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Find video protocols related to scientific articles indexed in Pubmed.
Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan.
Orig Life Evol Biosph
PUBLISHED: 09-25-2014
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At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words).
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Identification of a Multipotent Self-Renewing Stromal Progenitor Population during Mammalian Kidney Organogenesis.
Stem Cell Reports
PUBLISHED: 08-11-2014
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The mammalian kidney is a complex organ consisting of multiple cell types. We previously showed that the Six2-expressing cap mesenchyme is a multipotent self-renewing progenitor population for the main body of the nephron, the basic functional unit of the kidney. However, the cellular mechanisms establishing stromal tissues are less clear. We demonstrate that the Foxd1-expressing cortical stroma represents a distinct multipotent self-renewing progenitor population that gives rise to stromal tissues of the interstitium, mesangium, and pericytes throughout kidney organogenesis. Fate map analysis of Foxd1-expressing cells demonstrates that a small subset of these cells contributes to Six2-expressing cells at the early stage of kidney outgrowth. Thereafter, there appears to be a strict nephron and stromal lineage boundary derived from Six2-expressing and Foxd1-expressing cell types, respectively. Taken together, our observations suggest that distinct multipotent self-renewing progenitor populations coordinate cellular differentiation of the nephron epithelium and renal stroma during mammalian kidney organogenesis.
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Novel urushiols with human immunodeficiency virus type 1 reverse transcriptase inhibitory activity from the leaves of Rhus verniciflua.
J Nat Med
PUBLISHED: 06-28-2014
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Two novel urushiols, 1 and 2, and two known urushiols, 3 and 4, were isolated from the leaves of Rhus verniciflua and were examined for their human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitory activity. The novel urushiols were found to be 1,2-dihydroxyphenyl-3-[7'(E),9'(Z),11'(Z)-pentadecatrienyl]-14'-ol (1) and 1,2-dihydroxyphenyl-3-[8'(Z),10'(E),12'(E)-pentadecatrienyl]-14'-ol (2) by spectroscopic analyses. The absolute configuration at C-14' in 1 and 2 was determined to be a racemic mixture of (R) and (S) isomers by ozonolysis. Compound 2 (IC50: 12.6 µM) showed the highest HIV-1 RT inhibitory activity among the four urushiols, being 2.5-fold more potent than the positive control, adriamycin (IC50: 31.9 µM). Although the known urushiols were isolated from the sap and leaves of R. verniciflua, 1 was exclusively present in the leaves, and higher amounts of 2 were found in the leaves than in the sap. Present findings indicate that the leaves of R. verniciflua represent a new biological resource from which novel and known urushiols may be prepared, and the possible use of novel urushiols as bioactive products.
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Discovery of 6-phenylpyrimido[4,5-b][1,4]oxazines as potent and selective acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) inhibitors with in vivo efficacy in rodents.
J. Med. Chem.
PUBLISHED: 04-08-2014
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The discovery and optimization of a series of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) inhibitors based on a pyrimido[4,5-b][1,4]oxazine scaffold is described. The SAR of a moderately potent HTS hit was investigated resulting in the discovery of phenylcyclohexylacetic acid 1, which displayed good DGAT1 inhibitory activity, selectivity, and PK properties. During preclinical toxicity studies a metabolite of 1 was observed that was responsible for elevating the levels of liver enzymes ALT and AST. Subsequently, analogues were synthesized to preclude the formation of the toxic metabolite. This effort resulted in the discovery of spiroindane 42, which displayed significantly improved DGAT1 inhibition compared to 1. Spiroindane 42 was well tolerated in rodents in vivo, demonstrated efficacy in an oral triglyceride uptake study in mice, and had an acceptable safety profile in preclinical toxicity studies.
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Differentiated kidney epithelial cells repair injured proximal tubule.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-14-2013
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Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreER(T2) cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair.
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Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 08-09-2013
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The origin of cells that make pathologic fibrillar collagen matrix in lung disease has been controversial. Recent studies suggest mesenchymal cells may contribute directly to fibrosis.
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Effects of elevated pressure on rate of photosynthesis during plant growth.
J. Biotechnol.
PUBLISHED: 07-08-2013
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The aim of this study is to investigate the effects of an artificially controlled environment, particularly elevated total pressure, on net photosynthesis and respiration during plant growth. Pressure directly affects not only cells and organelles in leaves but also the diffusion coefficients and degrees of solubility of CO2 and O2. In this study, the effects of elevated total pressure on the rates of net photosynthesis and respiration of a model plant, Arabidopsis thaliana, were investigated in a chamber that newly developed in this study to control the total pressure. The results clearly showed that the rate of respiration decreased linearly with increasing total pressure at a high humidity. The rate of respiration decreased linearly with increasing total pressure up to 0.2 MPa, and increased with increasing total pressure from 0.3 to 0.5 MPa at a low humidity. The rate of net photosynthesis decreased linearly with increasing total pressure under a constant partial pressure of CO2 at 40 Pa. On the other hand, the rate of net photosynthesis was clearly increased by up to 1.6-fold with increasing total pressure and partial pressure of CO2.
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Wnt4/?-catenin signaling in medullary kidney myofibroblasts.
