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Sleep pressure, the driving force of the homeostatic sleep regulation, is accumulated during wakefulness and dissipated during sleep. Sleep deprivation (SD) has been used as a method to acutely increase animal’s sleep pressure for investigating the molecular changes under high sleep pressure. However, SD induces changes not only reflecting increased sleep pressure but also inevitable stresses and prolonged wake state itself. The Sik3Sleepy mutant mice (Sleepy) exhibit constitutively high sleep pressure despite sleeping longer, and have been useful as a model of increased sleep pressure. Here we conducted a cross-comparison of brain metabolomic profiles between SD versus ad lib slept mice, as well as Sleepy mutant versus littermate wild-type mice. Targeted metabolome analyses of whole brains quantified 203 metabolites in total, of which 43 metabolites showed significant changes in SD, whereas three did in Sleepy mutant mice. The large difference in the number of differential metabolites highlighted limitations of SD as methodology. The cross-comparison revealed that a decrease in betaine and an increase in imidazole dipeptides are associated with high sleep pressure in both models. These metabolites may be novel markers of sleep pressure at the whole-brain level. Furthermore, we found that intracerebroventricular injection of imidazole dipeptides increased subsequent NREM sleep time, suggesting the possibility that imidazole dipeptides may participate in the regulation of sleep in mice.

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1-3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8⁺ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses., GABAを標的とする抗腫瘍免疫機構 --代謝産物を介した免疫細胞間制御の一端を解明--. 京都大学プレスリリース. 2021-11-10.

Despite the success of cancer immunotherapies such as programmed cell death–1 (PD-1) and PD-1 ligand 1 (PD-L1) inhibitors, patients often develop resistance. New combination therapies with PD-1/PD-L1 inhibitors are needed to overcome this issue. Bezafibrate, a ligand of peroxisome proliferator–activated receptor–γ coactivator 1α/peroxisome proliferator–activated receptor complexes, has shown a synergistic antitumor effect with PD-1 blockade in mice that is mediated by activation of mitochondria in T cells. We have therefore now performed a phase 1 trial (UMIN000017854) of bezafibrate with nivolumab in previously treated patients with advanced non–small cell lung cancer. The primary end point was the percentage of patients who experience dose-limiting toxicity, and this combination regimen was found to be well tolerated. Preplanned comprehensive analysis of plasma metabolites and gene expression in peripheral cytotoxic T cells indicated that bezafibrate promoted T cell function through up-regulation of mitochondrial metabolism including fatty acid oxidation and may thereby have prolonged the duration of response. This combination strategy targeting T cell metabolism thus has the potential to maintain antitumor activity of immune checkpoint inhibitors and warrants further validation.

Spermidine (SPD) delays age-related pathologies in various organisms. SPD supplementation overcame the impaired immunotherapy against tumors in aged mice by increasing mitochondrial function and activating CD8 + T cells. Treatment of naïve CD8 + T cells with SPD acutely enhanced fatty acid oxidation. SPD conjugated to beads bound to the mitochondrial trifunctional protein (MTP). In the MTP complex, synthesized and purified from Escherichia coli , SPD bound to the α and β subunits of MTP with strong affinity and allosterically enhanced their enzymatic activities. T cell–specific deletion of the MTP α subunit abolished enhancement of programmed cell death protein 1 (PD-1) blockade immunotherapy by SPD, indicating that MTP is required for SPD-dependent T cell activation.

Macropinocytosis is among ubiquitous cellular uptake mechanisms of peptide-based intracellular delivery. Due to its capability of engulfing large macromolecules, macropinocytosis shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. We previously reported SN21, a peptide derived from the N-terminus of stromal cell-derived growth factor 1α (SDF-1α), as a potent macropinocytosis inducer. In this work, we obtained the 8-residue analog P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A with membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins.

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1–3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8+ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses., A paper in Nature demonstrates that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages able to limit anti-tumour T cell cytotoxicity.

