Cleavage of the Jaw1 C-terminal region enhances its augmentative effect on the Ca2+ release via IP3 receptors.

Jaw1 (also known as IRAG2), a tail-anchored protein with 39 carboxyl (C)-terminal amino acids, is oriented to the lumen of the endoplasmic reticulum and outer nuclear membrane. We previously reported that Jaw1, as a member of the KASH protein family, plays a role in maintaining nuclear shape via its C-terminal region. Furthermore, we recently reported that Jaw1 functions as an augmentative effector of Ca2+ release from the endoplasmic reticulum by interacting with the inositol 1,4,5-trisphosphate receptors (IP3Rs). Intriguingly, the C-terminal region is partially cleaved, meaning that Jaw1 exists in the cell in at least two forms - uncleaved and cleaved. However, the mechanism of the cleavage event and its physiological significance remain to be determined. In this study, we demonstrate that the C-terminal region of Jaw1 is cleaved after its insertion by the signal peptidase complex (SPC). Particularly, our results indicate that the SPC with the catalytic subunit SEC11A, but not SEC11C, specifically cleaves Jaw1. Furthermore, using a mutant with a defect in the cleavage event, we demonstrate that the cleavage event enhances the augmentative effect of Jaw1 on the Ca2+ release ability of IP3Rs.

Royal Jelly Enhances the Ability of Myoblast C2C12 Cells to Differentiate into Multilineage Cells.

Royal jelly (RJ) is recognized as beneficial to mammalian health. Multilineage differentiation potential is an important property of mesenchymal stem cells (MSCs). C2C12 cells have an innate ability to differentiate into myogenic cells. Like MSCs, C2C12 cells can also differentiate into osteoblast- and adipocyte-lineage cells. We recently reported that RJ enhances the myogenic differentiation of C2C12 cells. However, the effect of RJ on osteoblast or adipocyte differentiation is still unknown. Here in this study, we have examined the effect of RJ on the osteoblast and adipocyte differentiation of C2C12 cells. Protease-treated RJ was used to reduce the adverse effects caused by RJ supplementation. To induce osteoblast or adipocyte differentiation, cells were treated with bone morphogenetic proteins (BMP) or peroxisome proliferator-activated receptor γ (PPARγ) agonist, respectively. RNA-seq was used to analyze the effect of RJ on gene expression. We found that RJ stimulates osteoblast and adipocyte differentiation. RJ regulated 279 genes. RJ treatment upregulated glutathione-related genes. Glutathione, the most abundant antioxidative factor in cells, has been shown to promote osteoblast differentiation in MSC and MSC-like cells. Therefore, RJ may promote osteogenesis, at least in part, through the antioxidant effects of glutathione. RJ enhances the differentiation ability of C2C12 cells into multiple lineages, including myoblasts, osteoblasts, and adipocytes.

Chemical synthesis of per-selenocysteine human epidermal growth factor.

Human seleno-epidermal growth factor (seleno-EGF), a 53-residue peptide where all six cysteine residues of the parent human EGF sequence were replaced by selenocysteines, was synthesized and the oxidative folding of a polypeptide containing three diselenide bonds was compared to that of the parent cysteine peptide. The crude high performance liquid chromatography (HPLC) profiles clearly showed that both the native EGF and its selenocysteine-analogue fold smoothly, yielding a single sharp peak, proving that even in the case of three disulfide-bonded polypeptides the disulfide-to-diselenide bond substitution is highly isomorphous, as confirmed by conformational circular dichroism measurements and particularly by the biological assays.

Identification of the ternary complex of ribonuclease HI:RNA/DNA hybrid:metal ions by ESI mass spectrometry.

Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme-substrate complex in the presence of divalent metal ions (Mn2+ or Zn2+) using electrospray ionization-mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn2+], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate.

Royal Jelly Protects against Epidermal Stress through Upregulation of the NQO1 Expression.

