Applications of Plant-Made Fibroblast Growth Factor for Human Pluripotent Stem Cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold great potential in regenerative medicine. These cells can be expanded indefinitely in theory and are able to differentiate into different types of cells for cell therapies, drug screening, and basic biology studies. The reliable and effective propagation of hESCs and hiPSCs is important for their downstream applications. Basic fibroblast growth factor (bFGF) is critical to hESCs and hiPSCs for maintaining their pluripotency. Plant-produced growth factors are safe to use without potential contamination of infectious viruses and are less expensive to produce. In this study, we used rice cell-made basic fibroblast growth factor (RbFGF) to propagate hESCs and hiPSCs for at least eight passages. Both hESCs and hiPSCs cultured with RbFGF not only maintained the morphology but also the specific expression (OCT4, SSEA4, SOX2, and TRA-1-60) of PSCs, similar to those cultured with the commercial Escherichia coli-produced bFGF. Furthermore, both gene chip-based PluriTest and TaqMan hPSC Scorecard pluripotency analysis demonstrated the pluripotent expression profile of the hESCs cultured with RbFGF. In vitro trilineage assays further showed that these hESCs and hiPSCs cultured on RbFGF were capable of giving rise to cell derivatives of ectoderm, mesoderm, and endoderm, further demonstrating their pluripotency. Finally, chromosome stability was also maintained in hESCs cultured with RbFGF as demonstrated by normal karyotypes. This study suggests broad applications for plant-made growth factors in stem cell culture and regenerative medicine.

Detection of homogentisic acid by electrospray ionization mass spectrometry.

OBJECTIVE: Homogentisic acid (HGA) is excreted in excessive amounts in the urine of patients with alkaptonuria, which is a hereditary metabolic disorder of phenylalanine and tyrosine. Therefore, the detection of HGA in urine is useful for the diagnosis of alkaptonuria. To evaluate the detection of HGA, we confirmed the color shift of HGA solutions and analyzed them by electrospray ionization mass spectrometry (ESI-MS). METHODS: We observed the color change of the HGA solutions under different pH conditions (pH 6.0, 7.0, and 8.0) and examined the influences of adding potassium hydroxide (KOH) and ascorbic acid (AA) to the HGA solutions. Then, we analyzed the chemical reaction in HGA solutions using ESI-MS. RESULTS: The HGA solution at pH 8.0 became brown after incubation at room temperature for 24 h and became darker brown with the addition of KOH; however, HGA solutions at pH 6.0 and 7.0 showed no color changes. The brown color change of the HGA solution at pH 8.0 was also inhibited by AA. Moreover, all HGA sample solutions showed the deprotonated molecular ion peak at m/z 167.035 in the negative ion mode after incubation at room temperature for 24 h and with the addition of KOH and AA. CONCLUSION: We identified the molecular ion of HGA in all sample solutions by ESI-MS, regardless of different pH conditions, color changes, or the presence of AA. These results suggest that spectral analysis by ESI-MS is suitable for the detection of HGA and the diagnosis of alkaptonuria.

12,20-Epoxypregnane Glycosides from the Roots of Oxypetalum caeruleum.

The MeOH extract from dried roots of Oxypetalum caeruleum (Apocynaceae, formerly known as Asclepiadaceae) plants yielded twenty new pregnane glycosides, some of which had a new 12,20-epoxy type aglycone. The structures of these compounds were established using NMR, MS spectroscopic analysis and chemical evidence.

Synthesis of Dimethyldisilabicycloalkanes: Cage-Size Effects on the Relative Stabilities between In,Out and Twist-Out,Out Forms.

In large bicycloalkanes, several in/out forms exist, wherein substituents on the bridgehead atoms are oriented either outside or inside the cage. The relative stability between the in,out and twist-out,out forms, which can interconvert through homeomorphic conversion, was found to depend upon the cage size. The in,out form demonstrated thermodynamic stability in the smaller C10 derivative, whereas the twist-out,out form prevailed in the larger derivatives of C14 and C18 plausibly as a result of dispersion forces among the chains.

Isolation and Reactions of Imidoyl Fluorides Generated from Oxime Using the Diethylaminosulfur Trifluoride/Tetrahydrofuran (DAST-THF) System.

