Assessment of ESR-CT imaging by comparison with autoradiography for the distribution of a blood-brain-barrier permeable spin probe, MC-PROXYL, to rodent brain

Blood-brain-barrier (BBB)-permeable, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MC-PROXYL) and BBB-impermeable carbamoyl-PROXYL were used to assess the ESR imaging technique by comparing with autoradiography. For this purpose, spin probes, 14C-labeled at their five-membered ring, [14C]MC-PROXYL and [14C]carbamoyl-PROXYL, were newly synthesized. These probes were i.p. or i.v. injected into rats and autoradiograms were recorded. The autoradiograms of rat head showed that [14C]MC-PROXYL distributed well in the brain compared to [14C]carbamoyl-PROXYL. In vivo ESR spectra and two-dimensional ESR images of isolated rat brain treated with MC- or carbamoyl-PROXYL also indicated the extensive distribution of MC-PROXYL but not carbamoyl-PROXYL in the rat brain. The three-dimensional ESR images of the head of rats and mice were consistent with the fact that MC-PROXYL but not carbamoyl-PROXYL is incorporated into the brain. The ESR-CT images were better for mice than rats. However, the quality of the ESR-CT images was still not satisfactory. Although the resolution and sensitivity of the ESR-CT images were worse than those of the autoradiographic images, the former technique has unique features and advantages; e.g., functional, noninvasive and three-dimensional detection.     

Supramolecular Chemistry of Cyclodextrin-Peptide Hybrids: Azobenzene-Tagged Peptides

AC17, which is composed of 17 amino acids and has an azobenzene moiety but has no cyclodextrin (CD) unit in the side chain, exhibits 54% helix content. However, AC17, which has both trans-azobenzene and -CD, shows 82% helix content. This result suggests that the helix structure is stabilized by host (CD)-guest (azobenzene) bridge in the side chain of the peptide. The helix content changed by trans-cis photoisomerization as shown by 64% helix content for AC17 in its cis form. This result suggests that cis-azobenzene unit is excluded from the -CD cavity, thus resulting in the smaller helix content. The helix contents for AC17, which has both azobenzene and -CD, are 94% in the cis form and 87% in the trans form, suggesting that the cis form is included in the -CD cavity. Azobenzene-tagged CD-peptide hybrids with histidine unit were also prepared and photoregulation of catalytic activity in ester hydrolysis was examined.     

Synthesis of methylcellulose model copolymers with heterogeneous distribution and their solution properties

In order to elucidate the characteristic features of commercial methylcellulose precisely, O-methylcellulose model copolymers consisting of 2,3,6-tri-O-methylanhydroglucose unit (236MeAGU) and 2-O-methylanhydroglucose unit (2MeAGU) with various composition ratios were synthesized via cationic ring-opening copolymerization of the corresponding glucose orthoester derivatives, subsequent removal of pivaloyl and allyl groups, and methylation. The structure of the obtained copolymers was confirmed by ¹H-, ¹³C-NMR, and FT-IR. Temperature-dependent turbidity measurement verified their thermoresponsive behavior in aqueous solution. The lower critical solution temperature was tuned from 63 to 45 °C above 47 mol-% 236MeAGU content. The hydrophobicity along the cellulose chain was dominant to determine their physical properties. However, the aqueous properties of the MC model copolymers were strongly affected by the slight difference of the composition ratio. The present method would provide further details of the structure–property relationship of O-methylcellulose.     

3-Nitro-l,2,4-triazol-l-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as a condensing reagent for solid-phase peptide synthesis

Solid-phase oligopeptide synthesis has been well developed and most short oligopeptides can now be easily synthesized. However, when a desired oligopeptide forms a secondary structure or includes less reactive amino acids such as aminoisobutyric acid, its terminal amino groups become less reactive and synthesis of the desired oligopeptides becomes difficult. To expand the number of synthetic peptide sequences, we have developed efficient coupling conditions using 3-nitro-l,2,4-triazol-l-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as a highly reactive condensing reagent on an unswellable solid support. PyNTP demonstrated higher reactivity than conventional condensing reagents and the optical purity of the synthesized oligopeptides was sufficiently high for application to general oligopeptide synthesis.     

Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain

Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure‐based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N‐abietoyl‐l ‐tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild‐type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near‐atomic resolution of 1.22 Å. Using these crystals as seeds in a cross‐microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem‐domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.     

Development of exposure assessment method based on the analysis of urinary heterocyclic amines as biomarkers by on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry

Heterocyclic amines (HCAs) formed in cooked meats and fish are mutagens and carcinogens in rodents and nonprimates. Exposure to HCAs may also be a risk factor for human tumors, but the association between dietary intake and human cancer risk has not been determined. To assess recent exposure to HCAs, we developed a simple and sensitive method for measuring HCAs in urine by automated on-line in-tube solid-phase microextraction (SPME) using a Supel-Q PLOT capillary column as an extraction device, in combination with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Thirteen HCAs were separated within 15 min using a ZORBAX Eclipse XDB-C8 column and detected selectively by multiple reaction monitoring using MS/MS. This method can be applied easily to the analysis of small amounts of urine samples without any other pretreatment except for alkaline hydrolysis of bound forms of HCAs. The quantification limits of HCAs in 0.2 mL of urine samples were about 1.7–4.1 pg/mL (S/N?=?10). Using this method, we evaluated the exposure to HCAs in persons who consumed well-done pan-fried beef and the suitability of using urinary HCAs as exposure biomarkers. We also analyzed the ability of vegetable consumption to prevent carcinogenic risks from exposure to HCAs by measuring free and bound forms of HCAs in urine.

Prediction of the layered ink layout for 3D printers considering a desired skin color and line spread function

Optical Review - We propose a layout estimation method for multi-layered ink using a measurement of the line spread function (LSF) and machine learning. The three-dimensional printing market for...