Royal jelly (RJ) is secreted by honeybees and has been used as an apitherapy to obtain healthy skin since ancient times. However, the mechanism of the protective effects of RJ against skin aging and skin diseases caused by skin stress and its components have not been clarified. In this study, we attempted to understand the effect of RJ on epidermal function and observed that NAD(P)H quinone dehydrogenase 1 (NQO1) is significantly induced by RJ in keratinocytes. The expression of NQO1 was also increased in the 3D epidermal skin model. NQO1 is involved in antioxidation and detoxification metabolism, and we found that RJ protects against the epidermal stress caused by UVB and menadione through the upregulation of NQO1. We identified 10-hydroxy-2-decenoic acid (10H2DA), a major fatty acid in RJ, as an active compound in this reaction as it induced the expression of NQO1 and protected the skin against oxidative stress. We demonstrated that the protective effect of RJ against epidermal stress is mediated through the upregulation of NQO1 by 10H2DA.

Total Synthesis and Structural Characterization of Caveolin-1.

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, is triply palmitoylated at the C-terminal region, and is inserted into the lipid bilayer to form a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into five peptide segments, each of which was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, O-acyl isopeptide structures were incorporated. After ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted into the native peptide bond. Finally, the obtained polypeptide was successfully inserted into bicelles, thus showing the success of the synthesis.

Metastable decomposition at the peptide C-terminus: Possible use in protein identification.

RATIONALE: The b n-1 ion of a peptide, as well as a [b n-1 + 18] ion, can be observed not only as normal product ions, but also as prominent metastable ions in a reflectron-embedded matrix-assisted laser desorption ionization time-of-flight spectrometer. The m/z values for the peaks are slightly shifted compared with the ordinary product ions and appear as relatively broad peaks, which permits them to be discriminated from other ions. METHODS: A standard protein mixture and gel-derived proteins digested with LysN protease, which cleaves peptide linkages in proteins at the N-terminal side of Lys residues, were examined. The collected data were used for protein identification using in-house software, iD-plus (http://coco.protein.osaka-u.ac.jp/id-plus/), which was developed for searching for proteins in the peptide database, based on enzyme specificity (N-terminal Lys in this study), peptide masses and C-terminal amino acids. RESULTS: The b n-1 as well as [b n-1 + 18] ions were observed as broad ion peaks for all of the peptides (86 peptides) examined in this study. In silico calculations using the database of LysN digested peptides (11 969 470), created from 553 941 protein sequences (SwissProt: 2017_03), indicate that the use of no less than four peptides permits a protein to be identified without the need of any probability-based scoring. CONCLUSIONS: The preference for b n-1 ion formation is probably due to the higher propensity of the C-terminal peptide bond to be cleaved than other internal bonds. The fact that such C-terminal fragmentation takes place for most of the peptides examined suggests that the use of an N-terminal specific enzyme would allow the C-terminal amino acids to be more reliably read out than other internal sequences, information that could be efficiently used for protein identification.

Phospholipid methylation controls Atg32-mediated mitophagy and Atg8 recycling.

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy-inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria-anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8-PMME, a mitophagy-incompetent lipid conjugate of the autophagy-related ubiquitin-like modifier. Amelioration of Atg32 expression and attenuation of Atg8-PMME conjugation markedly rescue mitophagy in opi3-null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32-mediated mitophagy.

Heterosis extends the reproductive ability in aged female mice†.

Heterosis is the beneficial effect of genetical heterogeneity in animals and plants. Although heterosis induces changes in the cells and individual abilities, few reports have described the effect of heterosis on the female reproductive ability during aging. In this study, we investigated the reproductive capability of genetically heterogeneous (HET) mice established by the four-way crossing of C57BL/6N, BALB/c, C3H/He, and DBA/2. We found the HET females naturally and repeatedly produced offspring, even in old age (14-18 months of age). We also found that HET females showed a significantly enlarged body and organ sizes in both youth and old age. In histological analyses, the numbers of primordial follicles, primary follicles, secondary follicles, and corpora lutea were significantly increased in the old ovaries of HET females compared with those in inbred C57BL/6 mice of the same age. In vitro fertilization experiments revealed that aged HET oocytes showed identical rates of fertilization, early development, and birth compared to those of young and old C57BL/6 oocytes. We further found the significantly increased expression of sirtuin genes concomitant with the up-regulation of R-spondin2 in old HET ovaries. These results confirm the novel phenotype, characterized by fertility extension and follicular retention due to heterosis, in old HET females. The HET female will be a valuable model for clarifying the mechanism underlying the effect of heterosis in the field of reproduction.