Fluorination of oximes with the relatively mild diethylaminosulfur trifluoride/tetrahydrofuran (DAST-THF) system affords imidoyl fluorides. These compounds were isolated, and their structures were confirmed by X-ray single-crystal structure analysis. Reaction of imidoyl fluorides with various nucleophiles efficiently afforded amides, amidines, thioamides, and amine derivatives in high yields. Furthermore, one-pot reaction of in situ generated imidoyl fluorides from oximes was also applicable to efficient synthesis of these products. The oxime stereochemistry and acid-labile protecting group remained intact in this system.

N-acetylglutamic acid alleviates oxidative stress based on histone acetylation in plants.

Oxidative stress causes cellular damage and genomic instability through the accumulation of reactive oxygen species (ROS) in plants, resulting in reduced crop production. Chemical priming, which can enhance plant tolerance to environmental stress using functional chemical compounds, is expected to improve agricultural yield in various plants without genetic engineering. In the present study, we revealed that non-proteogenic amino acid N-acetylglutamic acid (NAG) can alleviate oxidative stress damage in Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice). Exogenous treatment with NAG prevented chlorophyll reduction induced by oxidative stress. The expression levels of ZAT10 and ZAT12, which are regarded as master transcriptional regulators in response to oxidative stress, increased following NAG treatment. Additionally, Arabidopsis plants treated with NAG showed enhanced levels of histone H4 acetylation at ZAT10 and ZAT12 with the induction of histone acetyltransferases HAC1 and HAC12. The results suggest that NAG could enhance tolerance to oxidative stress through epigenetic modifications and contribute to the improvement of crop production in a wide variety of plants under environmental stress.

A spectrophotometric method for the determination of tryptophan following oxidation by the addition of sodium hypochlorite pentahydrate.

Tryptophan (Trp) is an essential amino acid that functions in various biological processes and human daily health. As the significant functions of Trp become more apparent, its measurement is becoming increasingly important in various situations. Herein, we improved the Trp color reaction based on the Hopkins-Cole reaction and established a simple colorimetric method for Trp determination using several different reagents, including sodium hypochlorite pentahydrate and monosodium glutamate. The detection method can be performed using safe materials, rather than conventional toxic substances, and induces a crimson color change with an absorption peak at 525 nm, enabling the quantification of Trp by simple spectrophotometry in just 10 min. This assay exhibited a linear detection range from 10 to 100 mg/L (R2 = 0.9996). The average recoveries in the spiked cerebrospinal fluid ranged from 90.5% to 104.3%, with a relative standard deviation of 0.27% (n = 3, 29.40 mg/L Trp) to 1.19% (n = 3, 72.90 mg/L Trp). This novel spectrophotometric method may enable many researchers and laboratory technicians to detect Trp in various sample solutions without expensive analytical instruments or complicated operations.

Synthesis and rotational dynamics of diazamacrocycles having bridged 1,4-naphthylene as framed molecular rotors.

The rotation of a part of a molecule has attracted attention because of its possible role in the development of an artificial molecular rotor. In this study, 1,4-naphthylene bridged diazamacrocycles, in which the 1,4-diaminonaphthalene moiety is bridged by two long alkyl chains, were designed as novel framed molecular rotors, and the dependence of the rotation on the frame size were investigated. Framed rotors CnNp (n = 12, 14, 16, 18), where the number in the compound name indicates the length of a side chain, were synthesized by direct cyclization of 1,4-naphthalenediamine with α,ω-dihaloalkane in the presence of Na2CO3 as the base. The rotation of the naphthylene rotor in C12Np, C14Np, and C16Np was nearly suppressed in solution, whereas the rotor in C18Np showed rotation, as confirmed by the temperature-dependent coalescence of the NMR signals of the α-CH2.

C21 Steroidal Glycosides from the Roots of Oxypetalum caeruleum.

The MeOH extract from dried roots of Oxypetalum caeruleum (Apocynaceae) plants yielded seventeen new pregnane glycosides, some of which had the acylated-ramanone or -isoramanone type aglycone. The structures of these compounds were established using NMR, MS spectroscopic analysis and chemical evidence.

Analysis of nucleotide insertion opposite urea and translesion synthesis across urea by DNA polymerases.