J. Am. Soc. Nephrol.
PUBLISHED: 06-13-2013
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Injury to the adult kidney induces a number of developmental genes thought to regulate repair, including Wnt4. During kidney development, early nephron precursors and medullary stroma both express Wnt4, where it regulates epithelialization and controls smooth muscle fate, respectively. Expression patterns and roles for Wnt4 in the adult kidney, however, remain unclear. In this study, we used reporters, lineage analysis, and conditional knockout or activation of the Wnt/?-catenin pathway to investigate Wnt4 in the adult kidney. Proliferating, medullary, interstitial myofibroblasts strongly expressed Wnt4 during renal fibrosis, whereas tubule epithelia, except for the collecting duct, did not. Exogenous Wnt4 drove myofibroblast differentiation of a pericyte-like cell line, suggesting that Wnt4 might regulate pericyte-to-myofibroblast transition through autocrine signaling. However, conditional deletion of Wnt4 in interstitial cells did not reduce myofibroblast proliferation, cell number, or myofibroblast gene expression during fibrosis. Because the injured kidney expresses multiple Wnt ligands that might compensate for the absence of Wnt4, we generated a mouse model with constitutive activation of canonical Wnt/?-catenin signaling in interstitial pericytes and fibroblasts. Kidneys from these mice exhibited spontaneous myofibroblast differentiation in the absence of injury. Taken together, Wnt4 expression in renal fibrosis defines a population of proliferating medullary myofibroblasts. Although Wnt4 may be dispensable for myofibroblast transformation, canonical Wnt signaling through ?-catenin stabilization is sufficient to drive spontaneous myofibroblast differentiation in interstitial pericytes and fibroblasts, emphasizing the importance of this pathway in renal fibrosis.
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Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvatochromism in the rubber-producing plant, Eucommia ulmoides Oliver.
Planta
PUBLISHED: 03-27-2013
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A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry.
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Porous tantalum tibial component prevents periprosthetic loss of bone mineral density after total knee arthroplasty for five years-a matched cohort study.
J Arthroplasty
PUBLISHED: 02-27-2013
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In 21 knees receiving porous tantalum tibial component and 21 knees receiving a cemented cobalt-chromium tibial component, dual x-ray absorptiometry scans were performed for five years post-operatively. The postoperative decrease in the bone mineral density in the lateral aspect of the tibia was significantly less in knees with porous tantalum tibial components (11.6%) than in knees with cemented cobalt-chromium tibial components (29.6%) at five years (p < 0.05). No prosthetic migration or periprosthetic fracture was detected in either group. The present study is one of the studies with the longest follow-up period on bone mineral density after total knee arthroplasty. Porous tantalum tibial component has a favorable effect on the bone mineral density of the proximal tibia after total knee arthroplasty up to five years.
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LRP-6 is a coreceptor for multiple fibrogenic signaling pathways in pericytes and myofibroblasts that are inhibited by DKK-1.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-09-2013
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Fibrosis of vital organs is a major public health problem with limited therapeutic options. Mesenchymal cells including microvascular mural cells (pericytes) are major progenitors of scar-forming myofibroblasts in kidney and other organs. Here we show pericytes in healthy kidneys have active WNT/?-catenin signaling responses that are markedly up-regulated following kidney injury. Dickkopf-related protein 1 (DKK-1), a ligand for the WNT coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP-5 and LRP-6) and an inhibitor of WNT/?-catenin signaling, effectively inhibits pericyte activation, detachment, and transition to myofibroblasts in vivo in response to kidney injury, resulting in attenuated fibrogenesis, capillary rarefaction, and inflammation. DKK-1 blocks activation and proliferation of established myofibroblasts in vitro and blocks pericyte proliferation to PDGF, pericyte migration, gene activation, and cytoskeletal reorganization to TGF-? or connective tissue growth factor. These effects are largely independent of inhibition of downstream ?-catenin signaling. DKK-1 acts predominantly by inhibiting PDGF-, TGF-?-, and connective tissue growth factor-activated MAPK and JNK signaling cascades, acting via LRP-6 with associated WNT ligand. Biochemically, LRP-6 interacts closely with PDGF receptor ? and TGF-? receptor 1 at the cell membrane, suggesting that it may have roles in pathways other than WNT/?-catenin. In summary, DKK-1 blocks many of the changes in pericytes required for myofibroblast transition and attenuates established myofibroblast proliferation/activation by mechanisms dependent on LRP-6 and WNT ligands but not the downstream ?-catenin pathway.
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TLR-2/TLR-4 TREM-1 signaling pathway is dispensable in inflammatory myeloid cells during sterile kidney injury.
PLoS ONE
PUBLISHED: 01-01-2013
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Inflammatory macrophages are abundant in kidney disease, stimulating repair, or driving chronic inflammation and fibrosis. Damage associated molecules (DAMPs), released from injured cells engage pattern recognition receptors (PRRs) on macrophages, contributing to activation. Understanding mechanisms of macrophage activation during kidney injury may lead to strategies to alleviate chronic disease. We identified Triggering-Receptor-in-Myeloid-cells (TREM)-1, a regulator of TLR signaling, as highly upregulated in kidney inflammatory macrophages and tested the roles of these receptors in macrophage activation and kidney disease. Kidney DAMPs activated macrophages in vitro, independently of TREM-1, but partially dependent on TLR-2/-4, MyD88. In two models of progressive interstitial kidney disease, TREM-1 blockade had no impact on disease or macrophage activation in vivo, but TLR-2/-4, or MyD88 deficiency was anti-inflammatory and anti-fibrotic. When MyD88 was mutated only in the myeloid lineage, however, there was no bearing on macrophage activation or disease progression. Instead, TLR-2/-4 or MyD88 deficiency reduced activation of mesenchyme lineage cells resulting in reduced inflammation and fibrosis, indicating that these pathways play dominant roles in activation of myofibroblasts but not macrophages. To conclude, TREM-1, TLR2/4 and MyD88 signaling pathways are redundant in myeloid cell activation in kidney injury, but the latter appear to regulate activation of mesenchymal cells.
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Leukocytapheresis in pediatric patients with ulcerative colitis.
J. Pediatr. Gastroenterol. Nutr.
PUBLISHED: 06-23-2011
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Leukocytapheresis (LCAP) is a nonpharmacologic therapy that has recently been used to treat ulcerative colitis (UC). This multicenter open-label study prospectively assessed the efficacy and safety of LCAP in pediatric patients with UC.
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Effects of spaced feeding on gene expression of hepatic transaminase and gluconeogenic enzymes in rats.