Blood eosinophil count is a useful measure in asthma or COPD management. Recent epidemiological studies revealed that body mass index (BMI) is positively associated with eosinophil counts. However, few studies focused on the role of adiposity and fatty acid-related metabolites on eosinophil counts, including the effect of genetic polymorphism. In this community-based study involving 8265 participants (30–74 year old) from Nagahama city, we investigated the relationship between eosinophil counts and serum levels of fatty acid-related metabolites. The role of MDC1, a gene that is related to eosinophil counts in our previous study and encodes a protein that is thought to be involved in the repair of deoxyribonucleic acid damage, was also examined taking into account its interaction with adiposity. Serum levels of linoleic acid (LA) and β-hydroxybutyric acid (BHB) were negatively associated with eosinophil counts after adjustment with various confounders; however, there were positive interactions between serum LA and BMI and between serum BHB and BMI/body fat percentages in terms of eosinophil counts. In never-smokers, there was positive interaction for eosinophil counts between the CC genotype of MDC1 rs4713354 and BMI/body fat percentages. In conclusion, both serum LA and BHB have negative impacts on eosinophil counts, while adiposity shows robust positive effects on eosinophil counts, partly via genetic background in never-smokers.

Metabolites underlying brain function and pathology are not as well understood as genes. Here, we applied a novel metabolomics approach to further understand the mechanisms of memory processing in sleep. As hippocampal dentate gyrus neurons are known to consolidate contextual fear memory, we analyzed real-time changes in metabolites in the dentate gyrus in different sleep–wake states in mice. Throughout the study, we consistently detected more than > 200 metabolites. Metabolite profiles changed dramactically upon sleep–wake state transitions, leading to a clear separation of phenotypes between wakefulness and sleep. By contrast, contextual fear memory consolidation induced less obvious metabolite phenotypes. However, changes in purine metabolites were observed upon both sleep–wake state transitions and contextual fear memory consolidation. Dietary supplementation of certain purine metabolites impaired correlations between conditioned fear responses before and after memory consolidation. These results point toward the importance of purine metabolism in fear memory processing during sleep.

BACKGROUND: Current clinical biomarkers for the programmed cell death 1 (PD-1) blockade therapy are insufficient because they rely only on the tumor properties, such as programmed cell death ligand 1 expression frequency and tumor mutation burden. Identifying reliable, responsive biomarkers based on the host immunity is necessary to improve the predictive values. METHODS: We investigated levels of plasma metabolites and T cell properties, including energy metabolism markers, in the blood of patients with non-small cell lung cancer before and after treatment with nivolumab (n = 55). Predictive values of combination markers statistically selected were evaluated by cross-validation and linear discriminant analysis on discovery and validation cohorts, respectively. Correlation between plasma metabolites and T cell markers was investigated. RESULTS: The 4 metabolites derived from the microbiome (hippuric acid), fatty acid oxidation (butyrylcarnitine), and redox (cystine and glutathione disulfide) provided high response probability (AUC = 0.91). Similarly, a combination of 4 T cell markers, those related to mitochondrial activation (PPARγ coactivator 1 expression and ROS), and the frequencies of CD8(+)PD-1(hi) and CD4(+) T cells demonstrated even higher prediction value (AUC = 0.96). Among the pool of selected markers, the 4 T cell markers were exclusively selected as the highest predictive combination, probably because of their linkage to the abovementioned metabolite markers. In a prospective validation set (n = 24), these 4 cellular markers showed a high accuracy rate for clinical responses of patients (AUC = 0.92). CONCLUSION: Combination of biomarkers reflecting host immune activity is quite valuable for responder prediction. FUNDING: AMED under grant numbers 18cm0106302h0003, 18gm0710012h0105, and 18lk1403006h0002; the Tang Prize Foundation; and JSPS KAKENHI grant numbers JP16H06149, 17K19593, and 19K17673.

(1)H nuclear magnetic resonance (NMR) spectroscopy-based metabolic phenotyping is now widely used for large-scale epidemiological applications. To minimize signal overlap present in 1D (1)H NMR spectra, we have investigated the use of 2D J-resolved (JRES) (1)H NMR spectroscopy for large-scale phenotyping studies. In particular, we have evaluated the use of the 1D projections of the 2D JRES spectra (pJRES), which provide single peaks for each of the J-coupled multiplets, using 705 human plasma samples from the FGENTCARD cohort. On the basis of the assessment of several objective analytical criteria (spectral dispersion, attenuation of macromolecular signals, cross-spectral correlation with GC-MS metabolites, analytical reproducibility and biomarker discovery potential), we concluded that the pJRES approach exhibits suitable properties for implementation in large-scale molecular epidemiology workflows.

