Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.

Characterization of the enzymatic properties of human RNPEPL1/aminopeptidase Z.

It is now evident that the M1 family of aminopeptidases play important roles in many pathophysiological processes. Among them, the enzymatic properties of arginyl aminopeptidase-like 1 (RNPEPL1) are characterized only by its truncated form. No peptide substrate has been identified. To characterize the enzymatic properties of RNPEPL1 in more detail, the full-length protein was expressed in Escherichia coli and purified to homogeneity. The full-length RNPEPL1 showed rather restricted substrate specificity and basic amino acid preference towards synthetic substrates, which was different from the previously reported specificity characterized by the truncated form. Searching for peptide substrates, we found that several peptides, such as Met-enkephalin and kallidin, were cleaved. RNPEPL1 cleaved bradykinin to de-[Arg]-bradykinin despite the presence of proline at the P2'-position. The enzyme cleaved Met-enkephalin but not dynorphin A1-17. Similar to aminopeptidase B, the full-length RNPEPL1 showed basic amino acid preference towards both synthetic and peptide substrates. In addition to the unusual cleavage of bradykinin, this enzyme shows chain length-dependent cleavage of peptide substrates sharing N-terminal amino acid sequence. This is the first study to report the enzymatic properties of the full-length human RNPEPL1 as an aminopeptidase enzyme.

Characteristic and complementary chiral recognition ability of four recently developed immobilized chiral stationary phases based on amylose and cellulose phenyl carbamates and benzoates.

To date, various immobilized chiral stationary phases (CSPs) have been developed. The immobilized CSPs have opened up possibilities not only maintaining the high chiral recognition abilities as well as corresponding coated ones but also affording high durability to various mobile phase. This report directed to investigate enantioseparation of recently launched four immobilized CSPs with cellulose and amylose backbones under normal phase liquid chromatography conditions. Their chiral recognition abilities were compared with previously developed six immobilized CSPs. Particularly, we focused on the complementarity for chiral recognitions. Among them, amylose tris(3-chloro-5-methylphenylcarbamate) CSP, namely, CHIRALPAK IG, showed notable chiral recognition abilities to various racemates. As expected, the investigated immobilized CSPs represented remarkable durability to wide range of mobile phases, whereas the corresponding coated CSPs could not be run due to the irreversible degradation. Taking advantage of unrestricted solvent compatibility, chiral separation selectivities were improved for some racemates.

Application of Polysaccharide-Based Chiral High-Performance Liquid Chromatography Columns for the Separation of Regio-, E/Z-, and Enantio-Isomeric Mixtures of Allylic Compounds.

Daicel Chiralpak IA, IB, and IC, which are the polysaccharide-based chiral stationary phase (CSP) columns for high-performance liquid chromatography (HPLC), were applied in the separation of the non-enantiomeric isomeric mixtures obtained by the various allylation reactions and were highly effective in separating the regio- and (E)/(Z)-isomers in the allylation products. Due to the close structural similarity of the isomeric allylic compounds in the reaction mixtures, separations of the isomers are laborious and could not be accomplished by the conventional methods such as silica gel column chromatography, silica gel HPLC, preparative GPC, distillation, and so forth. This study has shown potential advantages of using the polysaccharide-based CSP columns in the separation of not only enantiomeric but also non-enantiomeric isomeric mixtures.

Achiral Molecular Recognition of Substituted Aniline Position Isomers by Crown Ether Type Chiral Stationary Phase.

To understand the selectivity of the crown ether type chiral stationary phase (CSP), the retention selectivity for aniline and the positional isomers of substituted anilines were studied. In various substituted isomers, except nitroaniline, a remarkable decrease of retention due to steric hindrance was observed for the 2-substituted isomer. To determine the detailed molecular recognition mechanism, quantum chemical calculations were performed for the aggregates between the crown ether and the anilines. The results suggested that the 20-Crown-6, which includes a phenyl-substituted 1,1'-binaphthyl moiety, interacts with alkyl and aryl amines in an unconventional form different from the proposed one for 18-Crown-6.