J Toxicol Sci
PUBLISHED: 06-02-2011
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Blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities are widely used as sensitive markers of liver toxicity. However, these activities are also recognized to be altered by hormonal and nutritional modifications. We investigated the relationships between the activity and gene expression of the hepatic transaminases and the state of hepatic amino acid/glucose/fatty acid metabolism in the ad libitum fed (ALF) and spaced-fed (SF) rats. Acceleration of hepatic gluconeogenesis and fatty acid oxidation was noted in the SF rats. Expression of hepatic clock gene was also altered in the SF rats. Hepatic transaminase activities in the SF rats were higher than those in the ALF rats. These alterations were due to increases in the synthesis of hepatic ALT and AST proteins. In conclusion, the increased transaminase protein synthesis in the liver of the SF rats was considered to be related to the acceleration of hepatic gluconeogenesis under the conditions of spaced feeding.
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Mid-term clinical results of alumina medial pivot total knee arthroplasty.
Knee Surg Sports Traumatol Arthrosc
PUBLISHED: 05-12-2011
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The medial pivot total knee prosthesis has been designed to reproduce physiological knee kinematics. It has been reported that alumina ceramic femoral components reduce polyethylene wear. Thus, medial pivot total knee prostheses with alumina ceramic femoral components were introduced. The purpose of this study was to evaluate the clinical results of patients who underwent newly introduced alumina medial pivot total knee arthroplasties (TKA).
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?-Catenin is essential for Müllerian duct regression during male sexual differentiation.
Development
PUBLISHED: 04-13-2011
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During male sexual differentiation, the transforming growth factor-? (TGF-?) signaling molecule anti-Müllerian hormone (AMH; also known as Müllerian inhibiting substance, MIS) is secreted by the fetal testes and induces regression of the Müllerian ducts, the primordia of the female reproductive tract organs. Currently, the molecular identity of downstream events regulated by the AMH signaling pathway remains unclear. We found that male-specific Wnt4 expression in mouse Müllerian duct mesenchyme depends upon AMH signaling, implicating the WNT pathway as a downstream mediator of Müllerian duct regression. Inactivation of ?-catenin, a mediator of the canonical WNT pathway, did not affect AMH signaling activation in the Müllerian duct mesenchyme, but did block Müllerian duct regression. These data suggest that ?-catenin mediates AMH signaling for Müllerian duct regression during male sexual differentiation.
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Construction of a genetic AND gate under a new standard for assembly of genetic parts.
BMC Genomics
PUBLISHED: 12-02-2010
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Appropriate regulation of respective gene expressions is a bottleneck for the realization of artificial biological systems inside living cells. The modification of several promoter sequences is required to achieve appropriate regulation of the systems. However, a time-consuming process is required for the insertion of an operator, a binding site of a protein for gene expression, to the gene regulatory region of a plasmid. Thus, a standardized method for integrating operator sequences to the regulatory region of a plasmid is required.
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Relationships between plasma and tissue transaminase activities in rats maintained under different feeding conditions.
J Toxicol Sci
PUBLISHED: 10-09-2010
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In order to verify the nutritional aspect of alterations of the plasma and tissue transaminase activities, rats were fed 4 hr per day for 35 days (the spaced-fed (SF) rats) and the time course of the alterations in plasma and tissue transaminase activity was compared with those in the ad libitum fed (ALF) rats. Plasma transaminase activities were stable throughout the experiment period in the ALF rats. In the SF rats there were alterations in the plasma alanine aminotransferase (ALT) activities, the direction of which was different between the early phase and late phase of the experiment period; plasma ALT activities decreased in the early phase and gradually increased in the late phase. Plasma aspartate aminotransferase (AST) activities were stable in the SF rats throughout the experiment period as well as the ALF rats. The decreases in plasma ALT activities in the early phase were considered to be related to decreases in ALT activities in the small intestinal mucosa (SI mucosa). On the other hand, the increases in plasma ALT activities in the late phase were considered to be related to increases in ALT activities in the liver. Multiple regression analyses (MRAs) revealed that plasma ALT activities in the SF rats could be estimated by the ALT activities in the SI mucosa and liver. From these results, the alterations of the plasma ALT activities in the SF rats could be explained by those in the SI mucosa and liver under the conditions in our study.
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Contribution of mevalonate and methylerythritol phosphate pathways to polyisoprenoid biosynthesis in the rubber-producing plant Eucommia ulmoides oliver.
Z. Naturforsch., C, J. Biosci.
PUBLISHED: 07-27-2010
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The biosynthetic origin of isopentenyl diphosphate in the polyisoprenoid biosynthesis of the rubber-producing plant Eucommia ulmoides Oliver was elucidated for the first time by feeding experiments using 13C-labeled isotopomers of (RS)-mevalonate, 1-deoxy-D-xylulose-3,4,5-triacetate, 2C-methyl-D-erythritol-1,2,3,4-tetraacetate, and pyruvate. After 13C-labeled isotopomers were fed to the young seedlings, the polyisoprenoid fractions were prepared and analyzed by 13C NMR. The NMR data showed that the isoprene units of polyisoprenoid derived from isopentenyl diphosphate, which was biosynthesized using both mevalonate and 1-deoxy-D-xylulose-5-phosphate in E. ulmoides. It is assumed that the cross-talk of isopentenyl diphosphate, derived from both pathways, occurs during the biosynthesis of polyisoprenoid; therefore, it was observed in the formation of low-molecular weight isoprenoids.
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Metabolomics-based systematic prediction of yeast lifespan and its application for semi-rational screening of ageing-related mutants.