Dietary salt restriction is essential for managing fluid retention in patients with chronic kidney disease (CKD). In this retrospective cohort study, we investigated weight loss from the perspective of fluid status in CKD patients during a 7-day hospitalization period while consuming a low-salt diet (5 g/day). Among 311 patients, the median weight loss (interquartile range, maximum) was 0.7 (0.0–1.4, 4.7) kg on Day 4 and 1.0 (0.3–1.7, 5.9) kg on Day 7. Patients were classified into quartiles based on pre-hospital urinary salt excretion (quartile [Q] 1, 1.2–5.7; Q2, 5.8–8.4; Q3, 8.5–11.3; Q4, 11.4–29.2 g/day). Weight loss was significantly greater in Q3 and Q4 than in Q1. The body mass index (BMI) and urinary salt excretion in the first 24 hours after admission were independently associated with rapid weight loss on Day 4 by multivariate logistic regression analysis. In conclusion, CKD patients with a high salt intake or high BMI exhibit rapid weight loss within a few days of consuming a low-salt diet. Dietary salt restriction is effective for reducing proteinuria in these patients, but long-term observation is needed to confirm the sustained effects.

SUMMARYGut microbiota changes are associated with increased risk of Type 2 diabetes (T2D) and obesity. Through serum metabolome profiling in patients with cardiometabolic disease (CMD) we identified significant inverse correlation between the microbial metabolite 4-cresol and T2D. Chronic administration of non toxic dose of 4-cresol in two complementary preclinical models of CMD reduced adiposity, glucose intolerance and liver triglycerides, and enhanced insulin secretion in vivo, which may be explained by markedly increased pancreas weight, augmented islet density and size, and enhanced vascularisation suggesting activated islet neogenesis. Incubation of isolated islets with 4-cresol enhanced insulin secretion, insulin content and cell proliferation. In both CMD models 4-cresol treatment in vivo was associated with altered expression of SIRT1 and the kinase DYRK1A, which may contribute to mediate its biological effects. Our findings identify 4-cresol as an effective regulator of β-cell function and T2D endophenotypes, which opens therapeutic perspectives in syndromes of insulin deficiency.

The rise in population aging worldwide is causing an unparalleled increase in death from many cancers, including glioblastoma (GBM). Here, we have explored the impact of aging and rejuvenation on GBM tumorigenesis. Compared with neuro-inflammatory old GBM, young GBM displayed elevated neuronal/synaptic signaling via brain-derived neurotrophic factor (BDNF) and SLIT and NTRK like-family member 6 (SLITRK6), promoting favorable survival rates. These effects were attributed to the rise in nicotinamide adenine dinucleotide (NAD+) levels, as brain rejuvenation by parabiosis or administration of nicotinamide mononucleotide (NMN) in mice elicited a younger phenotype with activated neuronal/synaptic signaling and improved outcomes. Our data indicate that age-associated NAD+ loss contributes to the highly aggressive GBM by the shift from neuronal/synaptic activity to neuro-inflammation in the elderly brain. These findings have therapeutic and preventive implications in GBM and provide mechanistic insights into the exacerbation of GBM tumorigenesis by aging.

The rise in population aging worldwide is causing an unparalleled increase in death from many cancers, including glioblastoma (GBM). Here, we have explored the impact of aging and rejuvenation on GBM tumorigenesis. Compared with old GBM, young GBM displayed elevated neuronal/synaptic signaling via brain-derived neurotrophic factor (BDNF) and SLIT and NTRK like-family member 6 (SLITRK6), promoting favorable survival rates. These effects were attributed to the rise in nicotinamide adenine dinucleotide (NAD+) levels, as brain rejuvenation by parabiosis or administration of nicotinamide mononucleotide (NMN) in mice elicited a younger phenotype with activated neuronal/synaptic signaling and improved outcomes. Our data indicate that age-associated NAD+ loss contributes to the highly aggressive GBM in the elderly. These findings have therapeutic implications in GBM and provide mechanistic insights into the exacerbation of GBM tumorigenesis with age.

