and chromosome 14 in T. muenninki. CBX2 is the candidate gene for the novel sex determination system in the two other species of Tokudaia, which lack a Y chromosome and SRY gene. Overall, these results indicated that the Y chromosome of T. muenninki avoided a loss event, which occurred in an ancestral lineage of T. osimensis and T. tokunoshimensis, through fusion with an autosome. Despite retaining the Y chromosome, sex determination in T. muenninki might not follow the usual mammalian pattern and deserves further investigation.

Multiple copies of SRY on the large Y chromosome of the Okinawa spiny rat, Tokudaia muenninki.

The Okinawa spiny rat, Tokudaia muenninki, is the only species with a Y chromosome in the genus Tokudaia. Its phylogenic relationship with two XO/XO species, Tokudaia osimensis and Tokudaia tokunoshimensis, lacking a Y chromosome and the mammalian sex-determining gene SRY, is unknown. Furthermore, there has been little cytogenetic analysis of the sex chromosomes in T. muenninki. Therefore, we constructed molecular phylogenetic trees with nucleotide sequences of cyt b, RAG1, and IRBP. All trees strongly supported that T. muenninki was the first to diverge from the Tokudaia ancestor, indicating that loss of the Y chromosome and SRY occurred in the common ancestor of the two XO/XO species after T. muenninki diverged. We found that the X and Y chromosomes of T. muenninki consisted of large euchromatic and heterochromatic regions by conducting G- and C-banding analyses. PCR, Southern blotting, and FISH revealed that T. muenninki males had multiple SRY copies on the long arm of the Y chromosome. At least three of 24 SRY sequences contained a complete open reading frame (ORF). A species-specific substitution from alanine to serine was found in all copies at the DNA-binding surface within the HMG-box, suggesting that it occurred in an original SRY.

Efficient identification and quantification of proteins using isotope-coded 1-(6-methylnicotinoyloxy)succinimides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We describe a convenient and useful method for the identification and relative quantification of proteins using light and heavy reagents, 1-(6-methylnicotinoyloxy)succinimides (6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS, respectively). This method is based on the chemical derivatization of amino groups of tryptic peptides with these reagents, i.e., the basic moiety of the reagents thus incorporated into both the N-terminal amino group and the epsilon-amino group of the lysine residue would improve the ionization efficiency of tryptic peptides. An increase in protein sequence coverage is achieved by derivatization with these reagents or by combination of mass values before and after derivatization. Since a combination of 6-CH(3)-Nic-NHS and d(3)-labeled reagent (6-CD(3)-Nic-NHS) generates a 3 Da mass difference per reaction site, the d(3)-labeled reagent shifts the mass values of d(0)-labeled peptides according to the number of reactive amino groups in the peptides. In the case of tryptic peptides, the mass values of C-terminal arginine and lysine peptides are shifted by 3 and 6 Da, respectively. Further, the 3 Da mass difference between 6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS offers a means for the relative quantification of protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.

Synaptic vesicles (SVs) are retrieved by clathrin-mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP-2 complex directly interacts with phosphatidylinositol 4-phosphate 5-kinase gamma661 (PIP5Kgamma661), the major PI(4,5)P2-producing enzyme in the brain. The beta2 subunit of AP-2 was found to bind to the C-terminal tail of PIP5Kgamma661 and cause PIP5Kgamma661 activation. The interaction is regulated by PIP5Kgamma661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kgamma661 C-terminal region in hippocampal neurons suppresses depolarization-dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kgamma661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.

PGAP2 is essential for correct processing and stable expression of GPI-anchored proteins.

Biosynthesis of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the ER has been extensively studied, whereas the molecular events during the transport of GPI-APs from the ER to the cell surface are poorly understood. Here, we established new mutant cell lines whose surface expressions of GPI-APs were greatly decreased despite normal biosynthesis of GPI-APs in the ER. We identified a gene responsible for this defect, designated PGAP2 (for Post-GPI-Attachment to Proteins 2), which encoded a Golgi/ER-resident membrane protein. The low surface expression of GPI-APs was due to their secretion into the culture medium. GPI-APs were modified/cleaved by two reaction steps in the mutant cells. First, the GPI anchor was converted to lyso-GPI before exiting the trans-Golgi network. Second, lyso-GPI-APs were cleaved by a phospholipase D after transport to the plasma membrane. Therefore, PGAP2 deficiency caused transport to the cell surface of lyso-GPI-APs that were sensitive to a phospholipase D. These results demonstrate that PGAP2 is involved in the processing of GPI-APs required for their stable expression at the cell surface.

Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization.

The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography-mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity.

Preparation of deuterated methyl and dimethyl substituted nicotinoylating agents for derivatization of the N-terminal of protein.

Methyl groups of 6-methylnicotinic acid and 2,6-dimethylnicotinic acid were deuterated by an H-D exchange reaction under conditions of 1% NaOD/D(2)O on heating. With a condensation reaction between the D-labeled nicotinic acid derivative and N-hydroxysuccinimide with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the nicotinoylating agents, 1-(6-methyl[D(3)]nicotinoyloxy)succinimide (2c) and 1-(2,6-dimethyl[D(6)]nicotinoyloxy)succinimide (2f) were prepared. Both D-labeled nicotinoylating agents and their unlabeled counterparts quantitatively modified the N-terminal of protein.