Aging Cell
PUBLISHED: 06-09-2010
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Metabolomics - the comprehensive analysis of metabolites - was recently used to classify yeast mutants with no overt phenotype using raw data as metabolic fingerprints or footprints. In this study, we demonstrate the estimation of a complicated phenotype, longevity, and semi-rational screening for relevant mutants using metabolic profiles as strain-specific fingerprints. The fingerprints used in our experiments are profiled data consisting of individually identified and quantified metabolites rather than raw spectrum data. We chose yeast replicative lifespan as a model phenotype. Several yeast mutants that affect lifespan were selected for analysis, and they were subjected to metabolic profiling using mass spectrometry. Fingerprinting based on the profiles revealed a correlation between lifespan and metabolic profile. Amino acids and nucleotide derivatives were the main contributors to this correlation. Furthermore, we established a multivariate model to predict lifespan from a metabolic profile. The model facilitated the identification of putative longevity mutants. This work represents a novel approach to evaluate and screen complicated and quantitative phenotype by means of metabolomics.
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Sagittal alignment of the lower extremity while standing in female.
Knee Surg Sports Traumatol Arthrosc
PUBLISHED: 03-29-2010
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There is only limited information available on the sagittal alignment of the lower extremity of normal subjects under weight-bearing conditions. Our aim was to determine the sagittal alignment of the lower extremity under such conditions. Anteroposterior and lateral radiographs were taken of the 20 lower extremities of 20 healthy female volunteers while standing. The coronal mechanical axis passed through 29% medial to the proximal tibial articulating surface. The sagittal mechanical axis passed through 44% anterior to the distal femoral condyle and 33% anterior to the proximal tibial articulating surface, and also passed 3 mm anterior to the intercondylar notch. Our study showed that the coronal and sagittal mechanical axes of the lower extremity do not always pass through the center of the knee. Our results will provide important information for alignment in surgery of lower extremity such as knee arthroplasty and osteotomy.
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Comparison of bone mineral density between porous tantalum and cemented tibial total knee arthroplasty components.
J Bone Joint Surg Am
PUBLISHED: 03-03-2010
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Porous tantalum was recently introduced as a metallic implant material for total knee arthroplasty. Its porosity, low modulus of elasticity, and high frictional characteristics were expected to provide physiologic load transfer and relative preservation of bone stock. However, to our knowledge, the effect of a Trabecular Metal tibial component on bone mineral density has not been reported. The purpose of the present study was to compare the periprosthetic bone mineral density between patients managed with uncemented Trabecular Metal and cemented tibial components.
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Efficacy and safety of pulse steroid therapy in Japanese pediatric patients with ulcerative colitis: a survey of the Japanese Society for Pediatric Inflammatory Bowel Disease.
Digestion
PUBLISHED: 01-19-2010
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We have evaluated the therapeutic efficacy and safety of pulse steroid therapy for ulcerative colitis (UC) in a Japanese pediatric population by means of a survey.
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High-throughput and highly sensitive analysis method for polyisoprene in plants by pyrolysis-gas chromatography/mass spectrometry.
Biosci. Biotechnol. Biochem.
PUBLISHED: 01-07-2010
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Natural polyisoprene is a biopolymer consisting of isoprene units (C(5)H(8)) that is used commercially in household, medical, and industrial materials. For the management of natural polyisoprene production, the selection of high-yield polyisoprene-producing trees, and an understanding of polyisoprene biosynthesis, a high-throughput and highly sensitive screening method for the quantification of polyisoprene is required. In this study, we examined pyrolysates from polyisoprenes, polyprenols, carotenoids, ubiquinone (CoQ-10), and sterols by pyrolysis gas chromatography/mass spectrometry (PyGC/MS) and determined that the amounts of isoprene and limonene released from polyprenols and polyisoprenes were dependent upon their molecular weights. Based on these results, we developed a relative quantification method for polyisoprene in leaves by direct analysis of 1 mg of leaves using PyGC/MS. This novel quantification method eliminated extraction steps and can be used in the measurement of polyisoprene contents in Eucommia ulmoides and Hevea brasiliensis.
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Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
Am. J. Pathol.
PUBLISHED: 12-11-2009
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Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor beta(1) treatment. However, using either red fluorescent protein or beta-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive((+)) mesenchymal cells give rise to adult CD73(+), platelet derived growth factor receptor beta(+), smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin(+) myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease.
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Two Arabidopsis thaliana Golgi alpha-mannosidase I enzymes are responsible for plant N-glycan maturation.
Glycobiology
PUBLISHED: 11-12-2009
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N-Glycosylation is an important post-translational modification that occurs in many secreted and membrane proteins in eukaryotic cells. Golgi alpha-mannosidase I hydrolases (MANI) are key enzymes that play a role in the early N-glycan modification pathway in the Golgi apparatus. In Arabidopsis thaliana, two putative MANI genes, AtMANIa (At3g21160) and AtMANIb (At1g51590), were identified. Biochemical analysis using bacterially produced recombinant AtMANI isoforms revealed that both AtMANI isoforms encode 1-deoxymannojirimycin-sensitive alpha-mannosidase I and act on Man(8)GlcNAc(2) and Man(9)GlcNAc(2) structures to yield Man(5)GlcNAc(2). Structures of hydrolytic intermediates accumulated in the AtMANI reactions indicate that AtMANIs employ hydrolytic pathways distinct from those of mammalian MANIs. In planta, AtMANI-GFP/DsRed fusion proteins were detected in the Golgi stacks. Arabidopsis mutant lines manIa-1, manIa-2, manIb-1, and manIb-2 showed N-glycan profiles similar to that of wild type. On the other hand, the manIa manIb double mutant lines produced Man(8)GlcNAc(2) as the predominant N-glycan and lacked plant-specific complex and hybrid N-glycans. These data indicate that either AtMANIa or AtMANIb can function as the Golgi alpha-mannosidase I that produces the Man(5)GlcNAc(2) N-glycan structure necessary for complex N-glycan synthesis.
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Metabolic engineering of lignan biosynthesis in Forsythia cell culture.
Plant Cell Physiol.