To assess the use of plasma free amino acids (PFAAs) as biomarkers for metabolic disorders, it is essential to identify genetic factors that influence PFAA concentrations. PFAA concentrations were absolutely quantified by liquid chromatography–mass spectrometry using plasma samples from 1338 Japanese individuals, and genome-wide quantitative trait locus (QTL) analysis was performed for the concentrations of 21 PFAAs. We next conducted a conditional QTL analysis using the concentration of each PFAA adjusted by the other 20 PFAAs as covariates to elucidate genetic determinants that influence PFAA concentrations. We identified eight genes that showed a significant association with PFAA concentrations, of which two, SLC7A2 and PKD1L2, were identified. SLC7A2 was associated with the plasma levels of arginine and ornithine, and PKD1L2 with the level of glycine. The significant associations of these two genes were revealed in the conditional QTL analysis, but a significant association between serine and the CPS1 gene disappeared when glycine was used as a covariate. We demonstrated that conditional QTL analysis is useful for determining the metabolic pathways predominantly used for PFAA metabolism. Our findings will help elucidate the physiological roles of genetic components that control the metabolism of amino acids.

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono-hexsosyl ceramide groups with good peak area repeatability (RSD% 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co-existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.

Although immunotherapy by PD-1 blockade has dramatically improved the survival rate of cancer patients, further improvement in efficacy is required to reduce the fraction of less sensitive patients. In mouse models of PD-1 blockade therapy, we found that tumor-reactive cytotoxic T lymphocytes (CTLs) in draining lymph nodes (DLNs) carry increased mitochondrial mass and more reactive oxygen species (ROS). We show that ROS generation by ROS precursors or indirectly by mitochondrial uncouplers synergized the tumoricidal activity of PD-1 blockade by expansion of effector/memory CTLs in DLNs and within the tumor. These CTLs carry not only the activation of mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) but also an increment of their downstream transcription factors such as PPAR-gamma coactivator 1α (PGC-1α) and T-bet. Furthermore, direct activators of mTOR, AMPK, or PGC-1α also synergized the PD-1 blockade therapy whereas none of above-mentioned chemicals alone had any effects on tumor growth. These findings will pave a way to developing novel combinatorial therapies with PD-1 blockade.

Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.

Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.

Klotho is a circulating protein, and Klotho deficiency disturbs endothelial integrity, but the molecular mechanism is not fully clarified. We report that vascular endothelium in Klotho-deficient mice showed hyperpermeability with increased apoptosis and down-regulation of vascular endothelial (VE)-cadherin because of an increase in VEGF-mediated internal calcium concentration ([Ca 2+ ]i) influx and hyperactivation of Ca 2+ -dependent proteases. Immunohistochemical analysis, the pull-down assay using Klotho-fixed agarose, and FRET confocal imaging confirmed that Klotho protein binds directly to VEGF receptor 2 (VEGFR-2) and endothelial, transient-receptor potential canonical Ca 2+ channel 1 (TRPC-1) and strengthens the association to promote their cointernalization. An in vitro mutagenesis study revealed that the second hydrolase domain of Klotho interacts with sixth and seventh Ig domains of VEGFR-2 and the third extracellular loop of TRPC-1. In Klotho-deficient endothelial cells, VEGF-mediated internalization of the VEGFR-2/TRPC-1 complex was impaired, and surface TRPC-1 expression increased 2.2-fold; these effects were reversed by supplementation of Klotho protein. VEGF-mediated elevation of [Ca 2+ ]i was sustained at higher levels in an extracellular Ca 2+ -dependent manner, and normalization of TRCP-1 expression restored the abnormal [Ca 2+ ]i handling. These findings provide evidence that Klotho protein is associated with VEGFR-2/TRPC-1 in causing cointernalization, thus regulating TRPC-1–mediated Ca 2+ entry to maintain endothelial integrity.

