A survey of metabolic changes in potato leaves by NMR‐based metabolic profiling in relation to resistance to late blight disease under field conditions

Non‐targeted nuclear magnetic resonance (NMR)‐based metabolic profiling was applied to potato leaves to survey metabolic changes associated with late blight resistance under field conditions. Potato plants were grown in an experimental field, and the compound leaves with no visible symptoms were collected from 20 cultivars/lines at two sampling time points: (i) the time of initial presentation of symptoms in susceptible cultivars and (ii) 12 days before this initiation. ¹H NMR spectra of the foliar metabolites soluble in deuterium oxide‐ or methanol‐d₄‐based buffers were measured and used for multivariate analysis. Principal component analysis for six cultivars at symptom initiation showed a class separation corresponding to their levels of late blight resistance. This separation was primarily explained by higher levels of malic acid, methanol, and rutin and a lower level of sucrose in the resistant cultivars than in the susceptible ones. Partial least squares regression revealed that the levels of these metabolites were strongly associated with the disease severity measured in this study under field conditions. These associations were observed only for the leaves harvested at the symptom initiation stage, but not for those collected 12 days beforehand. Subsequently, a simple, alternative enzymatic assay for l ‐malic acid was used to estimate late blight resistance, as a model for applying the potential metabolic marker obtained. This study demonstrated the potential of metabolomics for field‐grown plants in combination with targeted methods for quantifying marker levels, moving towards marker‐assisted screening of new cultivars with durable late blight resistance. Copyright © 2016 John Wiley & Sons, Ltd.     

In situ observation of cell-detachment process initiated by femtosecond laser-induced stress wave

When a stress wave generated by focusing a femtosecond laser is loaded on an animal cell adhered on a substrate, the cell is detached from the substrate. There are two possible mechanisms for the cell detachment: (a) The cell is detached from a scaffold coated on a glass plate, and (b) the cell is detached from the glass plate with the scaffold. In this work, we have studied the cell-detachment mechanism by visualizing the scaffold with a fluorescence probe of quantum dots. When the cell was detached from the substrate, fluorescence from the scaffold simultaneously disappeared from the glass plate, although the scaffold was not irradiated by the laser. This indicates that detachment due to the stress wave is attributed to mechanism (a). On the other hand, when the cell was detached from the substrate by a trypsin treatment, the fluorescence from the scaffold remained, suggesting mechanism (b). By comparing both results, it is considered that physiological damage of the cell membrane during the detachment process by femtosecond laser-induced stress wave is less than that due to the trypsin treatment.     

Bioactive saponins and glycosides. XXVIII. New triterpene saponins, foliatheasaponins I, II, III, IV, and V, from Tencha (the leaves of Camellia sinensis)

New triterpene saponins, foliatheasaponins I-V, were isolated from the methanolic extract of Tencha [the leaves of Camellia sinensis (L.) O. KUNTZE (Theaceae)]. The chemical structures of these new saponins were elucidated on the basis of chemical and physicochemical evidence. Among the new saponins, foliatheasaponins II and III, were found to inhibit release of beta-hexosaminidase, as a marker of antigen-induced degranulation, in RBL-2H3 cells.     

Primary cultures of chick osteocytes retain functional gap junctions between osteocytes and between osteocytes and osteoblasts.

The inaccessibility of osteocytes due to their embedment in the calcified bone matrix in vivo has precluded direct demonstration that osteocytes use gap junctions as a means of intercellular communication. In this article, we report successfully isolating primary cultures of osteocytes from chick calvaria, and, using anti-connexin 43 immunocytochemistry, demonstrate gap junction distribution to be comparable to that found in vivo. Next, we demonstrate the functionality of the gap junctions by (1) dye coupling studies that showed the spread of microinjected Lucifer Yellow from osteoblast to osteocyte and between adjacent osteocytes and (2) analysis of fluorescence replacement after photobleaching (FRAP), in which photobleaching of cells loaded with a membrane-permeable dye resulted in rapid recovery of fluorescence into the photobleached osteocyte, within 5 min postbleaching. This FRAP effect did not occur when cells were treated with a gap junction blocker (18alpha-glycyrrhetinic acid), but replacement of fluorescence into the photobleached cell resumed when it was removed. These studies demonstrate that gap junctions are responsible for intercellular communication between adjacent osteocytes and between osteoblasts and osteocytes. This role is consistent with the ability of osteocytes to respond to and transmit signals over long distances while embedded in a calcified matrix.     