Molecular and functional diversity of the oxytocinase subfamily of M1 aminopeptidases.

The placental leucine aminopeptidase/insulin-regulated aminopeptidase, endoplasmic reticulum aminopeptidase 1 and endoplasmic reticulum aminopeptidase 2 are part of a distinct subfamily of M1 aminopeptidases termed the 'oxytocinase subfamily'. The subfamily members show molecular diversity due to differential usage of translation initiation sites, alternative splicing and multiple single nucleotide polymorphisms. It is becoming evident that, depending on their intracellular or extracellular location, members of the oxytocinase subfamily play important roles in the maintenance of homeostasis, including the regulation of blood pressure, maintenance of normal pregnancy, retention of memory and trimming of antigenic peptides presented to major histocompatibility complex class I molecules, by acting as either aminopeptidases or binding partners of specific functional proteins in the cells. Based on their molecular diversity and moonlighting protein-like properties, it is conceivable that the subfamily members exert pleiotropic effects during evolution, to become important players in the regulation of homeostasis.

Importance of Tyr409 and Tyr414 in constructing the substrate pocket of human aminopeptidase B.

Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes basic amino acids of synthetic substrates and requires a physiological concentration of chloride anions for optimal activity. Several amino acid residues of APB responsible for its enzymatic activity have been elucidated. In this study, we further searched for residues critical to its enzymatic activity, especially toward peptide substrates. APB residues Tyr409 (Y409) and Tyr414 (Y414), both of which were critical to its hydrolytic activity toward synthetic substrates, were predicted by molecular modeling to be involved in cleaving peptide substrates via its interaction with amino acids in the P1' cleavage site. Using site-directed mutagenesis, several mutant APBs were prepared. In contrast to synthetic substrates, wild-type and Y409F/Y414F double mutant enzymes showed P1'-dependent cleavage of peptide substrates, indicating that both tyrosine residues were not indispensable for hydrolytic activity toward peptide substrates. Moreover, the Y409F/Y414F double mutant enzyme cleaved peptides with a Pro residue at the P1' site, which is uncommon among the M1 family of aminopeptidases. These results suggested that Tyr409 and Tyr414 of APB play important roles in enzymatic function and characteristic properties of APB via proper formation of the S1' site.

Endoplasmic Reticulum Aminopeptidase 1 beyond Antigenic Peptide-Processing Enzyme in the Endoplasmic Reticulum.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is well known as a processing enzyme of antigenic peptides, which are presented to major histocompatibility complex (MHC) class I molecules in the lumen of endoplasmic reticulum. Besides antigen processing, ERAP1 performs multiple functions in various cells depending on its intracellular and extracellular localization. Of note is the secretion of ERAP1 into the extracellular milieu in response to inflammatory stimuli, which further activates immune cells including macrophages and natural killer cells. Furthermore, secreted ERAP1 enhances the expression of pro-inflammatory cytokines like tumor necrosis factor-α, interleukin-1ß, and interleukin-6. Such findings indicate that ERAP1 plays a significant role in the field of innate and acquired immunity. This review summarizes the functional analyses of ERAP1 that support our current understanding of its role as more than an antigenic peptide-processing enzyme, specifically emphasizing on its secretory form.

Poly(4-vinylpyridine) based novel stationary phase investigated under supercritical fluid chromatography conditions.

A novel poly(4-vinylpyridine) based stationary phase was investigated for its performance under supercritical fluid chromatography (SFC) mode. Due to its unique structure, this stationary phase has high molecular planarity recognition ability for aromatic samples possessing the same number of aromatic rings and π-electrons. Taking advantage of the planarity recognition ability observed, separations of structurally similar polycyclic aromatic hydrocarbons and steroids were achieved. This novel stationary phase afforded good peak symmetry for both acidic and basic active pharmaceutical ingredients even when excluding the use of additives such as acids, bases, and salts. These findings may be attributed to the polymeric pyridyl groups covalently-attached on silica gel, which will effectively shield the undesirable interaction between residual silanol groups on the surface and the analytes. Moreover, the properties of pyridyl group on the selector can be reversibly tuned to cationic pyridinium form by eluting trifluoroacetic acid containing modifier. Column robustness toward cycle durability testing was also confirmed.