PUBLISHED: 11-03-2009
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Lignans are a large class of secondary metabolites in plants, with numerous biological effects in mammals, including antitumor and antioxidant activities. Sesamin, the most abundant furofuran-class lignan in sesame seeds (Sesamum plants), is produced by the cytochrome P450 enzyme CYP81Q1 from the precursor lignan, pinoresinol. In contrast, Forsythia plants produce dibenzylbutyrolactone-class lignans, such as matairesinol, from pinoresinol via the catalysis of pinoresinol/lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase. Here we present the engineering of lignan biosynthesis in Forsythia cell suspension cultures for the development of an efficient production method of beneficial lignans. A suspension cell culture prepared from leaves of Forsythia koreana produced lignans, mainly pinoresinol and matairesinol glucosides, at levels comparable with that obtained from the leaves. In an attempt to increase the pinoresinol content in Forsythia, we generated a transgenic cell line overexpressing an RNA interference (RNAi) construct of PLR (PLR-RNAi). Down-regulation of PLR expression led to a complete loss of matairesinol and an accumulation of approximately 20-fold pinoresinol in its glucoside form in comparison with the non-transformant. Moreover, the Forsythia transgenic cells co-expressing CYP81Q1 and PLR-RNAi exhibited production of sesamin as well as accumulation of pinoresinol glucoside. These data suggest Forsythia cell suspension to be a promising tool for the engineering of lignan production. To the best of our knowledge, this is the first report on transgenic production of an exogenous lignan in a plant species.
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Effects of fenofibrate on plasma and hepatic transaminase activities and hepatic transaminase gene expression in rats.
J Toxicol Sci
PUBLISHED: 08-05-2009
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Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are widely used as sensitive markers of possible tissue damage, particularly liver toxicity. Lipid-lowering drugs, such as fibrates, slightly increase serum transaminase levels in humans, but there is little evidence that the phenomenon is related to drug-induced liver injury (DILI). Some in vitro studies have indicated that the elevations of serum transaminase activities after treatment of humans with fenofibrate, one of the fibrates, are related to increased transaminase synthesis in the hepatocytes rather than to transaminase leakage from the hepatocytes associated with cell lysis. In this study, male F344/DuCrlCrlj (Fischer) rats were treated once with fenofibrate at a dose level of 400 mg/kg and the relationships between the pharmacological effects, blood and hepatic transaminase activities and the gene expression of the transaminases in the liver were investigated. Fenofibrate treatment slightly increased plasma transaminase activities in rats with the findings directly related to the pharmacological action of the drug. The increases were in parallel with increases in hepatic transaminase activities associated with increases in the transaminase genes in the liver and were not considered to be a consequence of hepatotoxicity from the drug. The modification in transaminase gene expression is likely to be secondary to the pharmacological action of fenofibrate. The evidence obtained in our study underlines the importance of gene regulation as a possible alternative mechanism for increased blood transaminase activities.
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In vivo comparison of wear particles between highly crosslinked polyethylene and conventional polyethylene in the same design of total knee arthroplasties.
J. Biomed. Mater. Res. Part B Appl. Biomater.
PUBLISHED: 07-29-2009
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Reduction of wear with highly crosslinked polyethylene (HXLPE) has been reported in in vitro and in vivo studies of total hip prostheses. However, use of HXLPE in total knee prostheses is still controversial. The aim of this study was to compare in vivo polyethylene wear particle generation of HXLPE with that of conventional polyethylene in total knee prostheses of the same design. Synovial fluid was obtained from four knees with HXLPE inserts and three knees with conventional polyethylene inserts at 1 year after operation. Polyethylene particles were isolated and examined using a scanning electron microscope and image analyzer. The total number of particles in each knee was 0.28 +/- 0.12 x 10(6) in HXPLE group (mean +/- standard error) and 6.87 +/- 2.85 x 10(6) in conventional polyethylene group (p = 0.040). Particle size (equivalent circle diameter) was 0.64 +/- 0.07 microm in HXPLE group and 1.21 +/- 0.21 microm in conventional polyethylene group (p = 0.030). Particle shape (aspect ratio) was 1.33 +/- 0.10 in HXLPE and 1.88 +/- 0.19 in conventional polyethylene (p = 0.035). Thepercentage of particles of submicron size was greater than 90% in HXLPE group and 55% in conventional polyethylene group. Except for the material of the polyethylene insert, the design and material of prostheses were completely the same in both groups. The HXLPE insert generated fewer, smaller, and rounder polyethylene wear particles than the conventional polyethylene insert in the early stage after surgery.
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The risk of notching the anterior femoral cortex with the use of navigation systems in total knee arthroplasty.
Knee Surg Sports Traumatol Arthrosc
PUBLISHED: 06-08-2009
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Use of navigation systems has recently been introduced in total knee arthroplasty (TKA) to achieve more reliable prosthetic alignment. In the sagittal plane, there are two important requirements for navigation systems: (1) perpendicular cut to the femoral mechanical axis and (2) prevention of notching of anterior femoral cortex. These two requirements, however, may conflict. The angles between the line of the anterior femoral cortex and four sagittal femoral mechanical axes for navigation systems using radiographs of the entire lower extremity, while standing were measured and compared. These four sagittal axes simulated on the radiographs in navigation systems were in extension relative to the line of the anterior femoral cortex in 40-85% of cases in male and 65-100% in elderly female. The present study showed that navigation systems have the potential risk for notching of anterior femoral cortex.
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A novel application of metabolomics in vertebrate development.
Biochem. Biophys. Res. Commun.
PUBLISHED: 05-31-2009
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Many studies have demonstrated the functions of individual genes associated with embryogenesis and have determined the genome sequences of several organisms. Despite the availability of enormous amount of genetic information, dynamic changes that occur during embryogenesis have not yet been completely understood. In order to understand the dynamic processes involved in embryogenesis, we employed the metabolomic approach. The results of our study indicated that there is a close correlation between metabolomes and developmental stages. Our method enables the identification of embryonic stages using metabolomes as "fingerprints." In this manner, we could successfully predict embryonic development on the basis of metabolomic fingerprints. This is the first report describing a model for predicting vertebrate development by using metabolomics.
