The disease burden from diabetic kidney disease is large and growing. Effective therapies are lacking, despite an urgent need. Hyperbaric oxygen therapy (HBOT) activates Nrf2 and cellular antioxidant defenses; therefore, it may be generally useful for treating conditions that feature chronic oxidative tissue damage. Herein, we determined how periodic exposure to oxygen at elevated pressure affected type 2 diabetes mellitus-related changes in the kidneys of db/db mice. Two groups of db/db mice, designated 2.4 ATA and 1.5 ATA, were treated four times per week with 100 % oxygen at either 1.5 or 2.4 ATA (atmospheres absolute) followed by tests to assess kidney damage and function. The sham group of db/db mice and the Hets group of db/+ mice were handled but did not receive HBOT. Several markers of kidney damage were reduced significantly in the HBOT groups including urinary biomarkers neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C (CyC) along with significantly lower levels of caspase-3 activity in kidney tissue extracts. Other stress biomarkers also showed trends to improvement in the HBOT groups, including urinary albumin levels. Expressions of the stress response genes NRF2, HMOX1, MT1, and HSPA1A were reduced in the HBOT groups at the end of the experiment, consistent with reduced kidney damage in treated mice. Urinary albumin/creatinine ratio (ACR), a measure of albuminuria, was significantly reduced in the db/db mice receiving HBOT. All of the db/db mouse groups had qualitatively similar changes in renal histopathology. Glycogenated nuclei, not previously reported in db/db mice, were observed in these three experimental groups but not in the control group of nondiabetic mice. Overall, our findings are consistent with therapeutic HBOT alleviating stress and damage in the diabetic kidney through cytoprotective responses. These findings support an emerging paradigm in which tissue oxygenation and cellular defenses effectively limit damage from chronic oxidative stress more effectively than chemical antioxidants.

Understanding how colon cancer cells survive within the inflammatory milieu of a tumor, and developing approaches that increase their sensitivity to inflammatory cytokines, may ultimately lead to novel approaches for colon cancer therapy and prevention. Analysis of a number of chemopreventive and therapeutic agents reveal that HDAC inhibitors are particularly adept at sensitizing colon cancer cells TNF or TRAIL mediated apoptosis. In vivo data are consistent with an interaction between SAHA and TNF in inducing apoptosis, as AOM-induced colon tumors express elevated levels of TNF and are more sensitive to SAHA administration. Cell cycle analysis and time-lapse imaging indicated a close correspondence between SAHA-induced prophase arrest and TNF or TRAIL-induced apoptosis. Prophase arrest induced by the Aurora kinase inhibitor VX680 likewise sensitized cells to TNF and TRAIL, with siRNA analysis pointing to Aurora kinase A (and not Aurora kinase B) as being the relevant target for this sensitization. We propose that agents that promote prophase arrest may help sensitize cancer cells to TNF and other inflammatory cytokines. We also discuss how circumvention of an early mitotic checkpoint may facilitate cancer cell survival in the inflammatory micro-environment of the tumor.

The p53 tumor suppressor protein performs a number of cellular functions, ranging from the induction of cell cycle arrest and apoptosis to effects on DNA repair. Modulating p53 activity with Mdm2 inhibitors is a promising approach for treating cancer; however, it is presently unclear how the in vivo application of Mdm2 inhibitors impact the myriad processes orchestrated by p53. Since approximately half of all colon cancers (predominately cancers with microsatellite instability) are p53‐normal, we assessed the anticancer activity of the Mdm2 inhibitor Nutlin‐3 in the mouse azoxymethane (AOM) colon cancer model, in which p53 remains wild type. Using a cell line derived from an AOM‐induced tumor, we found that four daily exposures to Nutlin‐3 induced persistent p53 stabilization and cell cycle arrest without significant apoptosis. A 4‐day dosing schedule in vivo generated a similar response in colon tumors; growth arrest without significantly increased apoptosis. In adjacent normal colon tissue, Nutlin‐3 treatment reduced both cell proliferation and apoptosis. Surprisingly, Nutlin‐3 induced a transient DNA damage response in tumors but not in adjacent normal tissue. Nutlin‐3 likewise induced a transient DNA damage response in human colon cancer cells in a p53‐dependent manner, and enhanced DNA strand breakage and cell death induced by doxorubicin. Our findings indicate that Mdm2 inhibitors not only trigger growth arrest, but may also stimulate p53's reported ability to slow homologous recombination repair. The potential impact of Nutlin‐3 on DNA repair in tumors suggests that Mdm2 inhibitors may significantly accentuate the tumoricidal actions of certain therapeutic modalities. Mol. Carcinog. © 2011 Wiley Periodicals, Inc.

