The effect of fluence rate and wavelength on the formation of protoporphyrin IX photoproducts.

Photodynamic diagnosis and therapy (PDD and PDT) are emerging techniques for diagnosing and treating tumors and malignant diseases. Photoproducts of protoporphyrin IX (PpIX) used in PDD and PDT may be used in the diagnosis and treatment, making a detailed analysis of the photoproduct formation under various treatment and diagnosis conditions important.Spectroscopic and mass spectrometric analysis of photoproduct formation from PpIX dissolved in dimethyl sulfoxide were performed under commonly used irradiation conditions for PDD and PDT, i.

A Data Set of Ion Mobility Collision Cross Sections and Liquid Chromatography Retention Times from 71 Pyridylaminated N-Linked Oligosaccharides.

Determination of the glycan structure is an essential step in understanding structure-function relationships of glycans and glycoconjugates including biopharmaceuticals. Mass spectrometry, because of its high sensitivity and mass resolution, is an excellent means of analyzing glycan structures. We previously proposed a method for rapid and precise identification of -glycan structures by ultraperformance liquid chromatography-connected ion mobility mass spectrometry (UPLC/IM-MS).

Expression of TAS2R14 in the intestinal endocrine cells of non-human primates.

Background: Recent studies have demonstrated that genes related to bitter taste receptors (TAS2Rs) on various chromosomes are expressed in extra-oral organs of various animals. The bitter taste receptor TAS2R14 is conserved among primate species and shows broad ligand sensitivity. Mice have a number of orthologues to primate TAS2R14 located in tandem on chromosome 16; however, their expression patterns are not unique.

Discovery of natural TRPA1 activators through pharmacophore-based virtual screening and a biological assay.

Transient receptor potential cation channel subfamily A member 1 (TRPA1), a member of the transient receptor potential family, detects a wide range of environmental stimuli, such as low temperature, abnormal pH, and reactive irritants. TRPA1 is of great interest as a target protein in fields related to pharmaceuticals and foods. In this study, a library of natural products was explored to identify TRPA1 activators by pharmacophore screening of known TRPA1 agonists and biological assays for agonist activity.

Hydrophobic interactions at subsite S1' of human dipeptidyl peptidase IV contribute significantly to the inhibitory effect of tripeptides.

Functional inhibitory peptides of human dipeptidyl peptidase 4 (hDPP4) have been highly anticipated as the active ingredient of functional food for type II diabetes; however, the molecular mechanism of hDPP4 inhibition remains unclear. In this study, we focused on dipeptides and tripeptides, which display structure-function correlations that are relatively easy to analyze, and examined their interactions with hDPP4 on an atomic level using a combination of docking studies and an hDPP4 inhibition assay. First, we performed comprehensive binding mode analysis of the dipeptide library and demonstrated that the formation of a tight interaction with the S1 subsite composing part of the substrate pocket is essential for dipeptides to compete with the substrate and strongly inhibit hDPP4.

Identification of a new class of non-electrophilic TRPA1 agonists by a structure-based virtual screening approach.

Recent work has gradually been clarifying the binding site of non-electrophilic agonists on the transient receptor potential A1 (TRPA1). This study searched for non-electrophilic TRPA1 agonists by means of in silico drug discovery techniques based on three-dimensional (3-D) protein structure. First, agonist-bound pocket structures were explored using an advanced molecular dynamics simulation starting from the cryo-electron microscopic structure of TRPA1, and several pocket structures suitable for virtual screening were extracted by structure evaluation using known non-electrophilic TRPA1 agonists.

Correction to: Cell-free synthesis of functionally active HSPB5.

In the original publication, the given name of the last author was incorrectly displayed as the name must read: Katsuyoshi Masuda.

Expression of Bitter Taste Receptors in the Intestinal Cells of Non-Human Primates.

(1) Background: Recent studies have investigated the expression of taste-related genes in the organs of various animals, including humans; however, data for additional taxa are needed to facilitate comparative analyses within and among species. (2) Methods: We investigated the expression of taste-related genes in the intestines of rhesus macaques, the non-human primates most commonly used in experimental models. (3) Results: Based on RNAseq and qRT-PCR, genes encoding bitter taste receptors and the G-protein gustducin were expressed in the gut of rhesus macaques.

Amino acid residues of bitter taste receptor TAS2R16 that determine sensitivity in primates to β-glycosides.

In mammals, bitter taste is mediated by TAS2Rs, which belong to the family of seven transmembrane G protein-coupled receptors. Since TAS2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species-specific diets during mammalian evolution. Here, we analyzed the amino acids responsible for the difference in sensitivities of TAS2R16s of various primates using a cultured cell expression system.

Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose.

Lactisole inhibits the glucose-sensing receptor T1R3 expressed in mouse pancreatic β-cells.