The zinc form of carnosine dipeptidase 2 (CN2) has dipeptidase activity but its substrate specificity is different from that of the manganese form.

Carnosine dipeptidase II (CN2), a metallopeptidase present in the cytosol of various vertebrate tissues, catalyzes the hydrolysis of carnosine and several other dipeptides in the presence of Mn2+. Although the metal-binding center of mouse CN2 is also able to associate with Zn2+in vitro, it was not known whether the zinc form of CN2 has any enzymatic activity. In the present study, we show that Zn2+ has a higher affinity for binding to CN2 than Mn2+, as evidenced by native mass spectrometry. The issue of whether the zinc form of CN2 has enzymatic activity was also examined using various dipeptides as substrates. The findings indicate that the zinc form of CN2 catalyzes the hydrolysis of several different dipeptides including Leu-His, Met-His and Ala-His at a reaction rate comparable to that for its manganese form. On the other hand, the zinc form of CN2 did not catalyze the hydrolysis of carnosine and several other dipeptides that are hydrolyzed by the manganese form of CN2. Substrate specificity was also examined in HEK293T cells expressing CN2, and the findings indicate that Leu-His, Met-His, but not carnosine, were hydrolyzed in the cell culture. These results suggest that the zinc form of CN2 is an active enzyme, but with a different substrate specificity from that of the manganese form.

Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitro transport system and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics.

The human importin-ß family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. (1) Cells are cultured in a medium containing a stable isotope. (2) The cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-ß family carriers and then supplemented with a particular importin-ß family carrier of interest. (3) Proteins in the nuclei are extracted and analyzed quantitatively via LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-ß family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes as well as new potential cargoes, which we corroborated via in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possessed at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin, and Lys/Arg-rich segments capable of binding to both transportin and importin-ß. Thus, our method will be useful for linking a carrier to features shared among its cargoes and to specific nuclear localization signals.

Identification of cargo proteins specific for importin-ß with importin-α applying a stable isotope labeling by amino acids in cell culture (SILAC)-based in vitro transport system.

The human importin (Imp)-ß family consists of 21 nucleocytoplasmic transport carrier proteins, which transport thousands of proteins (cargoes) across the nuclear envelope through nuclear pores in specific directions. To understand the nucleocytoplasmic transport in a physiological context, the specificity of cargoes for their cognate carriers should be determined; however, only a limited number of nuclear proteins have been linked to specific carriers. To address this biological question, we recently developed a novel method to identify carrier-specific cargoes. This method includes the following three steps: (i) the cells are labeled by stable isotope labeling by amino acids in cell culture (SILAC); (ii) the labeled cells are permeabilized, and proteins in the unlabeled cell extracts are transported into the nuclei of the permeabilized cells by a particular carrier; and (iii) the proteins in the nuclei are quantitatively identified by LC-MS/MS. The effectiveness of this method was demonstrated by the identification of transportin (Trn)-specific cargoes. Here, we applied this method to identify cargo proteins specific for Imp-ß, which is a predominant carrier that exclusively utilizes Imp-α as an adapter for cargo binding. We identified candidate cargoes, which included previously reported and potentially novel Imp-ß cargoes. In in vitro binding assays, most of the candidate cargoes bound to Imp-ß in one of three binding modes: directly, via Imp-α, or via other cargoes. Thus, our method is effective for identifying a variety of Imp-ß cargoes. The identified Imp-ß and Trn cargoes were compared, ensuring the carrier specificity of the method and illustrating the complexity of these transport pathways.

