Randomized clinical trial comparing two vessel-sealing devices with crush clamping during liver transection.

BACKGROUND: Previous RCTs have failed to demonstrate the usefulness of combining energy devices with the conventional clamp crushing method to reduce blood loss during liver transection. Here, the combination of an ultrasonically activated device (UAD) and a bipolar vessel-sealing device (BVSD) with crush clamping was investigated. METHODS: Patients scheduled to undergo hepatectomy at the University of Tokyo Hospital or Nihon University Itabashi Hospital were eligible for this parallel-group, single-blinded randomized study. Patients were assigned to a control group (no energy device used), an UAD group or a BVSD group. The primary endpoint was the volume of blood loss during liver transection. Outcomes of the control group and the combined energy device groups (UAD plus BVSD) were first compared. Pairwise comparisons among the three groups were made for outcomes for which the combined energy device group was superior to the control group. RESULTS: A total of 380 patients were enrolled between July 2012 and May 2014; 116 patients in the control group, 122 in the UAD group and 123 in the BVSD group were included in the final analysis. Median blood loss during liver transection was lower in the combined energy device group (245 patients) than in the control group (116 patients): median 190 (range 0-3575) versus 230 (range 3-1570) ml (P = 0·048). Pairwise comparison revealed that blood loss was lower in the BVSD group than in the control group (P = 0·043). CONCLUSION: The use of energy devices combined with crush clamping reduced blood loss during liver transection. Registration number: C000008372 (www.umin.ac.jp/ctr/index.htm).

Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum.

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.

Expression of a Cs(+)-resistant guard cell K+ channel confers Cs(+)-resistant, light-induced stomatal opening in transgenic arabidopsis.

Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+in channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+in channel, KAT1, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo.

Increased resistance to extracellular cation block by mutation of the pore domain of the Arabidopsis inward-rectifying K+ channel KAT1.

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K+ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K+ channel blockers are potent tools for studying the physiological functions and structural properties of K+ channels. In the present study the structural and biophysical mechanisms of Cs+ and TEA+ block of a cloned Arabidopsis inward-rectifying K+ channel (KAT1) were analyzed. Effects of the channel blockers Cs+ and TEA+ were characterized both extracellularly and intracellularly. Both external Cs+ and TEA+ block KAT1 currents. A mutant of KAT1 ("m2KAT1"; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs+ and TEA+ than the wild-type K+ channel. Internal TEA+ and Cs+ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K+ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K+ channels.

Multiple genes, tissue specificity, and expression-dependent modulationcontribute to the functional diversity of potassium channels in Arabidopsis thaliana.

K+ channels play diverse roles in mediating K+ transport and in modulating the membrane potential in higher plant cells during growth and development. Some of the diversity in K+ channel functions may arise from the regulated expression of multiple genes encoding different K+ channel polypeptides. Here we report the isolation of a novel Arabidopsis thaliana cDNA (AKT2) that is highly homologous to the two previously identified K+ channel genes, KAT1 and AKT1. This cDNA mapped to the center of chromosome 4 by restriction fragment length polymorphism analysis and was highly expressed in leaves, whereas AKT1 was mainly expressed in roots. In addition, we show that diversity in K+ channel function may be attributable to differences in expression levels. Increasing KAT1 expression in Xenopus oocytes by polyadenylation of the KAT1 mRNA increased the current amplitude and led to higher levels of KAT1 protein, as assayed in western blots. The increase in KAT1 expression in oocytes produced shifts in the threshold potential for activation to more positive membrane potentials and decreased half-activation times. These results suggest that different levels of expression and tissue-specific expression of different K+ channel isoforms can contribute to the functional diversity of plant K+ channels. The identification of a highly expressed, leaf-specific K+ channel homolog in plants should allow further molecular characterization of K+ channel functions for physiological K+ transport processes in leaves.

PF1: an A-T hook-containing DNA binding protein from rice that interacts with a functionally defined d(AT)-rich element in the oat phytochrome A3 gene promoter.

Phytochrome-imposed down-regulation of the expression of its own phytochrome A gene (PHYA) is one of the fastest light-induced effects on transcription reported in plants to date. Functional analysis of the oat PHYA3 promoter in a transfection assay has revealed two positive elements, PE1 and PE3, that function synergistically to support high levels of transcription in the absence of light. We have isolated a rice cDNA clone (pR4) encoding a DNA binding protein that binds to the AT-rich PE1 element. We tested the selectivity of the pR4-encoded DNA binding activity using linker substitution mutations of PE1 that are known to disrupt positive expression supported by the PHYA3 promoter in vivo. Binding to these linker substitution mutants was one to two orders of magnitude less than to the native PE1 element. Because this is the behavior expected of positive factor 1 (PF1), the presumptive nuclear transcription factor that acts in trans at the PE1 element in vivo, the data support the conclusion that the protein encoded by pR4 is in fact rice PF1. The PF1 polypeptide encoded by pR4 is 213 amino acids long and contains four repeats of the A-T hook DNA binding motif found in high-mobility group I-Y (HMGI-Y) proteins. In addition, PF1 contains an 11-amino acid-long hydrophobic region characteristic of HMG I proteins, its N-terminal region shows strong similarities to a pea H1 histone sequence and a short peptide sequence from wheat HMGa, and it shows a high degree of similarity along its entire length to the HMG Y-like protein encoded by a soybean cDNA, SB16. In vitro footprinting and quantitative gel shift analyses showed that PF1 binds preferentially to the PE1 element but also at lower affinity to two other AT-rich regions upstream of PE1. This feature is consistent with the binding characteristics of HMG I-Y proteins that are known to bind to most runs of six or more AT base pairs. Taken together, the properties of PF1 suggest that it belongs to a newly described family of nuclear proteins containing both histone H1 domains and A-T hook DNA binding domains.

A convenient evaluation of the stereoselectivity of lipase-catalyzed hydrolysis of tri-O-acylglycerols on a chiral-phase liquid chromatography.

A general method was developed using chiral-phase chromatography in order to evaluate the stereoselectivities of lipases-catalyzed hydrolysis of tri-O-acylglycerols independent of acyl groups. 1,2-Di-O-acyl-sn-glycerols or its enantiomer 2,3-di-O-acyl-sn-glycerols in the enzymatic reaction mixtures were derivatized to the key compound, 1,2-di-O-benzoyl-3-O-tert-butyldimethylsilyl-sn-glycerol 2 (+) or its enantiomer 2' (-), respectively. The enantiomers were separated on a chiral-phase HPLC, and the method was highly sensitive to determine the stereoselectivities of lipases.