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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.

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.

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.

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.

Jaw1, 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 deficit in the cleavage event, we demonstrate that the cleavage event enhances the augmentative effect of Jaw1 on the Ca2+release ability of IP3Rs.Summary statementThe C-terminal region of Jaw1, a tail-anchored protein, is cleaved by signal peptidase complex and this cleavage event enhances the augmentative effect of Jaw1 on the Ca2+release activity of inositol 1,4,5-trisphosphate receptors

The knockout (KO) of the cystine transporter xCT causes ferroptosis, a type of iron-dependent necrotic cell death, in mouse embryonic fibroblasts, but this does not occur in macrophages. In this study, we explored the gene that supports cell survival under a xCT deficiency using a proteomics approach. Analysis of macrophage-derived peptides that were tagged with iTRAQ by liquid chromatography-mass spectrometry revealed a robust elevation in the levels of carnosine dipeptidase II (CNDP2) in xCT KO macrophages. The elevation in the CNDP2 protein levels was confirmed by immunoblot analyses and this elevation was accompanied by an increase in hydrolytic activity towards cysteinylglycine, the intermediate degradation product of glutathione after the removal of the γ-glutamyl group, in xCT KO macrophages. Supplementation of the cystine-free media of Hepa1-6 cells with glutathione or cysteinylglycine extended their survival, whereas the inclusion of bestatin, an inhibitor of CNDP2, counteracted the effects of these compounds. We established CNDP2 KO mice by means of the CRISPR/Cas9 system and found a decrease in dipeptidase activity in the liver, kidney, and brain. An acetaminophen overdose (350 mg/kg) showed not only aggravated hepatic damage but also renal injury in the CNDP2 KO mice, which was not evident in the wild-type mice that were receiving the same dose. The aggravated renal damage in the CNDP2 KO mice was consistent with the presence of abundant levels of CNDP2 in the kidney, the organ prone to developing ferroptosis. These collective data imply that cytosolic CNDP2, in conjugation with the removal of the γ-glutamyl group, recruits Cys from extracellular GSH and supports redox homeostasis of cells, particularly in epithelial cells of proximal tubules that are continuously exposed to oxidative insult from metabolic wastes that are produced in the body.

Royal jelly (RJ), a natural product secreted by honeybees, is used in various topical products for skincare and aids in maintaining cutaneous homeostasis. However, the mechanism underlying the effect of RJ on the skin has not yet been fully explored. Our previous data indicated that the epidermal equivalents become thicker and contain more p63-expressing proliferative cells after the addition of RJ to the medium. Therefore, we examined the effect of RJ on the proliferative ability of human primary epidermal keratinocytes (HPEKs) in a two-dimensional culture here. We observed only a slight increase in the proliferation of cells with the addition of RJ, suggesting that it is not actively involved in the proliferation of HPEKs. However, population doubling was enhanced in the RJ-treated group in the long-term culture experiment, indicating that RJ inhibits senescence. Additionally, RJ suppressed cellular senescence by regulating the expression levels of ΔNp63, p16, and p21. These results were further confirmed by the presence of major fatty acids, such as 10-hydroxy-2-decenoic acid, in RJ. Overall, our data indicate that RJ can maintain epidermal stem cell properties by repressing senescence.

To evaluate the moisturizing function and other effects of royal jelly extract on the skin. The effects of applying an essence containing royal jelly extract on the skin of healthy Japanese males and females were examined.Thirty-five healthy Japanese men and women who were aware of their skin dryness applied an essence containing royal jelly extract or placebo for 4 weeks using the split-face method in a placebo-controlled, double-blind, parallel comparative study. The stratum corneum water content, transepidermal water evaporation, pigmentation, pores, and redness were evaluated.The stratum corneum water content significantly increased by the application of essence containing royal jelly extract to the cheeks for 4 weeks compared with placebo.The application of an essence containing royal jelly extract significantly improved the moisture content of the stratum corneum of the cheeks, confirming the improvement in the moisturizing function of the royal jelly extract. Furthermore, no adverse events were observed at the application site during the application period, and the test products and royal jelly extract contained in the test product were considered highly safe.

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.