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Immunoglobulin G4-related sclerosing sialadenitis: report of two cases and review of the literature.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
PUBLISHED: 05-30-2009
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A new concept of IgG4-related disease characterized by a high serum IgG4 level and tissue infiltration of IgG4-positive plasmacytes that can involve salivary glands has been proposed. In this article, 2 patients with IgG4-related sclerosing sialadenitis involving the submandibular glands are reported. One patient presented with bilateral and painless swelling of the submandibular glands. He had already been treated with systemic prednisolone owing to the occurrence of retrobulbar neuritis. Laboratory examinations showed high serum IgG4 concentrations, and a biopsy of the submandibular gland revealed the infiltration of IgG4-positive plasmacytes. Abdominal computerized tomography demonstrated tumefaction in the tail of the pancreas, thus suggesting localized autoimmune pancreatitis. The other patient also showed bilateral and painless swelling of the submandibular glands, but there was no involvement of any other organs. These patients were effectively treated with corticosteroids, which resulted in a reduction of the swelling of the submandibular gland and increased saliva. When a patient is suspected of having sclerosing sialadenitis, it is important to consider that the patient may have a systemic IgG4-related plasmacytic disease.
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High-resolution gene expression analysis of the developing mouse kidney defines novel cellular compartments within the nephron progenitor population.
Dev. Biol.
PUBLISHED: 05-01-2009
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The functional unit of the kidney is the nephron. During its organogenesis, the mammalian metanephric kidney generates thousands of nephrons over a protracted period of fetal life. All nephrons are derived from a population of self-renewing multi-potent progenitor cells, termed the cap mesenchyme. However, our understanding of the molecular and cellular mechanisms underlying nephron development is at an early stage. In order to identify factors involved in nephrogenesis, we performed a high-resolution, spatial profiling of a number of transcriptional regulators expressed within the cap mesenchyme and early developing nephron. Our results demonstrate novel, stereotypic, spatially defined cellular sub-domains within the cap mesenchyme, which may, in part, reflect induction of nephron precursors. These results suggest a hitherto unappreciated complexity of cell states that accompany the assembly of the metanephric kidney, likely reflecting diverse regulatory actions such as the maintenance and induction of nephron progenitors.
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Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry.
Anal. Chim. Acta
PUBLISHED: 04-27-2009
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In recent plant hormone research, there is an increased demand for a highly sensitive and comprehensive analytical approach to elucidate the hormonal signaling networks, functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive and quantitative analytical method developed with nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring (MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and the detection limits (S/N=3) of several plant hormones were in the sub-fmol range. The results showed excellent linearity (R(2) values of 0.9937-1.0000) and reproducibility of elution times (relative standard deviations, RSDs, <1.1%) and peak areas (RSDs, <10.7%) for all target compounds. Further, sample purification using Oasis HLB and Oasis MCX cartridges significantly decreased the ion-suppressing effects of biological matrix as compared to the purification using only Oasis HLB cartridge. The optimized nanoflow-LC-ESI-IT-MS/MS method was successfully used to analyze endogenous plant hormones in Arabidopsis and tobacco samples. The samples used in this analysis were extracted from only 17 tobacco dry seeds (1mg DW), indicating that the efficiency of analysis of endogenous plant hormones strongly depends on the detection sensitivity of the method. Our analytical approach will be useful for in-depth studies on complex plant hormonal metabolism.
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Unique phytochrome responses of the holoparasitic plant Orobanche minor.
New Phytol.
PUBLISHED: 03-28-2009
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Holoparasitic plants such as Orobanche spp. have lost their photosynthetic ability, so photoresponses to optimize photosynthesis are not necessary in these plants. Photoresponses are also involved in the regulation of plant development but the photoresponses of holoparasites have not been characterized in detail. In this study, the phytochrome (phy)-related photoresponse of Orobanche minor was investigated. Its photoreceptor, phytochrome A (OmphyA), was also characterized. Light effects on germination, shoot elongation, anthocyanin biosynthesis, and OmphyA expression and subcellular localization were analyzed. Red light (R):far-red light (FR) reversible inhibition of O. minor seed germination demonstrated that phy-mediated responses are retained in this holoparasite. Shoot elongation was inhibited by FR but not by R. This pattern is unique among known patterns of plant photoresponses. Additionally, molecular analysis showed that OmphyA is able to respond to the light signals. Interestingly, the unique pattern of photoresponses in O. minor seems to have been modified for adaptation to its parasitic life cycle. We hypothesize that this alteration has resulted from the loss or alteration of some phy-signaling components. Elucidation of altered components in phy signaling in this parasite will provide useful information not only about its physiological characteristics but also about general plant photoreception systems.
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TKA sagittal alignment with navigation systems and conventional techniques vary only a few degrees.
Clin. Orthop. Relat. Res.
PUBLISHED: 03-20-2009
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Navigation systems have been developed to achieve more reliable prosthetic alignment in TKAs. However, the component alignment in the sagittal plane is reportedly less reliable than in the coronal plane even with navigation systems. We measured and compared sagittal prosthetic alignments for TKAs with the conventional technique and three navigation approaches to establish reference frames, using radiographs of the entire lower extremity while standing. The sagittal alignments simulated on the radiographs with the conventional technique and navigation systems differed by a mean of 2 degrees to 4 degrees . Use of navigation systems resulted in a mean of 1 degrees to 4 degrees hyperextension between the femoral and tibial components and use of the conventional technique resulted in a mean of 1 degrees flexion. Use of different reference points on the distal femoral condyle for the navigation systems resulted in differences of as much as 3 degrees alignment in the sagittal plane. Although optimal prosthetic alignment for TKA in the sagittal plane is unknown, surgeons and technicians using navigation systems should be aware of this difference in the sagittal plane and the risk of hyperextension between the femoral and tibial components, which might be associated with osteolysis and anterior post-cam impingement.
