Involvement of oxyradicals in promotion/inhibition of expansion growth in cucumber cotyledons

Cytokinin-induced expansion growth of cucumber cotyledons and its interaction with UV-B (280-320 nm) was studied with reference to oxyradicals. UV-B radiation enhanced the level of oxyradicals in the cotyledons measured by EPR spectroscopy. Cytokinin promoted expansion growth was inhibited by UV-B radiation. Cytokinins reduced the level of oxyradicals in dark grown cotyledons, while promoting growth. Overproduction of oxyradicals by UV-B could not be fully accounted for the inhibition of growth of cotyledons, since quenching of radicals by cytokinins did not fully restore inhibition of growth.     

Blood flow in stented arteries: a parametric comparison of strut design patterns in three dimensions

BACKGROUND: Restenosis after stent implantation varies with stent design. Alterations in secondary flow patterns and wall shear stress (WSS) can modulate intimal hyperplasia via their effects on platelet and inflammatory cell transport toward the wall, as well as direct effects on the endothelium. METHOD OF APPROACH: Detailed flow characteristics were compared by estimating the WSS in the near-strut region of realistic stent designs using three-dimensional computational fluid dynamics (CFD), under pulsatile high and low flow conditions. The stent geometry employed was characterized by three geometric parameters (axial strut pitch, strut amplitude, and radius of curvature), and by the presence or lack of the longitudinal connector. RESULTS: Stagnation regions were localized around stent struts. The regions of low WSS are larger distal to the strut. Under low flow conditions, the percentage restoration of mean axial WSS between struts was lower than that for the high flow by 10-12%. The largest mean transverse shear stresses were 30-50% of the largest mean axial shear stresses. The percentage restoration in WSS in the models without the longitudinal connector was as much as 11% larger than with the connector The mean axial WSS restoration between the struts was larger for the stent model with larger interstrut spacing. CONCLUSION: The results indicate that stent design is crucial in determining the fluid mechanical environment in an artery. The sensitivity of flow characteristics to strut configuration could be partially responsible for the dependence of restenosis on stent design. From a fluid dynamics point of view, interstrut spacing should be larger in order to restore the disturbed flow; struts should be oriented to the flow direction in order to reduce the area of flow recirculation. Longitudinal connectors should be used only as necessary, and should be parallel to the axis. These results could guide future stent designs toward reducing restenosis.     

Novel 70-kDa chondroitin sulfate/dermatan sulfate hybrid chains with a unique heterogeneous sulfation pattern from shark skin, which exhibit neuritogenic activity and binding activities for growth factors and neurotrophic factors

Chondroitin sulfate (CS) and dermatan sulfate (DS) hybrid chains of proteoglycans are critical in growth factor binding, neuritogenesis, and brain development. Here we isolated CS/DS hybrid chains from shark skin aiming to develop therapeutic agents. Digestion with various chondroitinases showed that both GlcUA- and IdoUA-containing disaccharides are scattered along the polysaccharide chains with an unusually large average molecular mass of 70 kDa. The CS/DS chains were separated into major (80%) and minor (20%) fractions by anion-exchange chromatography. Both fractions had relatively low degrees of sulfation (sulfate/disaccharide molar ratio=1.17 versus 0.87), showing a unique feature compared with the marine CS and DS isolated to date, most of which are oversulfated. They were highly heterogeneous and characterized by multiple disaccharides including GlcUA-GalNAc, GlcUA-GalNAc(6S), GlcUA-GalNAc(4S), IdoUA-GalNAc(4S), GlcUA-GalNAc(4S,6S), IdoUA-GalNAc(4S,6S), GlcUA(2S)-GalNAc(6S), and/or IdoUA(2S)-GalNAc(6S), IdoUA(2S)-GalNAc(4S) and novel GlcUA(2S)-GalNAc(4S), where 2S, 4S, and 6S represent 2-O-, 4-O- and 6-O-sulfate, respectively. The CS/DS chains bound two neurotrophic factors and various growth factors expressed in the brain with high affinity as evaluated for the major fraction by kinetic analysis using a surface plasmon resonance detector, and also promoted the outgrowth of neurites of both an axonic and a dendritic nature. The neuritogenic activity was abolished completely by digestion with chondroitinase ABC, AC-I, or B, suggesting the importance of both GlcUA- and IdoUA-containing moieties. It also showed anti-heparin cofactor II activity comparable to that exhibited by DS from porcine skin. Thus, by virtue of its unique structure and biological activities, DS will find a potential use in therapeutics.     

Differential effects of phthalates on the testis and the liver

Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) alpha is equally abundant in the liver and the testis, whereas PPAR gamma and retinoic acid receptor (RAR) alpha are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RAR alpha and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPAR alpha and PPAR gamma in Sertoli cells, but they decreased the nuclear localization of RAR alpha, as previously shown. Both PPAR alpha and PPAR gamma were in the nucleus and cytoplasm of liver cells, but RAR alpha was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RAR alpha, PPAR alpha, and PPAR gamma in these organs.     