Objectives. This study aimed to evaluate the effect of propolis on cognitive function in elderly Japanese with a placebo-controlled design. Material and Methods. This study was performed on 79 elderly Japanese. Participants orally received either a placebo or dietary supplement containing propolis extract for 24 weeks. Cognitive function assessed by Cognitrax and various blood or urine markers were measured at pre- and postadministration. Results and Conclusion. Eligible data from 68 subjects (placebo: 33, propolis: 35) who completed the study were analyzed. Compared to the placebo group, the propolis group showed significant improvement in verbal memory in Cognitrax ( P = 0.028 ). Total cholesterol, LDL cholesterol, urea nitrogen, creatinine, and uric acid were significantly improved in the propolis group compared to the placebo group ( P = 0.011 , P = 0.004 , P = 0.048 , P = 0.045 , and P = 0.005 , respectively). However, urea nitrogen, creatinine, and uric acid fluctuated within the normal level. Furthermore, a subgroup analysis was performed on those with higher than 100 of the standardized score of the neurocognitive index indicated by the overall Cognitrax score. Significant improvements in the propolis group compared to placebo were confirmed in verbal memory ( P = 0.007 ) and processing speed as indications for information processing ability, complex attention, and concentration ( P = 0.029 ). No side effects were observed in any of the groups. This study demonstrates that propolis is effective in improving cognitive functions such as memory, information processing, complex attention, and concentration in elderly Japanese.

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.

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.

Honeybees produce royal jelly (RJ) from their cephalic glands. Royal jelly is a source of nutrition for the queen honey bee throughout its lifespan and is also involved in fertility and longevity. Royal jelly has long been considered beneficial to human health. We recently observed that RJ delayed impairment of motor function during aging, affecting muscle fiber size. However, how RJ affects skeletal muscle metabolism and the functional component of RJ is as of yet unidentified. We demonstrate that feeding mice with RJ daily prevents a decrease in myofiber size following denervation without affecting total muscle weight. RJ did not affect atrophy-related genes but stimulated the expression of myogenesis-related genes, including IGF-1 and IGF receptor. Trans-10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), two major fatty acids contained in RJ. After ingestion, 10H2DA and 10HDAA are metabolized into 2-decenedioic acid (2DA) and sebacic acid (SA) respectively. We found that 10H2DA, 10HDAA, 2DA, and SA all regulated myogenesis of C2C12 cells, murine myoblast cells. These novel findings may be useful for potential preventative and therapeutic applications for muscle atrophy disease included in Sarcopenia, an age-related decline in skeletal muscle mass and strength.

Aging is associated with motor disorders that decrease the quality of life (QOL). Royal jelly (RJ), used as a dietary supplement, has shown various health benefits and, therefore, it has the potential to improve the QOL during aging. We have previously developed protease enzyme-treated RJ to avoid the anaphylactic response induced by RJ supplementation. However, the effects of a lifelong treatment with RJ on normal aging have not been fully clarified. In this study, we investigated the effects of enzyme-untreated RJ (NRJ) and enzyme-treated RJ (ERJ) on the aging process focusing on motor functions, by using a genetically heterogeneous (HET) mouse model experimentally endowed with genetic diversity. We performed four different physical performance tests (grip strength, wire hang, horizontal bar, and rotarod). We showed that the age-related impairment of the motor functions was significantly delayed in RJ-treated mice. Both NRJ and ERJ were similarly effective against these types of aging-associated declines. Histological analyses revealed that the RJ treatment affected the muscle fiber size at an advanced age. We also demonstrated that age-related changes in muscle satellite cell markers and catabolic genes were affected in RJ-treated mice. These results suggest that non-protein components of RJ improved the motor function in aging mice. These findings indicate that RJ has the potential to change the QOL during aging by regulating the motor function.

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.

Carnosine dipeptidase II (CN2/CNDP2) is an M20 family metallopeptidase that hydrolyses various dipeptides including β-alanyl-L-histidine (carnosine). Crystallographic analysis showed that CN2 monomer is composed of one catalytic and one dimerization domains, and likely to form homodimer. In this crystal, H228 residue of the dimerization domain interacts with the substrate analogue bestatin on the active site of the dimer counterpart, indicating that H228 is involved in enzymatic reaction. In the present study, the role of intradimer interaction of CN2 in its catalytic activity was investigated using electrospray-ionization time-of-flight mass spectrometry (ESI-TOF MS). First, a dimer interface mutant I319K was prepared and shown to be present as a folded monomer in solution as examined by using ESI-TOF MS. Since the mutant was inactive, it was suggested that dimer formation is essential to its enzymatic activity. Next, we prepared H228A and D132A mutant proteins with different N-terminal extended sequences, which enabled us to monitor dimer exchange reaction by ESI-TOF MS. The D132A mutant is a metal ligand mutant and also inactive. But the activity was partially recovered time-dependently when H228A and D132A mutant proteins were incubated together. In parallel, H228A/D132A heterodimer was formed as detected by ESI-TOF MS, indicating that interaction of a catalytic center with H228 residue of the other subunit is essential to the enzymatic reaction. These results provide evidence showing that intradimer interaction of H228 with the reaction center of the dimer counterpart is essential to the enzymatic activity of CN2.