Melinjo (Gnetum gnemon L.) seed extract for treatment of sleep/wake fragmentation in diet-induced obese mice.

Dietary supplementation with melinjo (Gnetum gnemon L.) seed extract (MSE) has been an integral part of an anti-obesity therapeutic regimen. To examine the relationship between anti-obesity and sleep, we explored the effect of MSE on sleep structure in high-fat diet (HFD)-induced obese mice. Although HFD did not alter the total amount of daily sleep, it significantly reduced the average duration of non-rapid eye movement (NREM) sleep and wakefulness episodes and significantly increased the number of these episodes. These findings indicate fragmented NREM sleep due to repeated brief awakenings in the HFD-fed mice. When 1% (w/v) MSE was given to HFD-fed mice, their weight or sleep structure were comparable to those of ND-fed mice, proving that dietary MSE completely hindered HFD-induced weight gain and sleep/wake fragmentation. Our data provide compelling evidence that MSE is a novel and promising dietary supplement that restores obesity-induced sleep architecture changes in mice.

A Study of Small Intestinal Epigenomic Changes Induced by Royal Jelly.

This study explores the impact of royal jelly (RJ) on small intestinal epigenomic changes. RJ, produced by honeybees, is known for its effects on metabolic diseases. The hypothesis is that RJ induces epigenomic modifications in small intestinal epithelial cells, affecting gene expression and contributing to metabolic health. Male db/m and db/db mice were used to examine RJ's effects through mRNA sequencing and CUT&Tag methods. This study focused on histone modifications and gene expression changes, with statistical significance set at p < 0.05. RJ administration improved insulin sensitivity and lipid metabolism without affecting body weight. GO and KEGG pathway analyses showed significant enrichment in metabolic processes, cellular components, and molecular functions. RJ altered histone modifications, increasing H3K27me3 and decreasing H3K23Ac in genes associated with the G2M checkpoint. These genes, including Smc2, Mcm3, Ccnd1, Rasal2, Mcm6, and Mad2l1, are linked to cancer progression and metabolic regulation. RJ induces beneficial epigenomic changes in small intestinal epithelial cells, improving metabolic health and reducing cancer-associated gene expression. These findings highlight RJ's potential as a therapeutic agent for metabolic disorders. Further research is needed to fully understand the mechanisms behind these effects and their implications for human health.

Identification of a urinary CD276 fragment for detecting resectable pancreatic cancer using a C-terminal proteomics strategy.

This study aimed to confirm urinary protein fragments in relation to the presence of pancreatic ductal adenocarcinoma (PDAC) via a C-terminal proteomics strategy using exploratory and validation cohorts. Urinary fragments were examined by iTRAQ-labelling of tryptic peptides and concentrations of C-terminal fragments were evaluated. Only the urinary CD276 fragment showed a fold change (FC) of > 1.5 with a significant difference of P < 0.01 between healthy (H) and PDAC participants in both the exploratory (H, n = 42; PDAC, n = 39) and validation cohorts (H, n = 36; resectable PDAC, n = 28). The sensitivity and specificity of the CD276 fragment for diagnosing resectable PDAC were 75% and 89%, respectively, in the validation cohort. Postoperative urinary levels of the CD276 fragment were low as compared to those before surgery (n = 18, P < 0.01). Comprehensive C-terminus proteomics identified an increase in the urinary CD276 fragment level as a feature of patients with PDAC. The urinary CD276 fragment is a potential biomarker for detecting resectable PDAC.

Brazilian green propolis prevent Alzheimer's disease-like cognitive impairment induced by amyloid beta in mice.