Twelve-year trends of increasing overweight and obesity in patients with diabetes: the Shiga Diabetes Clinical Survey.

The prevalence of obesity is increasing globally in patients with diabetes. This study aimed to examine 12-year trends of increasing obesity in Japanese patients with diabetes, and their clinical features. The study used results of the Shiga Diabetes Clinical Survey, which recorded medical performance in diabetic patients in 2000, 2006 and 2012. Data were analyzed from 14,205, 14,407 and 21,449 adult patients in these three years, respectively. Overweight and obesity prevalence and the clinical features of diabetes patients were examined, stratified by body mass index (BMI) and age. The prevalence of overweight (BMI 25-30 kg/m2) and obesity (BMI ≥30 kg/m2) were 27.0% and 5.1% in 2000, 28.9% and 7.3% in 2006 and 30.9% and 10.0% in 2012. Glycemic control, blood pressure and serum lipid profile improved over 12 years in all BMI categories. However, glycemic and triglyceride control were insufficient in obese patients aged <65 years (hemoglobin A1c 7.5 ± 1.4%, triglyceride 197.7 ± 178.4 mg/dL in 2012). The percentage of patients who used antihypertensive and lipid-lowering drugs increased and patients with higher BMI had increased frequency of using these drugs, both in young and old age groups. Higher BMI was significantly and positively associated with albuminuria. In summary, overweight and obesity have increased in Japanese diabetic patients, particularly for younger generations. Findings suggest that obesity may lead to poorer glycemic control, blood pressure and lipid profiles. Overweight and obesity are important modifiable risk factors for diabetes, suggesting that more active weight-control interventions are warranted.

Poly(butylene terephthalate) based novel achiral stationary phase investigated under supercritical fluid chromatography conditions.

Poly(butylene terephthalate) based novel stationary phase (SP), composed of planar aromatic phenyl group together with ester group monomer units, was designed for supercritical fluid chromatography (SFC) use. As expected from its structure, this phase shows planarity recognition of isomeric aromatics and closely similar compounds. Interestingly, for most analytes, the retention behavior of this SP is significantly distinct from that of the 2-ethylpyridine based SPs which is among the most well-known SFC dedicated phases. Although the poly(butylene terephthalate) is coated on silica gel, the performance of the column did not change by using extended range modifiers such as THF, dichloromethane or ethyl acetate and column robustness was confirmed by cycle durability testing.

Achiral Molecular Recognition of Aromatic Position Isomers by Polysaccharide-Based CSPs in Relation to Chiral Recognition.

Chromatographic separation of several sets of aromatic position isomers on three cellulose- and one amylose-based chiral stationary phases was performed to evaluate the potential of a polysaccharide-based chiral stationary phase (CSP) in the separation of isomeric or closely similar molecules, and to understand the interaction mechanism of this type of CSP with analytes. Their ability of molecular recognition was quite outstanding, but the selection rule was particular to each polysaccharide derivative. In the series of analytes, cellulose tris(4-methylbenzoate) and tris(3,5-dimethylphenylcarbamate) exhibited a contrasting selection rule, and the recognition mechanism was considered based on the computer-simulation of the former polymer.

Involvement of Phenylalanine 297 in the Construction of the Substrate Pocket of Human Aminopeptidase B.