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.

Hepatitis B virus (HBV) replication is controlled by liver-enriched transcriptional factors, including forkhead box protein A (FOXA) members. Here, we found that FOXA members are directly and indirectly involved in HBV replication in human hepatic cells. HBV replication was elevated in HuH-7 treated with individual FOXA members-specific siRNA. Reciprocally, the downregulation of HBV replication was observed in FOXA-induced HuH-7. However, the mechanism of downregulation is different among FOXA members at the level of HBV RNA transcription, such as precore/pg RNA and 2.1kb RNA. In addition, FOXA1 and FOXA2 suppressed nuclear hormone receptors, such as HNF4α, that are related to HBV replication.

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.

Background Intact kininogen (KNG), the precursor of kinin that originates from the liver, is fragmented by proteases. A recent research showed an increase in the serum levels of KNG fragments in cancer patients (pts), which were possibly produced by proteases in the tumor microenvironment. We hypothesized that urinary excretion of KNG fragments would also be elevated in pancreatic ductal adenocarcinoma (PDAC) pts. This study was conducted to investigate the diagnostic usefulness of urinary KNG fragment measurement by the multiple-reaction-monitoring technique (MRM). Methods Treatment-naive PDAC pts and healthy participants for whom pooled frozen urine specimens were available were enrolled in this study. Urine samples from resectable PDAC pts were collected prior to the surgery. The urinary proteins were digested with trypsin, and the resultant peptides were measured by MRM analysis; the degree of protein fragmentation was estimated by the ratio of the amount of a protein fragment to that of a constant region of the original protein. Fragments for which the fragmentation ratios were higher in resectable PDAC pts than in the healthy participants were defined as having abnormal protein fragments. The diagnostic capability of each abnormal protein fragment and CA19-9 for discriminating resectable PDAC pts from healthy participants was evaluated by receiver operating characteristic (ROC) curve analysis. Results A total of 19 pts with resectable PDAC (median age 73 years, male 74%) and 30 healthy participants (median age 34 years, male 40%) were analyzed. The fragmentation ratios for three fragments were found to be higher in the resectable PDAC pts than in the healthy participants, and these fragments were determined as abnormal protein fragments. The areas under the ROC curves were 0.73, 0.78 and 0.82, respectively (CA19-9: 0.82). Conclusion Measurement of the urinary fragmentation ratios of KNG enabled discrimination between resectable PDAC pts and healthy volunteers.

214 Background: Non PDAC tissue-originated proteins are cleaved by proteases derived from PDAC, which can result in abnormal cleavage patterns in the urine of PDAC patients. Urinary proteomic analysis for quantifying the ratios of the abnormal protein fragments to the non-fragmented protein levels in the urine may be useful to distinguish early PDAC from healthy controls. This proof-of-concept study was planned to determine the usefulness of measuring the protein fragments from non PDAC tissue-originated proteins in the urine using the multiple-reaction-monitoring technique (MRM) for discriminating resectable PDAC from healthy controls. Methods: Urinary proteins were digested with trypsin, and resultant peptides were measured by MRM analysis and the ratio of the level of each fragment to the non-fragmented protein level (fragmentation ratio) was calculated. Fragments for which the fragmentation ratios were higher in the PDAC group than those in the healthy group were defined as abnormal protein fragments. The diagnostic capability of each abnormal protein fragment for discriminating cases of PDAC from healthy controls was evaluated by receiver operating characteristic (ROC) curve analysis. Results: A total of 21 patients with resectable PDAC and 30 healthy control subjects were enrolled in this study. All the PDAC patients were treated by pancreatic resection. Urine samples for this study were collected prior to the surgery from the PDAC patients. The non PDAC tissue-originated protein was determined as a liver-originated protein. The fragmentation ratios for six fragments were found to be higher in the PDAC group as compared to those in the healthy control group, and these fragments were determined as abnormal protein fragments. ROC curve analysis was performed for each of the abnormal fragments to determine the areas under the curve (AUCs) for discriminating cases of PDAC from healthy controls. The best AUC was 0.81 (95% CI, 0.68-0.91). Conclusions: The urinary fragmentation ratios showed the ability to discriminate cases of resectable PDAC from a healthy control group; abnormal fragmentation ratios may be promising, noninvasively measurable biomarkers of early PDAC.