Mdm2 inhibitors represent a promising class of p53 activating compounds that may be useful in cancer treatment and prevention. However, the consequences of pharmacological p53 activation are not entirely clear. We observed that Nutlin-3 triggered a DNA damage response in azoxymethane-induced mouse AJ02-NM(0) colon cancer cells, characterized by the phosphorylation of H2AX (at Ser-139) and p53 (at Ser-15). The DNA damage response was highest in cells showing robust p53 stabilization, it could be triggered by the active but not the inactive Nutlin-3 enantiomer, and it was also activated by another pharmacological Mdm2 inhibitor (Caylin-1). Quantification of gamma H2AX-positive cells following Nutlin-3 exposure showed that approximately 17% of cells in late S and G2/M were mounting a DNA damage response (compared to a approximately 50% response to 5-fluorouracil). Nutlin-3 treatment caused the formation of double-strand DNA strand breaks, promoted the formation of micronuclei, accentuated strand breakage induced by doxorubicin and sensitized the mouse colon cancer cells to DNA break-inducing topoisomerase II inhibitors. Although the HCT116 colon cancer cells did not mount a significant DNA damage response following Nutlin-3 treatment, Nutlin-3 enhanced the DNA damage response to the nucleotide synthesis inhibitor hydroxyurea in a p53-dependent manner. Finally, p21 deletion also sensitized HCT116 cells to the Nutlin-3-induced DNA damage response, suggesting that cell cycle checkpoint abnormalities may promote this response. We propose that p53 activation by Mdm2 inhibitors can result in the slowing of double-stranded DNA repair. Although this effect may suppress illegitimate homologous recombination repair, it may also increase the risk of clastogenic events.

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogen-defense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca 2+ permeable channels in mesophyll cells, resulting in cytosolic Ca 2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes ( PDF1.2 , MPK3 , and WRKY33 ) is mediated by the Ca 2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca 2+ conductance and resulting cytosolic Ca 2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca 2+ -dependent manner.

UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.

Although plants lack the mobile sentry cells present in animal innate immune systems, plants have developed complex innate immune reactions triggering basal resistance and the hypersensitive response (HR). Cytosolic Ca2+ elevation is considered to be an important early event in this pathogen response signal transduction cascade. Plasma membrane (PM)-localized cyclic nucleotide gated channels (CNGCs) contribute to the cytosolic Ca2+ rise upon pathogen perception. Recent work suggests that some PM-localized leucine-rich-repeat receptor-like kinases (LRR-RLKs) may be involved in the perception of pathogen associated molecular pattern molecules and triggering some pathogen responses in plants, some of these LRR-RLKs might have cyclic nucleotide cyclase activity. The recognition of pathogens may be connected to cyclic nucleotide generation and the activation of CNGCs, followed by cytosolic Ca2+ increase and downstream signaling events (possibly involving nitric oxide, reactive oxygen species (ROS), calmodulin (CaM), CaM-like protein (CML), and protein kinases). Notably, CaM or CML could be the crucial sensor downstream from the early Ca2+ signal leading to nitric oxide (NO) production during plant innate immune responses.