Aminopeptidase B (APB, EC 3.4.11.6) preferentially hydrolyzes the N-terminal basic amino acids of synthetic and peptide substrates and requires a physiological concentration of NaCl for optimal activity. In this study, we used site-directed mutagenesis and molecular modeling to search for an amino acid residue that is critical for the enzymatic properties of human APB. Substitution of Phe297 with Tyr caused a significant decrease in hydrolytic activity toward synthetic and peptide substrates as well as chloride anion sensitivity. Molecular modeling suggests that Phe297 contributes to the construction of the substrate pocket of APB, which is wide enough to hold a chloride anion and allow the interaction of Gln169 with the N-terminal Arg residue of the substrate through bridging with the chloride anion. These results indicate that Phe297 is crucial for the optimal enzymatic activity and chloride anion sensitivity of APB via formation of the optimal structure of the catalytic pocket.

Role of glutamine-169 in the substrate recognition of human aminopeptidase B.

BACKGROUND: Aminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB. METHODS: Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency. RESULTS: Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC50 values of inhibitors that interact with the catalytic pocket of APB. The EC50 value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion. CONCLUSION: Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.

Complementary enantiorecognition patterns and specific method optimization aspects on immobilized polysaccharide-derived chiral stationary phases.

The immobilized polysaccharide-derived chiral stationary phases (CSPs) combine the benefits of broad application scope and high preparative potential with CSP robustness and universal solvent compatibility. Strategies for efficient and straightforward method development with these CSPs were previously overviewed. In the current study, six CSPs with different structural features in the immobilized series were examined for their complementary properties in separation of enantiomers. Some method development aspects related to polysaccharide-derived CSPs in general and certain specific features observed on these immobilized supports, such as the effects of mobile phase, sample solvent and mobile phase additive on chiral separation, are also discussed.

Characteristics common to a cytokine family spanning five orders of insects.

Growth-blocking peptide (GBP) is a member of an insect cytokine family with diverse functions including growth and immunity controls. Members of this cytokine family have been reported in 15 species of Lepidoptera, and we have recently identified GBP-like peptides in Diptera such as Lucilia cuprina and Drosophila melanogaster, indicating that this peptide family is not specific to Lepidoptera. In order to extend our knowledge of this peptide family, we purified the same family peptide from one of the tenebrionids, Zophobas atratus,(1) isolated its cDNA, and sequenced it. The Z. atratus GBP sequence together with reported sequence data of peptides from the same family enabled us to perform BLAST searches against EST and genome databases of several insect species including Coleoptera, Diptera, Hymenoptera, and Hemiptera and identify homologous peptide genes. Here we report conserved structural features in these sequence data. They consist of 19-30 amino acid residues encoded at the C terminus of a 73-152 amino acid precursor and contain the motif C-x(2)-G-x(4,6)-G-x(1,2)-C-[KR], which shares a certain similarity with the motif in the mammalian EGF peptide family. These data indicate that these small cytokines belonging to one family are present in at least five insect orders.

Drosophila growth-blocking peptide-like factor mediates acute immune reactions during infectious and non-infectious stress.

Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. We demonstrated that growth-blocking peptide (GBP) is a potent cytokine that regulates stressor-induced AMP expression in insects.GBP overexpression in Drosophila elevated expression of AMPs.GBP-induced AMP expression did not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket were essential,indicating that GBP signaling in Drosophila did not use the orthodox Toll or Imd pathway but used the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors was apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious.

Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1 (BmLsd1).

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via the fatty acid biosynthetic pathway. This pathway is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets, which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells. PBAN stimulates lipolysis of the stored lipid droplet triacylglycerols (TAGs) and releases the precursor for final modification. PBAN exerts its physiological function via the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family. The PBAN receptor-mediated signal is transmitted via a canonical store-operated channel activation pathway utilizing Gq-mediated phospholipase C activation (Hull, J. J., Kajigaya, R., Imai, K., and Matsumoto, S. (2007) Biosci. Biotechnol. Biochem. 71, 1993-2001; Hull, J. J., Lee, J. M., Kajigaya, R., and Matsumoto, S. (2009) J. Biol. Chem. 284, 31200-31213; Hull, J. J., Lee, J. M., and Matsumoto, S. (2010) Insect Mol. Biol. 19, 553-566). Little, however, is known about the molecular components regulating TAG lipolysis in PG cells. In the current study we found that PBAN signaling involves phosphorylation of an insect PAT family protein named B. mori lipid storage droplet protein-1 (BmLsd1) and that BmLsd1 plays an essential role in the TAG lipolysis associated with bombykol production. Unlike mammalian PAT family perilipins, however, BmLsd1 activation is dependent on phosphorylation by B. mori Ca(2+)/calmodulin-dependent protein kinase II rather than protein kinase A.