Urea (Ua) is produced in DNA as the result of oxidative damage to thymine and guanine. It was previously reported that Klenow fragment (Kf) exo- incorporated dATP opposite Ua, and that DNA polymerase ß was blocked by Ua. We report here the following nucleotide incorporations opposite Ua by various DNA polymerases: DNA polymerase α, dATP and dGTP (dATP > dGTP); DNA polymerase δ, dATP; DNA polymerase ζ, dATP; Kf exo-, dATP; Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), dGTP and dATP (dGTP > dATP); and DNA polymerase η, dCTP, dGTP, dATP, and dTTP (dCTP > dGTP > dATP > dTTP). DNA polymerases ß and ε were blocked by Ua. Elongation by DNA polymerases δ and ζ stopped after inserting dATP opposite Ua. Importantly, the elongation efficiency to full-length beyond Ua using DNA polymerase η and Dpo4 were almost the same as that of natural DNA.

Combined Analysis Based on a Crystalline Sponge Method.

The crystalline sponge (CS) method was developed as an X-ray crystallographic molecular structure analysis method that can be performed without the need for crystallization of the analyte. CS has strong molecular-recognition properties and a highly flexible framework. The amount of analyte can be reduced to a sub-milligram level. These features of the crystalline nano-space allow for determining the absolute structure of a trace analyte. In this review, we focus on the discovery of the CS method and its applications to biosynthetic products in combination with NMR spectroscopy. We also describe some examples of the CS method that are used mainly in combination with mass spectrometry (MS). Both approaches demonstrate the potential of microanalysis to determine the molecular structure of an unknown sample. Finally, we mention the use of a crystalline "nano-surface" rather than a crystalline nano-space in MS, which can detect small metabolites as well as post-translation biomolecules.

Preparation of cage-shaped hexakis(spiroborate)s.

In previous research studies, various types of prismatic cage-shaped molecular containers have been prepared and evaluated in terms of their guest inclusion properties. Basically, most of these molecular cages have a cationic or electron-deficient nature, and exhibit strong affinity mainly toward electron-rich aromatic guests. On the other hand, there is no report concerning anionic prismatic cages that are expected to recognize cationic polyaromatic guests with various structures and functions. In this manuscript, we present the preparation of hexakis(spiroborate)-type molecular cages, which was achieved by the reaction of phenylene- or biphenylenebis(dihydroxynaphthalene), hexahydroxytriphenylene, and boric acid in N,N-dimethylformamide. Their triangular prismatic hollow structures were confirmed by X-ray crystallographic analysis, and it was found that both phenylene- and biphenylene-bridged spiroborate cages have internal cavities of the corresponding size. It was also revealed that tetra(n-butylammonium) cations located inside the cavity and between the two adjacent spiroborate cages resulted in the formation of a one-dimensional columnar array. The molecular recognition behavior of the spiroborate cages was evaluated using tris(pyridinium)triazines as tricationic aromatic guests. 1H NMR measurement implied that a discrete 1 : 1 host-guest complex was formed when 1 equiv. of guest was added to the cage, whereas infinite one-dimensional aromatic stacks were constructed by the addition of 2 equiv. of guest.

Formation of a Ruthenium(V)-Imido Complex and the Reactivity in Substrate Oxidation in Water through the Nitrogen Non-Rebound Mechanism.

A RuII-NH3 complex, 2, was oxidized through a proton-coupled electron transfer (PCET) mechanism with a CeIV complex in water at pH 2.5 to generate a RuV═NH complex, 5. Complex 5 was characterized with various spectroscopies, and the spin state was determined by the Evans method to be S = 1/2. The reactivity of 5 in substrate C-H oxidation was scrutinized in acidic water, using water-soluble organic substrates such as sodium ethylbenzene-sulfonate (EBS), which gave the corresponding 1-phenylethanol derivative as the product. In the substrate oxidation, complex 5 was converted to the corresponding RuIII-NH3 complex, 3. The formation of 1-phenylethanol derivative from EBS and that of 3 indicate that complex 5 as the oxidant does not perform nitrogen-atom transfer, in sharp contrast to other high-valent metal-imido complexes reported so far. Oxidation of cyclobutanol by 5 afforded only cyclobutanone as the product, indicating that the substrate oxidation by 5 proceeds through a hydride-transfer mechanism. In the kinetic analysis on the C-H oxidation, we observed kinetic isotope effects (KIEs) on the C-H oxidation with use of deuterated substrates and remarkably large solvent KIE (sKIE) in D2O. These positive KIEs indicate that the rate-determining step involves not only cleavage of the C-H bond of the substrate but also proton transfer from water molecules to 5. The unique hydride-transfer mechanism in the substrate oxidation by 5 is probably derived from the fact that the RuIV-NH2 complex (4) formed from 5 by 1e-/1H+ reduction is unstable and quickly disproportionates into 3 and 5.