Ca2+ influx is an early signal initiating cytosolic immune responses to pathogen perception in plant cells; molecular components linking pathogen recognition to Ca2+ influx are not delineated. Work presented here provides insights into this biological system of non-self recognition and response activation. We have recently identified a cyclic nucleotide-activated ion channel as facilitating the Ca2+ flux that initiates immune signaling in the plant cell cytosol. Work in this report shows that elevation of cAMP is a key player in this signaling cascade. We show that cytosolic Ca2+ elevation, nitric oxide (NO) and reactive oxygen species generation, as well as immune signaling, lead to a hypersensitive response upon application of pathogens and/or conserved molecules that are components of microbes and are all dependent on cAMP generation. Exogenous cAMP leads to Ca2+ channel-dependent cytosolic Ca2+ elevation, NO generation, and defense response gene expression in the absence of the non-self pathogen signal. Inoculation of leaves with a bacterial pathogen leads to cAMP elevation coordinated with Ca2+ rise. cAMP acts as a secondary messenger in plants; however, no specific protein has been heretofore identified as activated by cAMP in a manner associated with a signaling cascade in plants, as we report here. Our linkage of cAMP elevation in pathogen-inoculated plant leaves to Ca2+ channels and immune signaling downstream from cytosolic Ca2+ elevation provides a model for how non-self detection can be transduced to initiate the cascade of events in the cell cytosol that orchestrate pathogen defense responses.

d-Apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of d-apiose involves UDP-d-apiose/UDP-d-xylose synthase catalyzing the conversion of UDP-d-glucuronate to a mixture of UDP-d-apiose and UDP-d-xylose. In this study we have analyzed the cellular effects of depletion of UDP-d-apiose/UDP-d-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-d-apiose/UDP-d-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of d-apiose as well as 2-O-methyl-l-fucose and 2-O-methyl-d-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a d-apiose deficiency.

OBJECTIVES We aimed to evaluate the performance of a newly designed temporary stent device as a percutaneous emergency treatment of pulmonary embolism.BACKGROUND If thrombolysis is contraindicated or recanalization by thrombolysis delayed in patients with severe pulmonary embolism who are threatened by acute circulatory failure, percutaneous temporary pulmonary stent placement may represent an additional option before surgical embolectomy is considered.METHODS The newly designed temporary pulmonary stent is made from woven Nitinol and has a distal blunt end and a proximal crimped end, which is firmly fixed to a 0.035-inch guidewire. It is delivered through a 9.5-F polytetrafluoroethylene sheath using a pusher tube. Stent placement and removal were examined in 9 anesthetized sheep with experimentally induced pulmonary embolism. Hemodynamic parameters were recorded in 7 animals.RESULTS Delivery and removal of the stent was uneventful and rapidly accomplished. Stent placement was associated with a significant decrease in Miller angiographic index (from 11.2 +/- 3.1 to 3.8 +/- 1.9; p = 0.0001), heart rate (from 139 +/- 35 beats/min to 92 +/- 11 beats/min; p = 0.0129), and mean pulmonary artery pressure (from 32 +/- 14 min Hg to 21 +/- 14 min Hg; p = 0.0029) and a significant increase in mean aortic pressure (from 48 +/- 14 min Hg to 61 8 mm Hg; p = 0.0080). Autopsy revealed neither wall damage nor parenchymal hemorrhage.CONCLUSIONS Our preliminary study proves the technical feasibility of temporary placement and removal of a newly designed dedicated pulmonary stent to recanalize centrally located embolic occlusions in severe pulmonary embolism. Animal experimental evaluation revealed rapid and significant circulatory improvement after stent placement.