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Comprehensive metabolite profiling of phyA phyB phyC triple mutants to reveal their associated metabolic phenotype in rice leaves.
J. Biosci. Bioeng.
PUBLISHED: 03-13-2009
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The phytochrome photoreceptors regulate plant growth and development throughout their life cycle. Rice (Oryza sativa) possesses three phytochromes, phyA, phyB, and phyC. Physiological, genetic, and biochemical analyses of null mutants of each phytochrome have revealed the function of each in rice. However, few studies have investigated the relationship between phytochrome signaling and metabolism. In the present study, non-targeted metabolite analysis by gas chromatography time-of-flight mass spectrometry (GC/TOF-MS) and targeted metabolite analysis by capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) were employed to investigate metabolic changes in rice phyA phyB phyC triple mutants. Distinct metabolic profiles between phyA phyB phyC triple mutants and the wild type (WT), as well as those between young and mature leaf blades, could be clearly observed by principal component analysis (PCA). The metabolite profiles indicated high accumulation of amino acids, organic acids, sugars, sugar phosphates, and nucleotides in the leaf blades of phyA phyB phyC triple mutants, especially in the young leaves, compared with those in the WT. Remarkable overaccumulation of monosaccharide, such as glucose (53.4-fold), fructose (42.5-fold), and galactose (24.5-fold), was observed in young leaves of phyA phyB phyC triple mutants. These metabolic phenotypes suggest that sugar metabolism, carbon partitioning, sugar transport, or some combination of these is impaired in the phyA phyB phyC triple mutants, and conversely, that phytochromes have crucial roles in sugar metabolism.
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A Wnt7b-dependent pathway regulates the orientation of epithelial cell division and establishes the cortico-medullary axis of the mammalian kidney.
Development
PUBLISHED: 02-26-2009
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The mammalian kidney is organized into a cortex where primary filtration occurs, and a medullary region composed of elongated tubular epithelia where urine is concentrated. We show that the cortico-medullary axis of kidney organization and function is regulated by Wnt7b signaling. The future collecting duct network specifically expresses Wnt7b. In the absence of Wnt7b, cortical epithelial development is normal but the medullary zone fails to form and urine fails to be concentrated normally. The analysis of cell division planes in the collecting duct epithelium of the emerging medullary zone indicates a bias along the longitudinal axis of the epithelium. By contrast, in Wnt7b mutants, cell division planes in this population are biased along the radial axis, suggesting that Wnt7b-mediated regulation of the cell cleavage plane contributes to the establishment of a cortico-medullary axis. The removal of beta-catenin from the underlying Wnt-responsive interstitium phenocopies the medullary deficiency of Wnt7b mutants, suggesting a paracrine role for Wnt7b action through the canonical Wnt pathway. Wnt7b signaling is also essential for the coordinated growth of the loop of Henle, a medullary extension of the nephron that elongates in parallel to the collecting duct epithelium. These findings demonstrate that Wnt7b is a key regulator of the tissue architecture that establishes a functional physiologically active mammalian kidney.
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Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in Hevea brasiliensis using spectral confocal laser scanning microscopy.
Planta
PUBLISHED: 02-13-2009
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In Hevea brasiliensis, laticifers produce and accumulate rubber particles. Despite observation using histochemical methods, development stage structure and structures with ceasing functions have rarely been described. Spectral confocal laser scanning microscopy with Nile red staining simplifies laticifer structure observation in tangential sections while enhancing the resolution. Laticifer and ray images were extracted from unmixed images and used to monitor changes during growth. A laticifer network structure developed from increased anastomoses between adjoining laticifers outside of the conducting phloem, but because of increased radial division and growth of rays, the network structure ruptured and disintegrated. We also investigated immunohistochemical localization of two rubber particle-associated proteins in the laticifers: small rubber particle protein (SRPP) and rubber elongation factor (REF). Mature bark test results show that SRPP is localized only in the laticifer layers in the conducting phloem; REF is localized in all laticifer layers. Because SRPP plays a positive role in rubber biosynthesis, results show that the rubber biosynthesis capability of laticifers is concentrated where rays and the sieve tube actively transport metabolites.
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High-resolution spatial and temporal analysis of phytoalexin production in oats.
Planta
PUBLISHED: 01-16-2009
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The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N-acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor.
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Effect of gallic acid on peptides released by trypsin digestion of bovine ?-casein.
J. Biosci. Bioeng.
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In this study, the effects of gallic acid (GA) on trypsin digestion of commercial ?-casein (?-CN), which contains ?(s1)-CN and ?(s2)-CN, and the peptides released during digestion were investigated. Gallic acid showed no effect on the initial rate of digestion. However, the apparent degree of hydrolysis achieved its maximum value after 1 h, then decreased in the presence of GA, suggesting the cross-linking between peptides once released from ?-CN during digestion. In the presence of GA, three peaks derived from ?(s1)-CN disappeared and three new peaks appeared in high-performance liquid chromatography (HPLC) analysis. In these peptides, two Met residues corresponding to the Met(135) and Met(196) in ?(s1)-CN were oxidized to Met sulfoxide residues. The oxidation of Met(196) was quicker than that of Met(135). The inhibitory activity of TTMPLW (?(s1)-CN 193-199) against angiotensin I-converting enzyme was reduced slightly by the oxidation of its Met residue.
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Germ cells are not required to establish the female pathway in mouse fetal gonads.
PLoS ONE
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The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells.
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Social isolation stress induces hepatic hypertrophy in C57BL/6J mice.