BACKGROUND: The increasing incidence of cognitive impairment has become a health problem in the aging society. Owing to its antioxidant and anti-inflammatory properties, Brazilian green propolis-derived from Baccharis dracunculifolia-is anticipated to possess anticognitive properties. However, the preventive effect of Brazilian green propolis on cognitive impairment remains unexplained. This study aimed to investigate the effect of Brazilian green propolis on cognitive impairment using a mouse model of Alzheimer's disease (AD) induced by intracerebroventricular injection of amyloid beta (Aß)25‒35. METHODS: Five-week-old male Slc:ddY mice were randomly divided into five groups (n = 8). The groups were pretreated with vehicle and propolis at a dose of 100, 300 and 900 mg/kg body weight for 8 days, then AD-like phenotypes were induced by intracerebroventricular (ICV) injection of Aß25‒35. A sham operation group was set as the control. Memory and learning ability were measured at 7 to 8 days after ICV injection. Gene expression and histological studies were performed at the endpoint of the study. RESULTS: In a passive avoidance test, the administration of Brazilian green propolis prevented the impairment of learning and memory function. Furthermore, comprehensive gene expression analysis in the hippocampus and forebrain cortex revealed that Brazilian green propolis suppressed Aß25-35-induced inflammatory and immune responses. In particular, Brazilian green propolis prevented alterations in gene expressions of microglial and astrocytic markers such as Trem2 and Lcn2 induced by Aß25‒35 injection, suggesting the suppression of excessive activation of glial cells in the brain. In addition, Brazilian green propolis suppressed the elevation of plasma interleukin (IL)-6 levels induced by Aß25‒35 injection. CONCLUSIONS: The results suggest that the prophylactic administration of Brazilian green propolis has a preventive effect against AD by suppressing excessive inflammation and immune response in glial cells. To our knowledge, this study is the first to demonstrate that Brazilian green propolis may inhibit the hyperactivation of microglia and astrocytes as a mechanism of action to prevent AD. Thus, it is a promising ingredient for preventing AD-type dementia.

Royal Jelly Increases Hematopoietic Stem Cells in Peripheral Blood: A Double-Blind, Placebo-Controlled, Randomized Trial in Healthy Subjects.

Objectives: Royal jelly (RJ), produced by honeybees, influences stem cell functions, such as pluripotency maintenance of mouse embryonic stem cells and prevention of aging-related muscle stem cell functional deterioration. Thus, we hypothesized that RJ administration has various health-promoting effects based on stem cells. However, its effects are unknown in humans. In this study, we have attempted for the first time to clarify whether the administration of RJ in humans affects stem cells. Materials and Methods: This randomized, double-blind, placebo-controlled study was performed on healthy subjects (n = 90) who received protease-treated RJ at a dose of 1200 mg/day or placebo daily for four weeks. Also, the participants with a low number of hematopoietic stem cells (HSCs) in peripheral blood were preferentially selected. HSC counts, endothelial progenitor cell (EPC) counts, blood cell counts in peripheral blood, cytokines in serum, and physical conditions were evaluated. Results and Conclusion. Eligible data from 86 subjects (placebo: 42, RJ: 44) who completed the study were analyzed. There were no significant differences between the two groups regarding the changes in peripheral HSC count (p=0.103), while diastolic blood pressure showed a significant improvement in the RJ group compared to that in the placebo group (p=0.032). The subgroup analysis excluded 14 subjects who complained of cold symptoms at baseline or within five days of the four-week study. The changes in the HSC populations were significantly higher in the RJ group than those in the placebo group (p=0.042). No adverse effects were observed in any of the groups. These results suggest that RJ administration affected the peripheral HSC count and may influence stem cell functions. Further research is needed to reveal the various health-promoting benefits of RJ based on stem cells.

Effects of Royal Jelly on Gut Dysbiosis and NAFLD in db/db Mice.