209 Background: There is a clear need to identify a non-invasive biomarker for early diagnosis of PaCa, in order to improve the overall survival of PaCa patients. Secretion of large amounts of proteases is a hallmark of PaCa, which results in an abundance of protease-induced cleavage products being excreted in the urine. This has led to speculation that measurement of PaCa-specific fragments in the urine might be useful as a tool for discrimination between PaCa patients and healthy controls. Herein, we introduce urinary KNG fragments as a promising biomarker for early diagnosis of PaCa. Methods: Urine samples were collected from PaCa patients and healthy volunteers, with the written informed consent, from January 2014 to July 2016. Urinary protein tryptic fragments derived from protein C-termini were measured using isobaric tags (iTRAQ) for their relative quantitation, and the diagnostic ability of the urinary levels of these fragments was evaluated by receiver operating characteristic (ROC) curve analysis. The fragments which showed an area-under-the-curve (AUC) of over 0.8 were selected as candidate fragments for further validation by the multiple-reaction-monitoring technique (MRM) combined with high-speed liquid chromatography. The urinary level of each candidate fragment was quantified by MRM, and the diagnostic capability of each for discriminating PaCa patients from healthy controls was evaluated by ROC curve analysis. Results: Urine samples of 39 PaCa patients (7 resectable, 32 unresectable) and 42 healthy controls were examined by iTRAQ to find 12,783 fragments. ROC curve analysis was carried out to select two candidate fragments (fragments A, B), both of which turned out to be KNG cleavage products. The urinary levels of the two fragments were measured in 23 resectable PaCa, 118 unresectable PaCa patients, and 42 healthy volunteers using high-speed-LC-/MRM. The AUCs of serum CA19-9 and urinary levels of fragments A and B for discriminating patients of PaCa from healthy controls were 0.89, 0.81 and 0.70, respectively. Conclusions: Urinary KNG fragments showed favorable diagnostic capability and were considered as promising, noninvasively measurable biomarkers of PaCa.

The human importin (Imp)-β 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-β, 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-β cargoes. In in vitro binding assays, most of the candidate cargoes bound to Imp-β 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-β cargoes. The identified Imp-β and Trn cargoes were compared, ensuring the carrier specificity of the method and illustrating the complexity of these transport pathways. Background: A limited number of cargoes specific for importin-β family nucleocytoplasmic transport carriers have been reported. We have developed a stable isotope labeling by amino acids in cell culture (SILAC)-based transport method to identify specific cargo. Results: Candidate importin-β (with or without importin-α) cargoes were identified by our method and corroborated with binding assays. Conclusion: New importin-β-specific cargoes were identified. Significance: Our method has the potential to identify cargoes specific for other carriers.

Visual Overview, Maintenance of neuropathic pain caused by peripheral nerve injury crucially depends on the phosphorylation of GluN2B, a subunit of the N-methyl-d-aspartate (NMDA) receptor, at Tyr1472 (Y1472) and subsequent formation of a postsynaptic density (PSD) complex of superficial spinal dorsal horn neurons. Here we took advantage of comparative proteomic analysis based on isobaric stable isotope tags (iTRAQ) between wild-type and knock-in mice with a mutation of Y1472 to Phe of GluN2B (Y1472F-KI) to search for PSD proteins in the spinal dorsal horn that mediate the signaling downstream of phosphorylated Y1472 GluN2B. Among several candidate proteins, we focused on brain-enriched guanylate kinase-associated protein (BEGAIN), which was specifically up-regulated in wild-type mice after spared nerve injury (SNI). Immunohistochemical analysis using the generated antibody demonstrated that BEGAIN was highly localized at the synapse of inner lamina II in the spinal dorsal horn and that its expression was up-regulated after SNI in wild-type, but not in Y1472F-KI, mice. In addition, alteration of the kinetics of evoked excitatory postsynaptic currents for NMDA but not those for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in spinal lamina II was demonstrated by BEGAIN deletion. We demonstrated that mechanical allodynia, a condition of abnormal pain induced by innocuous stimuli, in the SNI model was significantly attenuated in BEGAIN-deficient mice. However, there was no significant difference between naive wild-type and BEGAIN-knockout mice in terms of physiological threshold for mechanical stimuli. These results suggest that BEGAIN was involved in pathological pain transmission through NMDA receptor activation by the phosphorylation of GluN2B at Y1472 in spinal inner lamina II.