Hollow and Solid Spheres Assembled from Functionalized Macrocycles Containing Adamantane.

An adamantane-based macrocycle possessing eight hydroxyl groups (1) was synthesized, in which the macrocyclic framework comprises two disubstituted adamantane molecules bearing phenyl derivatives connected to two biphenylene spacers by oxygen atoms. Furthermore, functionalized macrocycles containing methyl (2) and methoxycarbonylmethyl (3) groups were prepared. From the X-ray crystallographic analysis, the backbone of the macrocycles in all crystals had a nearly hexagonal shape with a cavity and these macrocycles could be arranged into different tubular structures dependent on the substituents. In acetone, macrocycle (1) formed stable hollow spherical aggregates with multilayer membranes. In contrast, macrocycle (3) exhibited no production of self-assembled materials in chloroform. The addition of hexane into the solution caused the generation of solid spheres and their fused network aggregates, which were finally transformed into crystals owing to the solvent effects.

Crystalline sponge-laser desorption ionization (CS-LDI) of unsaturated cyclic organic compounds encapsulated in different electronic environments in pores.

We have developed laser desorption ionization mass spectrometry (LDI-MS) of organic molecules encapsulated in a crystalline sponge (CS). Cyclic organic compounds 1,2,3,4,5-pentamethylcyclopentadiene, zerumbone, and muscone were encapsulated in CS as guest regardless of guest crystallization. Single-crystal X-ray analysis closely scrutinized the position of the guest in the pore and the interaction between the guest and the CS framework. After single-crystal X-ray analysis, the same single crystal was subjected to LDI-MS. Ionization efficiency differed markedly depending on whether π-π interaction existed or not. It is obvious that π-π interaction is the key to transferring laser energy from the CS framework to the guest for the ionization of molecule encapsulated in CS. The results suggest the importance of controlling the positions of analyte and matrix as well as noncovalent interactions, such as hydrogen bonding interaction, electrostatic interaction, and so on.

ß-Eudesmol, an Oxygenized Sesquiterpene, Reduces the Increase in Saliva 3-Methoxy-4-Hydroxyphenylglycol After the "Trier Social Stress Test" in Healthy Humans: A Randomized, Double-Blind, Placebo-Controlled Cross-Over Study.

Hops, the immature inflorescences of the female hop plant (Humulus lupulus L.) are one of the main components of beer and provides flavor and bitterness. ß-Eudesmol, an oxygenated sesquiterpene, is reported to accumulate in a particular hop cultivar. Recently, we revealed that ß-Eudesmol ingestion affected autonomic nerve activity in an animal model. The effect on humans has not been elucidated, therefore, we investigated the effects of ß-Eudesmol on reducing objective and subjective markers related to sympathetic nerve activity after the application of mental stress in healthy participants. Fifty participants (male and female aged 20 to 50 years) were randomly assigned to two groups. Five minutes before taking the Trier Social Stress Test (TSST) as a mental stressor, participants in each group ingested a beverage containing ß-Eudesmol, the active beverage, or a placebo beverage that did not contain ß-Eudesmol. Saliva 3-methoxy-4-hydroxyphenylglycol (MHPG), a major product of noradrenaline breakdown and a representative marker of sympathetic nerve activity, was significantly lower just after the TSST in the active group compared with the placebo group. Saliva cortisol, a marker of the endocrine stress response system, was not significantly different between the two groups. No adverse events related to test beverage ingestion were observed. This is the first experimental evidence of ß-Eudesmol effect for mental stress in human.

ß-eudesmol, an oxygenized sesquiterpene, affects efferent adrenal sympathetic nerve activity via transient receptor potential ankyrin 1 in rats.

