Choline phospholipid metabolites of human vascular endothelial cells altered by cyclooxygenase inhibition,growth factor depletion,and paracrine factors secreted by cancer cells

Magnetic resonance studies have previously shown that solid tumors and cancer cells in culture typically exhibit high phosphocholine and total choline. Treatment of cancer cells with the anti-inflammatory agent, indomethacin (INDO), reverted the phenotype of choline phospholipid metabolites in cancer cells towards a less malignant phenotype. Since endothelial cells form a key component of tumor vasculature, in this study, we used MR spectroscopy to characterize the phenotype of choline phospholipid metabolites in human umbilical vein endothelial cells (HUVECs). We determined the effect of growth factors, the anti-inflammatory agent INDO, and conditioned media obtained from a malignant cell line, on choline phospholipid metabolites. Growth factor depletion or treatment with INDO induced similar changes in the choline phospholipid metabolites of HUVECs. Treatment with conditioned medium obtained from MDA-MB-231 cancer cells induced changes similar to the presence of growth factor supplements. These results suggest that cancer cells secrete growth factors and/or other molecules that influence the choline phospholipid metabolism of HUVECs. The ability of INDO to alter choline phospholipid metabolism in the presence of growth factor supplements suggests that the inflammatory response pathways of HUVECs may play a role in cancer cell-HUVEC interaction and in the response of HUVECs to growth factors.     

Role of sphingosine-1 phosphate in the enhancement of endothelial barrier integrity by platelet-released products

In vitro and in vivo evidence indicates that circulating platelets affect both vascular integrity and hemostasis. How platelets enhance the permeability barrier of the vascular endothelium is not well understood. We measured the effect of isolated human platelets on human pulmonary artery endothelial cell (EC) barrier integrity by monitoring transmonolayer electrical resistance. EC barrier function was significantly increased by the addition of platelets ( approximately 40% maximum, 2.5 x 106 platelets/ml). Platelet supernatants, derived from 2.5 x 106 platelets/ml, reproduced the barrier enhancement and reversed the barrier dysfunction produced by the edemagenic agonist thrombin, which implicates a soluble barrier-promoting factor. The barrier-enhancing effect of platelet supernatants was heat stable but was attenuated by either charcoal delipidation (suggesting a vasoactive lipid mediator) or pertussis toxin, implying involvement of a Gialpha-coupled receptor signal transduction pathway. Sphingosine-1-phosphate (S1P), a sphingolipid that is released from activated platelets, is known to ligate G protein-coupled EC differentiation gene (EDG) receptors, increase EC electrical resistance, and reorganize the actin cytoskeleton (Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, and English D. J Clin Invest 108: 689-701, 2001). Infection of EC with an adenoviral vector expressing an antisense oligonucleotide directed against EDG-1 but not infection with control vector attenuated the barrier-enhancing effect of both platelet supernatants and S1P. These results indicate that a major physiologically relevant vascular barrier-protective mediator produced by human platelets is S1P.     

A statistical approach to computer processing of cryo-electron microscope images: virion classification and 3-D reconstruction

The scattering density of the virus is represented as a truncated weighted sum of orthonormal basis functions in spherical coordinates, where the angular dependence of each basis function has icosahedral symmetry. A statistical model of the image formation process is proposed and the maximum likelihood estimation method computed by an expectation-maximization algorithm is used to estimate the weights in the sum and thereby compute a 3-D reconstruction of the virus particle. If multiple types of virus particle are represented in the boxed images then multiple 3-D reconstructions are computed simultaneously without first requiring that the type of particle shown in each boxed image be determined. Examples of the procedure are described for viruses with known structure: (1). 3-D reconstruction of Flockhouse Virus from experimental images, (2). 3-D reconstruction of the capsid of Nudaurelia Omega Capensis Virus from synthetic images, and (3). 3-D reconstruction of both the capsid and the procapsid of Nudaurelia Omega Capensis Virus from a mixture of unclassified synthetic images.     