Purpose There is no clear evidence of the factors that could improve implant biomechanics in the posterior maxilla. Thus, a finite element analysis was performed to investigate the effect of maxillary cortical bone thickness, implant design and diameter on stress around implants. Methods A total of 12 models of the posterior maxilla with implant were computer-simulated by varying the thickness of the alveolar cortical bone (1.5, 1.0, 0.5 or 0mm) and implant characteristics (cylindrical implant of 4.1-mm diameter, screw-type implants of 4.1-mm or 4.8-mm outer diameters). On top of each implant, forces were separately applied axially (100N) and buccolingually (50N), and the von Mises stresses were calculated. Results Regardless of load direction, implant design and diameter, cortical and cancellous bone stresses increased with the decrease of crestal cortical bone thickness. In the absence of crestal cortical bone, cancellous bone stresses were highest and, under axial load, were transferred to the sinus floor. Implant design and diameter influenced stress to a less extent, especially under buccolingual load and in the presence of crestal cortical bone. Conclusions From a biomechanical viewpoint, to improve implant success odds in the posterior maxilla, rather than implant selection, careful preoperative evaluation of the cortical bone at the planned implant site is recommended. If this cortical bone is very thin or even lacking, implant treatment should be carried on with caution by progressive loading in the range of functional loads.

Thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits anti-fibrinolytic activity by removing C-terminal lysine residues from fibrin or plasminogen receptor proteins on the cellular surface, and plays an important role in the regulation of fibrinolysis. In this study, we examined the regulation of TAFI in hepatocytes during liver regeneration, and revealed its pivotal role in hepatocyte proliferation. In rat models, partial hepatectomy or carbon tetrachloride (CCl4)-induced acute liver injury suppressed the levels of plasma TAFI activity and hepatic TAFI mRNA, whereas this operation markedly increased both the hepatic plasmin activity and the level of proliferating cell nuclear antigen. In primary cultures of rat hepatocytes, the TAFI mRNA level was decreased under growth-promoting culture conditions. Treatment of the hepatocytes with TAFI siRNA increased the amount of plasmin on the hepatocytes and promoted hepatocyte proliferation. We concluded that TAFI regulates plasmin activity through its enzymatic activity whereby it reduces the plasminogen-binding capacity of the hepatocytes. The TAFI gene expression is down-regulated in hepatocyte proliferation for producing a fibrinolytic microenvironment suitable for cell growth. This is the first report on the role of TAFI in the pericellular fibrinolysis necessary for cellular proliferation.

beta-Adducin is a cytoskeletal protein that interacts with the actin filaments to suppress actin polymerization and facilitate actin-spectrin binding. We have previously shown that beta-adducin is phosphorylated by Fyn at tyrosine489 in the rat brain and bound to its Src-homology 2 domain. In the present study, we examined the immunohistochemical localization of the tyrosine489-phosphorylated form of beta-adducin (pY489-beta-adducin) in the rat brain. Among brain regions, highest immunoreactivity was located in the hypothalamic tanycytes that are of glial origin lining around the third cerebral ventricle. Their immunoreactive processes extended into the arcuate nucleus, ventromedial hypothalamus and the median eminence. In addition, the pY489-beta-adducin immunoreactivity in the tanycytes was enhanced after fasting for 36-48 h, being associated with a morphological change of the DARPP-32-immunoreactivity. Intraperitoneal injection of 2-deoxy-d-glucose also enhances pY489-beta-adducin immunoreactivity in the tanycytes, along with increased food intake. These results suggest that tyrosine phosphorylation of beta-adducin in the tanycytes is involved in hypothalamic regulation of food intake and energy homeostasis.