Glucose activates the glucose-sensing receptor T1R3 and facilitates its own metabolism in pancreatic β-cells. An inhibitor of this receptor would be helpful in elucidating the physiological function of the glucose-sensing receptor. The present study was conducted to examine whether or not lactisole can be used as an inhibitor of the glucose-sensing receptor.

Atmospheric pressure laser desorption/ionization using a 6-7 µm-band mid-infrared tunable laser and liquid water matrix.

Due to the characteristic absorption peaks in the IR region, various molecules can be used as a matrix for infrared matrix-assisted laser desorption/ionization (IR-MALDI). Especially in the 6-7 µm-band IR region, solvents used as the mobile phase for liquid chromatography have absorption peaks that correspond to their functional groups, such as O-H, C=O, and CH3. Additionally, atmospheric pressure (AP) IR-MALDI, which is applicable to liquid-state samples, is a promising technique to directly analyze untreated samples.

Alternative one-pot synthesis of (trifluoromethyl)phenyldiazirines from tosyloxime derivatives: application for new synthesis of optically pure diazirinylphenylalanines for photoaffinity labeling.

Alternative one-pot synthesis of 3-(trifluoromethyl)-3-phenyldiazirine derivatives from corresponding tosyloximes is developed. The deprotonation of intermediate diaziridine by NH2(-) is a new approach for construction of diazirine. Moreover, a novel synthesis of optically pure (trifluoromethyl)diazirinylphenylalanine derivatives was attempted involving these methods.

Hydrogen/deuterium exchange of cross-linkable α-amino acid derivatives in deuterated triflic acid.

In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable α-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives.

Continuous flow atmospheric pressure laser desorption/ionization using a 6-7-µm-band mid-infrared tunable laser for biomolecular mass spectrometry.

A continuous flow atmospheric pressure laser desorption/ionization technique using a porous stainless steel probe and a 6-7-µm-band mid-infrared tunable laser was developed. This ion source is capable of direct ionization from a continuous flow with a high temporal stability. The 6-7-µm wavelength region corresponds to the characteristic absorption bands of various molecular vibration modes, including O-H, C=O, CH3 and C-N bonds.

Elucidating the sequence of intact bioactive peptides by using electron capture dissociation and hot electron capture dissociation in a linear radio-frequency quadrupole ion trap.

Rationale: Electron capture dissociation (ECD) is useful tool for sequencing of peptides and proteins with post-translational modifications. To increase the sequence coverage for peptides and proteins, it is important to develop ECD device with high fragmentation efficiency.

Methods: Sequence analysis of intact undigested bioactive peptides (3000-5000 Da) was performed by use of electron capture dissociation (rf-ECD) and collision-induced dissociation (CID) in a linear radio-frequency quadrupole ion trap that was coupled to a time-of-flight mass spectrometer.

Comprehensive synthesis of photoreactive (3-trifluoromethyl)diazirinyl indole derivatives from 5- and 6- trifluoroacetylindoles for photoaffinity labeling.

5- and 6-trifluoromethyldiazirinyl indoles were synthesized from corresponding bromoindole derivatives for the first time. They acted as mother skeletons for the comprehensive synthesis of various bioactive indole metabolites. These can be used in biological functional analysis as diazirine-based photoaffinity labels.

Isolation and biochemical characterization of rubelase, a non-hemorrhagic elastase from Crotalus ruber ruber (Red Rattlesnake) venom.

A novel non-hemorrhagic basic metalloprotease, rubelase, was isolated from the venom of Crotalus ruber ruber. Rubelase hydrolyzes succinyl-L-alanyl-L-alanyl-L-alanyl p-nitroanilide (STANA), a specific substrate for elastase, and the hydrolytic activity was inhibited by chelating agents. It also hydrolyzes collagen and fibrinogen.

Identification and modulation of the key amino acid residue responsible for the pH sensitivity of neoculin, a taste-modifying protein.

Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity.

Development of a stigmatic mass microscope using laser desorption∕ionization and a multi-turn time-of-flight mass spectrometer.

A novel stigmatic mass microscope using laser desorption∕ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG, has been developed. Stigmatic ion images of crystal violet masked by a fine square mesh grid with a 12.7 μm pitch as well as microdot patterns with a 5 μm dot diameter and a 10 μm pitch made with rhodamine B were clearly observed.

Mutational deglycosylation of the Fc portion of immunoglobulin G causes O-sulfation of tyrosine adjacently preceding the originally glycosylated site.

Mutagenesis directed to a specific glycosylation site has been widely used to examine biological roles of individual glycans. However, occurrence of any post-translational modification on such deglycosylated mutants has not yet been well characterized. Here we performed mass spectrometric analyses of the Fc fragment of an unglycosylated mutant of mouse immunoglobulin G2b, whose conserved N-glycosylation site, i.

Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16.

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited.

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.

Background: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown.

Methods: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships.

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives.

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.