Accumulation of LEA proteins in salt (NaCl) stressed young seedlings of rice (Oryza sativa L.) cultivar Bura Rata and their degradation during recovery from salinity stress

Germination and subsequent hydroponic growth under salt stress (100 mmol/L NaCl) triggered an accumulation of six major stress proteins and resulted in a growth arrest of young seedlings of rice (Oryza sativa L.) cv. Bura Rata. Based on two-dimensional electrophoretic resolution, partial amino acid sequencing and immunodetection techniques, four of the salt stress-induced polypeptides were identified as LEA proteins. Under all experimental conditions wherein seedlings exhibited superior halotolerance, salt stress-induced LEA proteins were expressed at low levels. In contrast, accumulation of LEA proteins was found associated with growth arrest. When returned to non-saline media, seedlings stressed with salt for four days recovered immediately. Longer exposure to 100 mmol/L NaCl, however, progressively delayed recovery and reduced the number of seedlings which could recover from salt stress. Recovery from salt stress was consistently accompanied by degradation of the salt stress-induced LEA proteins. The results of this study show that LEA proteins accumulate during the salinity-triggered growth arrest of young Bura Rata seedlings and are mobilised during the recovery of seedlings from salinity stress.     

Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures

Cultures of dissociated foetal and postnatal mouse gut gave rise to neurosphere-like bodies, which contained large numbers of mature neurons and glial cells. In addition to differentiated cells, neurosphere-like bodies included proliferating progenitors which, when cultured at clonal densities, gave rise to colonies containing many of the neuronal subtypes and glial cells present in the mammalian enteric nervous system. These progenitors were also capable of colonising wild-type and aganglionic gut in organ culture and had the potential to generate differentiated progeny that localised within the intrinsic ganglionic plexus. Similar progenitors were also derived from the normoganglionic small intestine of mice with colonic aganglionosis. Our findings establish the feasibility of expanding and isolating early progenitors of the enteric nervous system based on their ability to form distinct neurogenic and gliogenic structures in culture. Furthermore, these experiments provide the rationale for the development of novel approaches to the treatment of congenital megacolon (Hirschsprung's disease) based on the colonisation of the aganglionic gut with progenitors derived from normoganglionic bowel segments.     

Related Galpha subunits play opposing roles during Dictyostelium development

Of the several known Dictyostelium G protein subunits, the Galpha4 and Galpha5 subunits are the most closely related pair based on phylogenetic analysis and expression patterns, but these subunits perform different roles during development. To investigate potential relationships between these subunits with respect to cell differentiation, chimeric organisms composed of strains lacking or overexpressing either subunit were created and examined for developmental morphogenesis and spore production. Chimeras of galpha4 null and galpha5 null strains or Galpha4 and Galpha5 overexpression strains displayed compensatory morphogenesis, implying that the subunits promote complementary developmental processes. However, chimeras composed of galpha4 null and Galpha5 overexpression strains or galpha5 null and Galpha4 overexpression strains displayed distorted tip morphogenesis, suggesting the strains of these chimeras share common developmental deficiencies. Cells lacking the Galpha5 subunit localized to the prespore region of chimeras similar to the pattern observed for cells overexpressing the Galpha4 subunit, and cells overexpressing the Galpha5 subunit displayed localization patterns similar to galpha4 null mutants. A strain overexpressing both subunits displayed a partial suppression of morphology, gene expression, and cell localization phenotypes associated with the overexpression of the individual Galpha subunit genes, suggesting that each Galpha subunits can inhibit signaling mediated by the other subunit. Overexpression of the Galpha5 subunit inhibited chemotaxis and cGMP accumulation in response to folic acid, indicating that the Galpha5 subunit can inhibit early steps in the Galpha4-mediated signal transduction pathway. The contrasting phenotypes of the Galpha mutants suggest the Galpha4 and Galpha5 subunits provide opposing functions in cell differentiation, localization, and chemotactic responses to folic acid.     

Oxalate decarboxylase from Collybia velutipes. Molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato

Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mechanism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5'-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (Mehta, A., and Datta, A. (1991) J. Biol. Chem. 266, 23548-23553). By screening a genomic library from Collybia velutipes with this cDNA as a probe, a genomic clone has been isolated. Sequence analyses and comparison of the genomic sequence with the cDNA sequence revealed that the cDNA is interrupted with 17 small introns. The cDNA has been successfully expressed in cytosol and vacuole of transgenic tobacco and tomato plants. The transgenic plants show normal phenotype, and the transferred trait is stably inherited to the next generation. The recombinant enzyme is partially glycosylated and shows oxalate decarboxylase activity in vitro as well as in vivo. Transgenic tobacco and tomato plants expressing oxalate decarboxylase show remarkable resistance to phytopathogenic fungus Sclerotinia sclerotiorum that utilizes oxalic acid during infestation. The result presented in the paper represents a novel approach to develop transgenic plants resistant to fungal infection.