Hydration of unsaturated carbon compounds is one of the most straightforward and environmentally benign methods to form the carbon±oxygen bond. Synthesis of carbonyl compounds by the hydration of alkynes is an important variation in this category, which has been extensively studied. [1] Acidcatalyzed hydration of alkynes is long known. [2, 3] However, only electron-rich acetylene compounds, such as alkynyl ethers, alkynyl thioethers and ynamines react satisfactorily. [1d, 4] The reaction of simple alkynes is usually sluggish and needs cocatalysts, typically toxic mercury(ii) salts, to enhance the reactivity. [5] More recent interest lies in the use of transition-metal-complex catalysts containing Ru II , [6] Ru III , [7] Rh, [8] Pt, [9] Au III , [10] and other metal centers. [11] However, the process catalyzed by these complexes is not efficient either. The highest turnover frequency (TOF) is 550 h À1 claimed as the initial TOF for the hydration of 3-pentyn-1-ol catalyzed by [cis-PtCl 2 (tppts) 2 ] (tppts ¼ P(m-C 6 H 4 SO 3 Na) 3 ), but its overall TOF is no more than approximately 100 h À1 . [9c] Recently Teles and co-workers reported the addition of methanol to alkynes catalyzed by Au I species in conjunction with acidic cocatalysts. [12] Hydration of propargyl alcohol was also briefly mentioned in their patent application, [13] although the yield was quite low. [14] Herein we report that the Au I ±acid systems in aqueous methanol serve as powerful catalysts, [15] which promote the hydration of alkynes [Eq. (1)] and have turnover frequencies of at least two orders of magnitude higher than [cis-PtCl 2 (tppts) 2 ].In a preliminary experiment, a mixture of 1-octyne (1 mmol), [(Ph 3 P)AuCH 3 ] (0.01 mmol, 1 mol %) and concentrated sulfuric acid (0.5 mmol, 50 mol %) in aqueous methanol (1.5 mL, methanol:H 2 O ¼ 2:1 v/v) was heated for 1 h at 70 8C affording the corresponding Markovnikov hydration product, 2-octanone, in 95 % yield without anti-Markovnikov hydration, or possible methanol addition. [12] The reaction did not proceed in the absence of either the Au catalyst or sulfuric acid.The following aspects about the catalytic system are worth noting. First, the nature of the reaction medium significantly affects the reaction. The reaction run without using solvent (in otherwise the same conditions as the preliminary experiment) did not furnish 2-octanone. On the other hand, the use of 2propanol (71 %), dioxane (56 %), acetonitrile (53 %), or THF (11 %) resulted in a low yield, and the yield obtained with dichloromethane, DMF, or toluene was even lower. Thus methanol was the solvent of choice for this particular transformation. [16] Second, the efficiency of the catalyst was significantly enhanced by addition of appropriate ligands, which enabled the quantity of the precious catalyst used to be minimized. For instance, the control experiment run without ligand addition under the conditions shown in Table 1 (only 0.01 mol % catalyst) gave 2-octanone in only 35 % yield (TOF ¼ 3500 h À1 , entry 1), while th...

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[reaction: see text] Addition of aniline derivatives to aromatic and aliphatic alkynes proceeds efficiently in the presence of a gold(I) catalyst (0.01-1.0 mol %) to afford ketimines in good yields

In legumes, Ca2+/calmodulin-dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca2+ spiking, one of the earliest cellular responses to microbial signals. A gain-of-function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain-of-function CCaMKT265D suppresses loss-of-function mutations of common symbiosis genes required for the generation of Ca2+ spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca2+ spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMKT265D induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.

Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

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Although cytosolic free Ca 2+ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca 2+ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca 2+ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn 2+ -dependent protein kinase activity, which was enhanced both by deletion of their FISL/ NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbeassociated molecular pattern-induced defense signaling pathway in rice cultured cells.Calcium ions regulate diverse cellular processes in plants as a ubiquitous internal second messenger, conveying signals received at the cell surface to the inside of the cell through spatial and temporal concentration changes that are decoded by an array of Ca 2+

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.