Studies of sex pheromone production under neuroendocrine control by analytical and morphological means in the oriental armyworm, Pseudaletia separata, Walker (Lepidoptera: Noctuidae).

Most female moths produce species-specific sex pheromone blends in the modified epidermal pheromone gland (PG) cells generally located between the 8 and 9th abdominal segments. The biosynthesis is often regulated by pheromone biosynthesis activating neuropeptide (PBAN) either in or prior to de novo fatty acid synthesis or at the formation of oxygenated functional group. In Pseudaletia separata, information about life span, calling, PG morphology, daily fluctuation of pheromone production and its hormonal regulation is limited. We measured pheromone titer daily (16:8; L:D) at 2h intervals in scotophase. Blend ratio stabilized during the 2nd day (till 4-5th) at 6th hour of scotophase, with the ratio of 27.5:12.8:44.4:15.3 for Z-11-16OH:16OH:Z-11-16Ac:16Ac, respectively. Females showed calling behavior from this time. We found with light and fluorescence microscopy that PG consisted of intersegmental membrane (A part), and dorso-lateral region of 9th abdominal segment (B part), encountering for ∼ 35% of total production revealed by gas chromatography. Ratios did not reveal difference. We did not find precursor (triacylglycerols) accumulation in form of lipid droplets, implying that PBAN stimulates de novo biosynthesis of 16:acyl precursors. In vivoHez-PBAN injections (1-3 × 5 pmol, 2h intervals) into 3 days old 16-18 h decapitated females stimulated pheromone production, both in A and B parts. Blend analyses including ratios suggest stimulation of the initial phase of synthesis, but desaturation of fatty acyl intermediates do not follow proportionally. More saturated fatty acid is converted from the available pool to the final OH and Ac, compared to females kept intact in scotophase. In vitro studies (PGs incubated 4-6h in the presence of 0.25 or 0.5 µM Hez-PBAN, especially with surplus 2mM malonyl-CoA) revealed higher saturated component ratio than the unsaturated, compared to natural blend or in vivo injections.

Screening for the Genes Involved in Bombykol Biosynthesis: Identification and Functional Characterization of Bombyx mori Acyl Carrier Protein.

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via fatty acid synthesis (FAS). Biosynthesis of moth sex pheromones is usually regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-aa peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets (LDs), which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells prior to eclosion. PBAN activation of the PBAN receptor stimulates lipolysis of the stored LD triacylglycerols (TAGs) resulting in release of the bombykol precursor for final modification. While we have previously characterized a number of molecules involved in bombykol biosynthesis, little is known about the mechanisms of PBAN signaling that regulate the TAG lipolysis in PG cells. In the current study, we sought to further identify genes involved in bombykol biosynthesis as well as PBAN signaling, by using a subset of 312 expressed-sequence tag (EST) clones that are in either our B. mori PG cDNA library or the public B. mori EST databases, SilkBase and CYBERGATE, and which are preferentially expressed in the PG. Using RT-PCR expression analysis and an RNAi screening approach, we have identified another eight EST clones involved in bombykol biosynthesis. Furthermore, we have determined the functional role of a clone designated BmACP that encodes B. mori acyl carrier protein (ACP). Our results indicate that BmACP plays an essential role in the biosynthesis of the bombykol precursor fatty acid via the canonical FAS pathway during pheromonogenesis.