Local stimulation of cultured myocyte cells by femtosecond laser-induced stress wave

When an 800 nm femtosecond laser is tightly focused into cell culture medium a stress wave is generated at the laser focal point. Since the stress wave localizes in a few tens of μm, it is possible to locally stimulate single cells in vitro. In this work, several kinds of cultured mammalian cells, HeLa, PC12, P19CL6, and C2C12, were stimulated by the stress wave and the cell growth after the stress loading with the laser irradiation was investigated. In comparison with the control conditions, cell growth after the laser irradiation was enhanced for the cells of C2C12 and P19CL6, which can differentiate into myocytes, and suppressed for PC12 and HeLa cell lines. These results suggest a possibility of cell growth enhancement due to myogenic cells response to the femtosecond laser-induced stress.     

Cu-Catalyzed Desulfonylative Amination of Benzhydryl Sulfones

A new method for the synthesis of benzhydryl amines from the reaction of readily available sulfone derivatives with amines is described. The Cu-catalyzed desulfonylative amination not only provides structurally diverse benzhydryl amines in good yields, but is also applicable to iterative and intramolecular aminations. Control experiments suggested that the formation of a Cu-carbene intermediate generated from the sulfone substrate, which represents a new route for desulfonylative transformations.     

Preparation of cyclosophoraose-A and its complex-forming ability

Cyclosophoraoses (CySs) are unbranched cyclic (12)--D-glucans produced by many strains ofAgrobacterium andRhizobium. Pure CyS-A, the group member having the smallest molecular size (degree of polymerization 17), was efficiently prepared by liquid chromatography using charcoal and ODS columns from the culture fluid of the mutant strain RA-12 fromR. phaseoli AHU 1133. The complex-forming ability of CyS-A was estimated from its enhancement of the solubilities of slightly soluble guest molecules in water using methods [I], [II], and [III]. In [I], an aqueous solution of CyS-A was shaken with the guest molecule, while, in [II], it was shaken with an acetone solution of the guest compound. In method [III], freeze-dried CyS-A powder was stirred with an acetone solution of the guest compound. The CyS-A cavity is thought to be able to accommodate three-dimensionally extended guest molecules,e.g., indomethacin. Method [II] was the best for obtaining CyS-A inclusion complexes, while method [III] would be recommended if the guest molecule is labile in the presence of water. Crystalline CyS-A inclusion complexes have not been obtained, but CyS-A complexes are expected to greatly enhance the solubilities of slightly soluble or insoluble guest molecules in water, because CyS-A is much more soluble than -cyclodextrin. Method [II] or [III] may afford a useful means of obtaining oily drug,e. g., vitamin E and K₁, in an amorphous state.     

Fluorimetric determination of α-keto acids with 4,5-dimethoxy-1,2-diaminobenzene and its application to high-performance liquid chromatography

A highly sensitive fluorimetric method for the determination of α-keto acids of biological importance is described. The α-keto acids react in dilute hydrochloric acid with 4,5-dimethoxy-1,2-diaminobenzene to give a compound which fluoresces in neutral solution. The method is selective for α-keto acids and the limits of detection are 30–750 pmol ml^?1 of test solution. The fluorescent compounds in a reaction mixture of ten α-keto acids are separated within 18 min by high-performance liquid chromatography on a reversed-phase column with isocratic elution. The limits of detection for the acids are in the range 9–780 fmol in a 10-μl injection volume.     

The electronic structure of the excited states for B,C+, and C arising from the configuration 1s2 2s 2pn—the effect of removal of the “equivalence” restriction

In order to test the effect of a removal of an “equivalence” restriction, some excited states of B, C⁺ and C arising from the configuration 1s²2s2pⁿ (n = 2, 3) are calculated by the use of a one-diffuse-electron model with the optimal spin coupling (DO), in which the freedom of the spin coupling is fully used. The configuration employed is 1s²2s2p^n–12p′ (n = 2, 3). The DO method gives a good agreement with the term energies especially for boron (⁴P → ²P) and carbon (⁵S° → ³S°). A diffuse 3p natural orbital with a large occupation number is found for boron ²P and carbon ³S° by DO . This result is confirmed by configuration interaction calculations. Using the same configuration as above, we also examine a one-diffuse-electron model with a fixed spin coupling for boron. This method almost reproduces the results of restricted Hartree–Fock and the effect of a removal of an “equivalence” restriction is not found. The importance of utilizing the freedom of the spin coupling in a one-diffuse-electron model is strongly indicated in this article.     

Ab initio calculation of the electronic structure of the [Co(CN)6]3− ion

Ab initio LCAO SCF MO calculations were carried out on the ion [Co(CN)₆]^3? with double-zeta type wavefunctions. The results showed differences with previous semi-empirical calculations in the relative energy level of the mainly metal-dπ molecular orbital and in other respects.