One of the major sugars present in the plant cell wall is d-galacturonate, the dominant monosaccharide in pectic polysaccharides. Previous work indicated that one of the activated precursors necessary for the synthesis of pectins is UDP-d-galacturonate, which is synthesized from UDP-d-glucuronate by a UDP-d-glucuronate 4-epimerase (GAE). Here, we report the identification, cloning and characterization of a GAE6 from Arabidopsis thaliana. Functional analysis revealed that this enzyme converts UDP-d-glucuronate to UDP-d-galacturonate in vitro. An expression analysis of this epimerase and its five homologs in the Arabidopsis genome by quantitative RT-PCR and promoter::GUS fusions indicated differential expression of the family members in plant tissues and expression of all isoforms in the developing pollen of A. thaliana.

The mur4 mutant of Arabidopsis shows a 50% reduction in the monosaccharide l-Ara in leaf-derived cell wall material because of a partial defect in the 4-epimerization of UDP-d-Xyl to UDP-l-Ara. To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned by a map-based procedure and found to encode a type-II membrane protein with sequence similarity to UDP-d-Glc 4-epimerases. Enzyme assays of MUR4 protein expressed in the methylotropic yeast Pichia pastoris indicate that it catalyzes the 4-epimerization of UDP-d-Xyl to UDP-l-Ara, the nucleotide sugar used by glycosyltransferases in the arabinosylation of cell wall polysaccharides and wall-resident proteoglycans. Expression of MUR4–green fluorescent protein constructs in Arabidopsis revealed localization patterns consistent with targeting to the Golgi, suggesting that the MUR4 protein colocalizes with glycosyltransferases in the biosynthesis of arabinosylated cell wall components. The Arabidopsis genome encodes three putative proteins with >76% sequence identity to MUR4, which may explain why mur4 plants are not entirely deficient in the de novo synthesis of UDP-l-Ara.

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved α-l-fucosyl-(1→2)-β-d-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.

The mur4 mutant of Arabidopsis shows a 50% reduction in the monosaccharide L-Ara in leaf-derived cell wall material because of a partial defect in the 4-epimerization of UDP-D-Xyl to UDP-L-Ara. To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned by a map-based procedure and found to encode a type-II membrane protein with sequence similarity to UDP-D-Glc 4-epimerases. Enzyme assays of MUR4 protein expressed in the methylotropic yeast Pichia pastoris indicate that it catalyzes the 4-epimerization of UDP-D-Xyl to UDP-L-Ara, the nucleotide sugar used by glycosyltransferases in the arabinosylation of cell wall polysaccharides and wall-resident proteoglycans. Expression of MUR4-green fluorescent protein constructs in Arabidopsis revealed localization patterns consistent with targeting to the Golgi, suggesting that the MUR4 protein colocalizes with glycosyltransferases in the biosynthesis of arabinosylated cell wall components. The Arabidopsis genome encodes three putative proteins with >76% sequence identity to MUR4, which may explain why mur4 plants are not entirely deficient in the de novo synthesis of UDP-L-Ara.

The mur4 mutant of Arabidopsis shows a 50% reduction in the monosaccharide L -Ara in leaf-derived cell wall material because of a partial defect in the 4-epimerization of UDPD -Xyl to UDPL -Ara. To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned by a map-based procedure and found to encode a type-II membrane protein with sequence similarity to UDPD -Glc 4-epimerases. Enzyme assays of MUR4 protein expressed in the methylotropic yeast Pichia pastoris indicate that it catalyzes the 4-epimerization of UDPD -Xyl to UDPL -Ara, the nucleotide sugar used by glycosyltransferases in the arabinosylation of cell wall polysaccharides and wall-resident proteoglycans. Expression of MUR4–green fluorescent protein constructs in Arabidopsis revealed localization patterns consistent with targeting to the Golgi, suggesting that the MUR4 protein colocalizes with glycosyltransferases in the biosynthesis of arabinosylated cell wall components. The Arabidopsis genome encodes three putative proteins with 76% sequence identity to MUR4, which may explain why mur4 plants are not entirely deficient in the de novo synthesis of UDPL -Ara.

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl(1--> 2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltrans-ferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.