A novel method for selectively labeling and isolating N-terminal peptide from protein has been developed. An N(alpha)-amino group of protein was converted to a carbonyl group through transamination reaction and the resulting carbonyl group was modified with O-(4-nitrobenzyl)hydroxylamine (NBHA). After proteolytic digestion using Grifola frondosa metalloendopeptidase (LysN), the modified N-terminal peptide remained unbound in the following treatment using amino-reactive p-phenylenediisothiocyanate (DITC) glass, whereas peptides other than the N-terminal peptide were effectively scavenged from the supernatant solution. The modified N-terminal peptide was thus successfully isolated and sequenced by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis.

A novel method for isolating C-terminal peptides from proteolytic digests of proteins was developed. Proteins were digested with lysyl endopeptidase (LysC) and applied to metal-ion-catalyzed transamination reactions. This reaction enabled the selective conversion of an Nα-amino group to a carbonyl group. Subsequent incubation with p-phenylenediisothiocyanate (DITC) glass effectively scavenged the lysine-containing N-terminus and internal peptides. The obtained C-terminal peptide is open to modification with reagents having virtually any type of functionality owing to the reactive α-ketocarbonyl group. In this report, 2,4-dinitrophenylhydrazine (DNPH) was used as an example of a nucleophile to the carbonyl group. The isolated C-terminal peptide was modified with DNPH, which exhibited signal enhancement, and was sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Copyright © 2009 John Wiley & Sons, Ltd.

Background/Aims: Peritoneal fibrosis can lead to the discontinuation of continuous ambulatory peritoneal dialysis. The present study investigated the direct effect of aldosterone, which influences tissue fibrosis, and its cellular mechanism using cultured rat peritoneal mesothelial cells (RPMCs). Materials andMethods: The expression of aldosterone synthase (CYP11B2), mineralocorticoid receptors, 11β-hydroxysteroid dehydrogenase 2, serum- and glucocorticoid-inducible protein kinase 1 (SGK1) and connective tissue growth factor (CTGF) was evaluated using reverse transcriptase-polymerase chain reaction and Western blot. The ability of RPMCs to produce aldosterone was examined by enzyme immunoassay. Small interfering RNA of SGK1 was transfected to determine the role of SGK1. Results: CYP11B2, mineralocorticoid receptors and 11β-hydroxysteroid dehydrogenase 2 were expressed in RPMCs. The release of aldosterone from RPMCs into the culture medium was confirmed. Stimulation of RPMCs with the addition of aldosterone significantly increased SGK1 expression and phosphorylation and CTGF upregulation, and these effects were completely inhibited by the mineralocorticoid receptor antagonist spironolactone. SGK1 gene silencing abrogated aldosterone-induced CTGF expression. Conclusion: The local aldosterone system exists and acts directly as a profibrotic factor in the peritoneal mesothelium.

Background/Aims: Peritoneal fibrosis leads to discontinuation of peritoneal dialysis. Although aldosterone promotes tissue fibrosis in many organs, its contribution to peritoneal fibrosis and the underlying mechanism are poorly understood. The present study investigated the direct effect of aldosterone on cultured rat peritoneal fibroblasts (RPFs). Methods: The expression of aldosterone synthase (CYP11B2), mineralocorticoid receptors (MRs), 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), serum- and glucocorticoid-inducible protein kinase 1 (SGK1), and connective tissue growth factor (CTGF) mRNA was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). To determine the role of reactive oxygen species (ROS) induced by aldosterone, an active oxygen assay with several inhibitors was used. The ability of RPFs to produce aldosterone was examined by enzyme immunoassay. Small interfering RNA (siRNA) of SGK1 was transfected into cultured cells using lipofectamine. Results: CYP11B2, MRs, and 11β-HSD2 were expressed in RPFs. The release of aldosterone from RPFs into the culture medium was confirmed. Aldosterone increased the expression of SGK1 mRNA via ROS generation. Spironolactone, apocynin, and tempol significantly reduced SGK1 expression. Aldosterone upregulated CTGF transcripts significantly. SGK1 gene silencing suppressed aldosterone-induced CTGF expression. Conclusion: The local aldosterone system acts directly as a profibrotic factor via ROS-mediated SGK1 in RPFs.