Royal jelly (RJ) is a naturally occurring substance synthesized by honeybees and has various health benefits. Herein, we focused on the medium-chain fatty acids (MCFAs) unique to RJ and evaluated their therapeutic efficacy in treating non-alcoholic fatty liver disease (NAFLD). We examined db/m mice that were exclusively fed a normal diet, db/db mice exclusively fed a normal diet, and db/db mice fed varying RJ quantities (0.2, 1, and 5%). RJ improved NAFLD activity scores and decreased gene expression related to fatty acid metabolism, fibrosis, and inflammation in the liver. RJ regulated innate immunity-related inflammatory responses in the small intestine and decreased the expression of genes associated with inflammation and nutrient absorption transporters. RJ increased the number of operational taxonomic units, the abundance of Bacteroides, and seven taxa, including bacteria that produce short-chain fatty acids. RJ increased the concentrations of RJ-related MCFAs (10-hidroxy-2-decenoic acid, 10-hydroxydecanoic acid, 2-decenedioic acid, and sebacic acid) in the serum and liver. These RJ-related MCFAs decreased saturated fatty acid deposition in HepG2 cells and decreased the gene expression associated with fibrosis and fatty acid metabolism. RJ and RJ-related MCFAs improved dysbiosis and regulated the expression of inflammation-, fibrosis-, and nutrient absorption transporter-related genes, thereby preventing NAFLD.

Role of L-carnosine in the control of blood glucose, blood pressure, thermogenesis, and lipolysis by autonomic nerves in rats: involvement of the circadian clock and histamine.

L-carnosine (ß-alanyl-L-histidine; CAR) is synthesized in mammalian skeletal muscle. Although the physiological roles of CAR have not yet been clarified, there is evidence that the release of CAR from skeletal muscle during physical exercise affects autonomic neurotransmission and physiological functions. In particular, CAR affects the activity of sympathetic and parasympathetic nerves innervating the adrenal glands, liver, kidney, pancreas, stomach, and white and brown adipose tissues, thereby causing changes in blood pressure, blood glucose, appetite, lipolysis, and thermogenesis. CAR-mediated changes in neurotransmission and physiological functions were eliminated by histamine H1 or H3 receptor antagonists (diphenhydramine or thioperamide) and bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian clock. Moreover, a carnosine-degrading enzyme (carnosinase 2) was shown to be localized to histamine neurons in the hypothalamic tuberomammillary nucleus (TMN). Thus, CAR released from skeletal muscle during exercise may be transported into TMN-histamine neurons and hydrolyzed. The resulting L-histidine may subsequently be converted into histamine, which could be responsible for the effects of CAR on neurotransmission and physiological function. Thus, CAR appears to influence hypoglycemic, hypotensive, and lipolytic activity through regulation of autonomic nerves and with the involvement of the SCN and histamine. These findings are reviewed and discussed in the context of other recent reports, including those on carnosine synthetases, carnosinases, and carnosine transport.

Significance of alanine aminopeptidase N (APN) in bile in the diagnosis of acute cellular rejection after liver transplantation.

BACKGROUND: Allograft dysfunction after liver transplantation requires histopathologic examination for confirmation of the diagnosis, however, the procedure is invasive and its interpretation is not always accurate. The aim of this study was to find novel protein markers in bile for the diagnosis of acute cellular rejection (ACR) after liver transplantation. MATERIALS AND METHODS: Quantitative proteomic analysis using the (18)O labeling method was used to search for bile proteins of interest. Nine recipients were selected who had liver dysfunction, diagnosed by liver biopsy, either with ACR (ACR group, n = 5) or without (LD group, n = 4). Donor bile samples were obtained from nine independent live liver donors. Enzyme activity in bile samples was assayed and liver biopsy specimens were immunostained for candidate protein of ACR. RESULTS: The analysis identified 78 proteins, among which alanine aminopeptidase N (APN/CD13) was considered a candidate marker of ACR. Comparative analysis of the ACR and LD groups showed high APN enzyme activity in three (60%) of five cases of the ACR group, while it was as low as donor level in all patients of the LD group. APN enzyme activity in bile samples of liver dysfunction liver transplantation (LDLT) recipients of the ACR group collected within 3 d before biopsy-confirmed ACR (n = 10) was significantly higher (584 ± 434 U/g protein) than in those of recipients free of ACR (n = 96, 301 ± 271 U/g protein) (P = 0.004). APN overexpression along bile canaliculi was observed during ACR in all five cases of the ACR group. CONCLUSION: APN in bile seems to be a useful and noninvasive biomarker of ACR after liver transplantation.