The autonomic nervous system innervates various peripheral tissue functions. Various external stimuli affect autonomic nerve activity, however, there is little information about the involvement of sensory receptors in the responses. The TRPA1 is a calcium-permeable non-selective cation channel which plays a crucial role in the susceptibility to various stimuli. ß-Eudesmol, an oxygenated sesquiterpene found in hop essential oil and beer, activates the TRPA1. Intragastric administration of ß-eudesmol decreased efferent adrenal sympathetic nerve activity (ASNA) in rats, whereas subcutaneous administration did not. ASNA suppression by ß-eudesmol was not observed in TRPA1 knockout rats. The ß-eudesmol derived ASNA suppression was partially, but significantly, eliminated by subdiaphragmatic vagotomy in rats, suggesting the afferent vagal nerve from the gastrointestinal tract to the brain is involved in the effect of ß-eudesmol on ASNA. Our results indicate that ß-eudesmol suppresses ASNA, partly through TRPA1 and the afferent vagus nerve. These findings introduce the physiological significance of the TRPA1 in the control of ASNA.

Control over multiple molecular states with directional changes driven by molecular recognition.

Recently, ligand-metal coordination, stimuli-responsive covalent bonds, and mechanically interlinked molecular constructs have been used to create systems with a large number of accessible structural states. However, accessing a multiplicity of states in sequence from more than one direction and doing so without the need for external energetic inputs remain as unmet challenges, as does the use of relatively weak noncovalent interactions to stabilize the underlying forms. Here we report a system based on a bispyridine-substituted calix[4]pyrrole that allows access to six different discrete states with directional control via the combined use of metal-based self-assembly and molecular recognition. Switching can be induced by the selective addition or removal of appropriately chosen ionic guests. No light or redox changes are required. The tunable nature of the system has been established through a combination of spectroscopic techniques and single crystal X-ray diffraction analyses. The findings illustrate a new approach to creating information-rich functional materials.

Combined analysis of 1,3-benzodioxoles by crystalline sponge X-ray crystallography and laser desorption ionization mass spectrometry.

The crystalline sponge (CS) method, which employs single-crystal X-ray diffraction to determine the structure of an analyte present as a liquid or an oil and having a low melting point, was used in combination with laser desorption ionization mass spectrometry (LDI-MS). 1,3-Benzodioxole derivatives were encapsulated in CS and their structures were determined by combining X-ray crystallography and MS. After the X-ray analysis, the CS was subjected to imaging mass spectrometry (IMS) with an LDI spiral-time-of-flight mass spectrometer (TOF-MS). The ion detection area matched the microscopic image of the encapsulated CS. In addition, the accumulated 1D mass spectra showed that fragmentation of the guest molecule (hereafter, guest) can be easily visualized without any interference from the fragment ions of CS except for two strong ion peaks derived from the tridentate ligand TPT (2,4,6-tris(4-pyridyl)-1,3,5-triazine) of the CS and its fragment. X-ray analysis clearly showed the presence of the guest as well as the π-π, CH-halogen, and CH-O interactions between the guest and the CS framework. However, some guests remained randomly diffused in the nanopores of CS. In addition, the detection limit was less than sub-pmol order based on the weight and density of CS determined by X-ray analysis. Spectroscopic data, such as UV-vis and NMR, also supported the encapsulation of the guest through the interaction between the guest and CS components. The results denote that the CS-LDI-MS method, which combines CS, X-ray analysis and LDI-MS, is effective for structure determination.

ß-Eudesmol, an oxygenized sesquiterpene, stimulates appetite via TRPA1 and the autonomic nervous system.

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel, which is activated by various noxious or irritant substances in nature. TRPA1 activators have been generally recognized as noxious, however, foods and beverages containing TRPA1 activators are preferably consumed; the reasons for this discrepancy are not well understood. We demonstrate that TRPA1 is involved in the stimulatory appetite control mechanism. ß-Eudesmol is an oxygenated sesquiterpene contained in medicinal or edible plants which activates TRPA1. Oral administration of ß-eudesmol brought significant increments in food intake in rats and elevated plasma ghrelin levels. Gastric vagal nerve activity (GVNA) has been reported to affect feeding behavior. In vivo electrophysiological measurement of GVNA revealed that oral-ingestion of ß-eudesmol significantly increased GVNA. This GVNA elevation was eliminated by TRPA1 inhibitor (HC-030031) treatment prior to ß-eudesmol administration. The physiological effects of ß-eudesmol, for example, incremental increase in food intake, ghrelin elevation and activation of GVNA, were significantly reduced in TRPA1 knockout rats. Our results indicated that ß-eudesmol stimulates an increase in appetite through TRPA1, and suggests why TRPA1 activator containing foods and beverages are preferably consumed.