In the present study, using urethane-anesthetized rats, we examined the effects of intralateral cerebral ventricular (LCV) injection of various doses of l -carnosine on neural activity innervating brown adipose tissue (BAT-SNA) and body temperature (BT). We found that injection of a low dose of l -carnosine (0.01 μg) suppressed BAT-SNA significantly. Conversely, a high dose (100 μg) of l -carnosine significantly elevated BAT-SNA. In the light period (14:00), brown adipose tissue temperature (BAT-T) and BT were suppressed after low and elevated after high dose injection of l -carnosine whereas in the dark period (2:00), these parameters remained unchanged with l -carnosine treatment. Bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN) abolished the effects of low and high doses of l -carnosine on BAT-SNA, BAT-T and BT. Furthermore, high dose treatment with l -carnosine altered c-Fos induction in the SCN and the PVN. These results suggest that l -carnosine affects BAT-SNA, BAT-T and BT in a dose-dependent manner in the rat, and that the SCN may be involved in these effects.

Syntrophins are components of the dystrophin-glycoprotein complex of the plasma membrane in muscular and neuronal cells, and recruit signaling proteins such as neuronal nitric oxide synthase via their multiple protein-protein interaction motifs. In this study, we found that alpha1-syntrophin binds to various subtypes of guanine nucleotide-binding protein alpha subunits (Galpha). A pull-down analysis using full-length recombinant alpha1-syntrophin and MS analysis showed that alpha1-syntrophin was coprecipitated with several isoforms of Galpha proteins in addition to known binding partners such as dystrobrevin and neuronal nitric oxide synthase. Further analysis using recombinant Galpha isoforms showed that alpha1-syntrophin associates with at least Galphai, Galphao, Galphas and Galphaq subtypes. The region of alpha1-syntrophin required for its interaction with Galphas was determined as the N-terminal half of the first pleckstrin homology domain. In addition, the syntrophin unique domain of alpha1-syntrophin was suggested to contribute to this interaction. In COS-7 cells, downregulation of alpha1-syntrophin by RNAi resulted in enhanced cAMP production and cAMP response element-binding protein phosphorylation induced by isoproterenol treatment. These results suggest that alpha1-syntrophin provides a scaffold for the Galpha family of heterotrimeric G proteins in the brain to regulate the efficiency of signal transduction evoked by G-protein-coupled receptors.

Carnosine dipeptidase II (CN2/CNDP2) is an M20 family metallopeptidase that hydrolyses various dipeptides including β‐alanyl‐l‐histidine (carnosine). Crystallographic analysis showed that CN2 monomer is composed of one catalytic and one dimerization domains, and likely to form homodimer. In this crystal, H228 residue of the dimerization domain interacts with the substrate analogue bestatin on the active site of the dimer counterpart, indicating that H228 is involved in enzymatic reaction. In the present study, the role of intradimer interaction of CN2 in its catalytic activity was investigated using electrospray‐ionization time‐of‐flight mass spectrometry (ESI‐TOF MS). First, a dimer interface mutant I319K was prepared and shown to be present as a folded monomer in solution as examined by using ESI‐TOF MS. Since the mutant was inactive, it was suggested that dimer formation is essential to its enzymatic activity. Next, we prepared H228A and D132A mutant proteins with different N‐terminal extended sequences, which enabled us to monitor dimer exchange reaction by ESI‐TOF MS. The D132A mutant is a metal ligand mutant and also inactive. But the activity was partially recovered time‐dependently when H228A and D132A mutant proteins were incubated together. In parallel, H228A/D132A heterodimer was formed as detected by ESI‐TOF MS, indicating that interaction of a catalytic center with H228 residue of the other subunit is essential to the enzymatic reaction. These results provide evidence showing that intradimer interaction of H228 with the reaction center of the dimer counterpart is essential to the enzymatic activity of CN2.

Oral administration of Lactobacillus casei reportedly reduces blood glucose concentrations in a non-insulin-dependent diabetic KK-Ay mouse model. In order to determine if other lactobacillus strains affect glucose metabolism, we evaluated the effect of the probiotic strain Lactobacillus johnsonii La1 (LJLa1) strain on glucose metabolism in rats. Oral administration of LJLa1 via drinking water for 2 weeks inhibited the hyperglycemia induced by intracranial injection of 2-deoxy- d -glucose (2DG). We found that the hyperglucagonemic response induced by 2DG was also suppressed by LJLa1. Oral administration of LJLa1 for 2 weeks also reduced the elevation of blood glucose and glucagon levels after an oral glucose load in streptozotocin-diabetic rats. In addition, we recently observed that intraduodenal injection of LJLa1 reduced renal sympathetic nerve activity and enhanced gastric vagal nerve activity, suggesting that LJLa1 might affect glucose metabolism by changing autonomic nerve activity. Therefore, we evaluated the effect of intraduodenal administration of LJLa1 on adrenal sympathetic nerve activity (ASNA) in urethane-anesthetized rats, since the autonomic nervous system, including the adrenal sympathetic nerve, may be implicated in the control of the blood glucose levels. Indeed, we found that ASNA was suppressed by intraduodenal administration of LJLa1, suggesting that LJLa1 might improve glucose tolerance by reducing glucagon secretion via alteration of autonomic nerve activities.