An improved method for de novo sequencing of arginine-containing peptides modified with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) is reported. A tagging reagent, TMPP-Ac-OSu, was introduced to improve the sequence analysis of peptides owing to the simplified fragmentation pattern. However, peptides containing arginine residues did not fragment efficiently even after TMPP-Ac modification at their N-termini. This report describes how fragmentation efficiency of TMPP-Ac-modified arginine-containing peptides was significantly improved by modifying the guanidino group on the side chain of arginine with acetylacetone.

A novel method for isolation and de novo sequencing of N-terminal peptides from proteins is described. The method presented here combines selective chemical tagging using succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) at the Nα-amino group of peptides after digestion by metalloendopeptidase (from Grifola frondosa) and selective capture procedures using p-phenylenediisothiocyanate resin, by which the N-terminal peptide can be isolated, whether or not it is N-terminally blocked. The isolated N-terminal peptide modified N-terminally with TMPP-Ac-OSu reagent produces a simple fragmentation pattern under tandem mass spectrometric analysis to significantly facilitate sequencing.

A simple and efficient method for C-terminal sequencing of proteins has long been pursued because it would provide substantial information for identifying the covalent structure, including post-translational modifications. However, there are still significant impediments to both direct sequencing from C termini of proteins and specific isolation of C-terminal peptides from proteins. We describe here a highly successful, de novo C-terminal sequencing method of proteins by employing succinimidyloxycarbonylmethyl tris (2,4,6-trimethoxyphenyl) phosphonium bromide and mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been one of the most powerful tools for analyzing protein phosphorylation. However, it is frequently difficult to detect phosphopeptides with high sensitivity by MALDI-MS. In our investigation of matrix/matrix-additive substances for improving the phosphopeptide ion response in MALDI-MS, we found that the addition of low-concentration alkylphosphonic acid to the matrix/analyte solution significantly enhanced the signal of phosphopeptides. In this study, the combination of methanediphosphonic acid and 2,5-dihydroxybenzoic acid gave the best results. In addition to enhancing the signal of the phosphopeptides, alkylphosphonic acid almost completely eliminated the signals of sodium and potassium ion adducts. We report herein sensitive detection of phosphopeptides by MALDI-MS with the use of alkylphosphonic acids as matrix additives.

Interaction with Fe(III) induces the reversible conformational switch of the extramembrane segment in the artificial receptor channel, which is transmitted into membranes as an increase in channel current (ion flux).

The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.

Various etiologic factors have been identified in tubulointerstitial nephritis (TIN), including allergic drug reaction, infection, and immune-mediated disease. Immune-mediated TIN without significant glomerular involvement has been reported to occur as a renal complication secondary to Sjogren's syndrome, lupus nephritis, and antitubular basement membrane antibody-related disease. We present a first case of acute TIN associated with autoimmune-related pancreatitis (AIP). A 64-year-old man was referred to our division from a surgeon for the close examination of renal dysfunction. The pancreatic and biliary imaging showed segmental narrowing of the pancreatic duct with localized swelling of the pancreatic head, suggesting the carcinoma of the pancreatic head at that time. However, the laboratory findings also showed renal dysfunction with high level of serum immunoglobulin G and hypocomplemetemia. Renal biopsy was performed to investigate the etiology of the renal dysfunction. The renal biopsy specimen showed acute TIN. The patient had no drug history, which may cause TIN. Oral corticosteroid therapy improved the renal function as well as histological damage, the pancreatic imaging study, and the laboratory tests of pancreatic and hepatobiliary enzyme. Although the pancreatic biopsy has not performed in our patient, his clinical course confirmed us that AIP was the final diagnosis for his pancreatic lesion. Despite further examination, there was no evidence of other autoimmune-related diseases such as Sjogren's syndrome. To our knowledge, this is the first report of acute TIN associated with AIP. We suggest that AIP may be an etiologic factor in some cases of TIN.