J Toxicol Sci
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We previously reported that social isolation stimulated a stress response leading to increasing plasma corticosterone level and disruption of the hepatic lipid metabolism-related pathway, without changing body and organ weights, in mice after 4 weeks of social isolation stress, compared with the grouped-housing control (5 mice/cage). In this study, we evaluated the effects of social isolation stress for an extended period on physiologic changes in male C57BL/6J mice. Plasma corticosterone was reduced after 13 weeks, indicating mice might adapt to social isolation stress. However, body and visceral fat weights were significantly increased in combination with hepatic hypertrophy, and significant decreases in levels of triglyceride and adiponectin in plasma were observed. In conclusion, it is tempting to speculate that mice exposed to social isolation stress for 13 continuous weeks could be at an increased risk of overweight with hepatic hypertrophy. Our results also imply that physiological changes, at least fatty acid metabolism, under stress exposure might be an important factor when evaluating the chronic effects of environmental chemicals.
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Enhancement of acetaminophen-induced chronic hepatotoxicity in restricted fed rats: a nonclinical approach to acetaminophen-induced chronic hepatotoxicity in susceptible patients.
J Toxicol Sci
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Acetaminophen (APAP) is a commonly used and effective analgesic and antipyretic agent. However, some patients encounter hepatotoxicity after repeated APAP dosing at therapeutic doses. In the present study, we focused on the nutritional state as one of the risk factors of APAP-induced chronic hepatotoxicity in humans and investigated the contribution of undernourishment to susceptibility to APAP-induced chronic hepatotoxicity using an animal model mimicking undernourished patients. Rats were divided into 2 groups: the ad libitum fed (ALF) and the restricted fed (RF) rats and were assigned to 3 groups (n = 8/group) for each feeding condition. The animals were given APAP at 0, 300 and 500mg/kg for 99 days under each feeding condition. Plasma and urinary glutathione-related metabolites and liver function parameters were measured during the dosing period and hepatic glutathione levels were measured at the end of the dosing period. In the APAP-treated ALF rats hepatic glutathione levels were increased and hepatic function parameters were not changed, but in the APAP-treated RF rats hepatic glutathione levels were decreased at 500mg/kg and hepatic function parameters were increased at 300 and 500mg/kg. Moreover the urinary endogenous metabolite profile after long-term treatment with APAP in the ALF and RF rats was similar to that in human non-responders and responders to APAP-induced chronic hepatotoxicity, respectively. In conclusion, the RF rats were more sensitive to APAP-induced chronic hepatotoxicity than the ALF rats and were considered to be a useful model to estimate the contribution of the nutritional state of patients to APAP-induced chronic hepatotoxicity.
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Simplification of the genetic code: restricted diversity of genetically encoded amino acids.
Nucleic Acids Res.
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At earlier stages in the evolution of the universal genetic code, fewer than 20 amino acids were considered to be used. Although this notion is supported by a wide range of data, the actual existence and function of the genetic codes with a limited set of canonical amino acids have not been addressed experimentally, in contrast to the successful development of the expanded codes. Here, we constructed artificial genetic codes involving a reduced alphabet. In one of the codes, a tRNAAla variant with the Trp anticodon reassigns alanine to an unassigned UGG codon in the Escherichia coli S30 cell-free translation system lacking tryptophan. We confirmed that the efficiency and accuracy of protein synthesis by this Trp-lacking code were comparable to those by the universal genetic code, by an amino acid composition analysis, green fluorescent protein fluorescence measurements and the crystal structure determination. We also showed that another code, in which UGU/UGC codons are assigned to Ser, synthesizes an active enzyme. This method will provide not only new insights into primordial genetic codes, but also an essential protein engineering tool for the assessment of the early stages of protein evolution and for the improvement of pharmaceuticals.
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Green leaf volatiles enhance methyl jasmonate response in Arabidopsis.
J. Biosci. Bioeng.
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Plants emit green leaf volatiles (GLVs) in response to insect or pathogen damage. GLVs consist of C6 and C9 aldehydes, alcohols, and their acetate esters, and play important roles in the plant defense response. One of the functions of GLVs in the defense response is priming. Plants pretreated by GLVs can induce a defense response more rapidly and effectively than unpretreated plants when they are damaged by pathogens or insects. In this study, we focused on the priming effects of GLVs on jasmonic acid response involved in the defense response. When Arabidopsis was pretreated with aldehyde GLVs, especially with (E)-2-hexenal, the anthocyanin content was significantly increased by a subsequent methyl jasmonate (MeJA) treatment. On the other hand, no effect of anthocyanin accumulation was observed for plants pretreated with alcohol GLVs. These results suggest that aldehyde GLVs, especially (E)-2-hexenal, could enhance sensitivity to MeJA in Arabidopsis.
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Construction and analysis of EST libraries of the trans-polyisoprene producing plant, Eucommia ulmoides Oliver.
Planta
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Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.
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Gallic acid oxidizes Met residues in peptides released from bovine ?-lactoglobulin by in vitro digestion.
J. Biosci. Bioeng.
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Phenolic compounds (PCs) are frequently present in foods. However, little is known about the effect of PCs on enzymatic digestion process of food proteins and their products. In this study, the effect of gallic acid (GA) on in vitro digestion of ?-lactoglobulin (?-LG) was investigated as a model system for analysis of the interaction between PCs and food proteins. GA showed no effect on the initial rate of ?-LG digestion. However, after 1.5 h of digestion, the observed degree of hydrolysis of ?-LG was lower in the presence than in the absence of GA. The peptides released from ?-LG were characterized by LC/IT-TOF-MS and thirty peptides were identified. In particular, four new peaks were obtained following in vitro digestion of ?-LG in the presence of GA. Met(7), Met(24) and Met(145) in the peptides corresponding to these peaks were oxidized to methionine sulfoxide residues.
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Conventional or navigated total knee arthroplasty affects sagittal component alignment.
Knee Surg Sports Traumatol Arthrosc
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Navigation systems have recently been developed to achieve highly reliable prosthetic alignment in total knee arthroplasty (TKA). However, component alignment in the sagittal plane is less reliable than that in the coronal plane even when navigation systems are used. Previous reports examining men showed differences in targeted sagittal prosthetic alignments of TKA achieved using the conventional technique and various navigation systems. However, there have been few studies examining the use of this technique in women, who are the primary candidates for TKA.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.