Fyn is a Src-family tyrosine kinase involved in neuronal development, transmission, and plasticity in mammalian central nervous system. We have previously reported that Fyn binds to a cytoskeletal protein, beta-adducin, in a phosphorylation-dependent manner. In the present report, we show that Fyn phosphorylates beta-adducin at tyrosine 489 located in its C-terminal tail domain. Phosphorylation of beta-adducin at Y489 was required for its association with the Fyn-SH2 domain. An antibody specific to the phosphorylated form of beta-adducin was raised in rabbits and showed that Y489 of beta-adducin was phosphorylated in wild type, but not in Fyn(-/-) mice, suggesting that Y489 of beta-adducin is phosphorylated downstream of Fyn in vivo. After phosphorylation at Y489, beta-adducin was translocated to the cell periphery, and colocalized with Fyn. These results suggest that Fyn phosphorylates and binds to beta-adducin at Y489, resulting in translocation of beta-adducin to the Fyn-enriched regions in the plasma membrane.

The hypothalamus plays a central role in the homeostatic regulation of internal physiological conditions such as body temperature and energy balance. We have previously shown that cold exposure enhances tyrosine phosphorylation of BIT/SHPS-1 (brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate-1) in hypothalamic nuclei including the suprachiasmatic nucleus. In order to elucidate the function of BIT/SHPS-1 in the hypothalamus, we stimulated BIT/SHPS-1 in vivo by using the anti-BIT monoclonal antibody (mAb) 1D4, which reacts with the extracellular domain of BIT/SHPS-1 and induces its tyrosine phosphorylation. Administration of mAb 1D4 into the third cerebral ventricle enhanced the electrical activity of the renal sympathetic nerves, while it suppressed that of the gastric parasympathetic nerves. Similarly, blood pressure increased in response to the mAb 1D4 injection, and additionally, temperatures of the abdomen and brown adipose tissue increased. These results indicate that BIT/SHPS-1 is involved in the hypothalamic regulation of thermogenesis via the autonomic nervous system.

Glucose-regulated protein 58 (GRP58)-like immunoreactivity in rat liver obtained in the evening or after fasting underwent an electrophoretic band-shift, which disappeared after phosphatase-treatment. Since mass spectrometric analysis raised a possibility that Ser150 of GRP58 is phosphorylated, an antibody against the phosphoserine150 GRP58 was generated. Immunoreactivity to this antibody was increased in the evening and after fasting. Since GRP58 was shown to interact with signal transducer and activator of transduction 3 (STAT3), a leptin-related protein, the effect of leptin was examined. Immunoreactivity to the anti-phosphoGRP58 antibody was markedly elevated after the leptin injection, indicating that Ser150 of GRP58 is phosphorylated after fasting and leptin-treatment.

L-Carnosine is a bioactive dipeptide present in mammalian tissues including the central nervous system. We have recently shown that L-carnosine is involved in the regulation of energy homeostasis through the autonomic nervous system, but the mechanisms for its biosynthesis and degradation have not yet been fully elucidated. Here we report the biochemical and immunohistochemical characterization of a mammalian protein that has a 17% overall amino acid sequence homology with a Lactobacilus carnosinase, PepV. A recombinant protein expressed in E. coli has the enzymatic ability to digest L-carnosine and various other dipeptides, and this activity is inhibited by bestatin. It requires Mn 2 + for enzymatic activity and its effect is reversible. Immunohistochemical analysis showed that a few neuronal populations express this protein at very high levels. It is highly expressed in the parafascicular nucleus of the thalamus, tuberomammillary nucleus of the hypothalamus and the mitral cell layer of the olfactory bulb. In addition, neuronal processes, but not cell bodies, are stained in the striatum. In all these areas, the protein did not colocalize with the glial fibrilary acidic protein. These results suggest that a peptidase that digests L-carnosine is enriched in several specific neuronal populations in the central nervous system.