The Rv0805 gene from Mycobacterium tuberculosis encodes a 3',5'-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis

Mycobacterium tuberculosis is an important human pathogen and has developed sophisticated mechanisms to evade the host immune system. These could involve the use of cyclic nucleotide-dependent signaling systems, since the M. tuberculosis genome encodes a large number of functional adenylyl cyclases. Using bioinformatic approaches, we identify, clone, and biochemically characterize the Rv0805 gene product, the first cyclic nucleotide phosphodiesterase identified in M. tuberculosis and a homologue of the cAMP phosphodiesterase present in Escherichia coli (cpdA). The Rv0805 gene product, a class III phosphodiesterase, is a member of the metallophosphoesterase family, and computational modeling and mutational analyses indicate that the protein possesses interesting properties not reported earlier in this class of enzymes. Mutational analysis of critical histidine and aspartate residues predicted to be essential for metal coordination reduced catalytic activity by 90-50%, and several mutant proteins showed sigmoidal kinetics with respect to Mn in contrast to the wild-type enzyme. Mutation of an asparagine residue in the GNHD motif that is conserved throughout the metallophosphoesterase enzymes almost completely abolished catalytic activity, and these studies therefore represent the first mutational analysis of this class of phosphodiesterases. The Rv0805 protein hydrolyzes cAMP and cGMP in vitro, and overexpression in Mycobacterium smegmatis and E. coli reduces intracellular cAMP levels. The presence of an orthologue of Rv0805 in Mycobacterium leprae suggests that the Rv0805 protein could have an important role to play in regulating cAMP levels in these bacteria and adds an additional level of complexity to cyclic nucleotide signaling in this organism.     

Translation of duck hepatitis B virus reverse transcriptase by ribosomal shunting

The duck hepatitis B virus (DHBV) polymerase (P) is translated by de novo initiation from a downstream open reading frame (ORF) that partially overlaps the core (C) ORF on the bicistronic pregenomic RNA (pgRNA). The DHBV P AUG is in a poor context for translational initiation and is preceded by 14 AUGs that could intercept scanning ribosomes, yet P translation is unanticipatedly rapid. Therefore, we assessed C and P translation in the context of the pgRNA. Mutating the upstream C ORF revealed that P translation was inversely related to C translation, primarily due to occlusion of P translation by ribosomes translating C. Translation of the pgRNA was found to be cap dependent, because inserting a stem-loop (BamHI-SL) that blocked >90% of scanning ribosomes at the 5' end of the pgRNA greatly inhibited C and P synthesis. Neither mutating AUGs between the C and P start sites in contexts similar to that of the P AUG nor blocking ribosomal scanning by inserting the BamHI-SL between the C and P start codons greatly altered P translation, indicating that most ribosomes that translate P do not scan through these sequences. Finally, optimizing the P AUG context did not increase P translation. Therefore, the majority of the ribosomes that translate P are shunted from a donor region near the 5' end of the pgRNA to an acceptor site at or near the P AUG, and the shunt acceptor sequences may augment initiation at the P AUG.     

Oxyradicals under UV-b stress and their quenching by antioxidants

Formation of oxyradicals under UV-B stress was investigated using cucumber cotyledons. UV-B radiation induced production of free radicals which were analyzed by ESR spectroscopy. Evidence was obtained for the formation of superoxide and hydroxyl radicals in the tissues by comparing PBN-adducts formed with radicals obtained by chemical autooxidation of KO2 and Fenton's reaction. Addition of superoxide dismutase (SOD) to the reaction mixture partially reduced the intensity of signals confirming the production of superoxide radical as well as hydroxyl radicals. These radicals were quenched in vitro by the natural antioxidants alpha-tocopherol, ascorbic acid and benzoquinone. Changes in the level of antioxidants were also monitored under UV-B stress. The endogenous level of ascorbic acid was enhanced and alpha-tocopherol level was reduced in the tissue after exposure to UV-B radiation. The present report happens to be the first direct evidence obtained for the formation of superoxide and hydroxyl radicals in plant tissues exposed to UV-B radiation.     

Estrogen receptor ligands. Part 10: Chromanes: old scaffolds for new SERAMs

The discovery, synthesis, and SAR of chromanes as ER alpha subtype selective ligands are described. X-ray studies revealed that the origin of the ER alpha-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx'd adult rat models. The best antagonists 8F and 12F also exhibited potent inhibition of MCF-7 cell growth and were shown to be estrogen receptor down-regulators (SERDs).     

Inhibition of IKK-2 by 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides

A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of the production of inflammatory cytokine IL-8 in IL-1beta stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating their potential usefulness as NF-kappaB regulators.     

Proteome analysis of the rice etioplast: metabolic and regulatory networks and novel protein functions

We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.