Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment.
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It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction. (+info)
Asparagine-linked oligosaccharides protect Lamp-1 and Lamp-2 from intracellular proteolysis.
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Lysosomes contain several integral membrane proteins (termed Lamps and Limps) that are extensively glycosylated with asparagine-linked oligosaccharides. It has been postulated that these glycans protect the underlying polypeptides from the proteolytic environment of the lysosome. Previous attempts to test this hypothesis have been inconclusive because they utilized approaches that prevent initial glycosylation and thereby impair protein folding. We have used endoglycosidase H to remove the Asn-linked glycans from fully folded lysosomal membrane proteins in living cells. Deglycosylation of Lamp-1 and Lamp-2 resulted in their rapid degradation, whereas Limp-2 was relatively stable in the lysosome in the absence of high mannose Asn-linked oligosaccharides. Depletion of Lamp-1 and Lamp-2 had no measurable effect on endosomal/lysosomal pH, osmotic stability, or density, and cell viability was maintained. Transport of endocytosed material to dense lysosomes was delayed in endoglycosidase H treated cells, but the rate of degradation of internalized bovine serum albumin was unchanged. These data provide direct evidence that Asn-linked oligosaccharides protect a subset of lysosomal membrane proteins from proteolytic digestion in intact cells. (+info)
Cell cycling and cell enlargement in developing leaves of Arabidopsis.
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Cell cycling plays an important role in plant development, including: (1) organ morphogenesis, (2) cell proliferation within tissues, and (3) cell differentiation. In this study we use a cyclin::beta-glucuronidase reporter construct to characterize spatial and temporal patterns of cell cycling at each of these levels during wild-type development in the model genetic organism Arabidopsis thaliana (Columbia). We show that a key morphogenetic event in leaf development, blade formation, is highly correlated with localized cell cycling at the primordium margin. However, tissue layers are established by a more diffuse distribution of cycling cells that does not directly involve the marginal zone. During leaf expansion, tissue proliferation shows a strong longitudinal gradient, with basiplastic polarity. Tissue layers differ in pattern of proliferative cell divisions: cell cycling of palisade mesophyll precursors is prolonged in comparison to that of pavement cells of the adjacent epidermal layers, and cells exit the cycle at different characteristic sizes. Cell divisions directly related to formation of stomates and of vascular tissue from their respective precursors occur throughout the period of leaf extension, so that differing tissue patterns reflect superposition of cycling related to cell differentiation on more general tissue proliferation. Our results indicate that cell cycling related to leaf morphogenesis, tissue-specific patterns of cell proliferation, and cell differentiation occurs concurrently during leaf development and suggest that unique regulatory pathways may operate at each level. (+info)
An imperfect heat shock element and different upstream sequences are required for the seed-specific expression of a small heat shock protein gene.
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Chimeric constructs containing the promoter and upstream sequences of Ha hsp17.6 G1, a small heat shock protein gene, reproduced in transgenic tobacco (Nicotiana tabacum) its unique seed-specific expression patterns previously reported in sunflower. These constructs did not respond to heat shock, but were expressed without exogenous stress during late zygotic embryogenesis coincident with seed desiccation. Site-directed mutagenesis of its distal and imperfect heat shock element strongly impaired in vitro heat shock transcription factor binding and transgene expression in seeds. Deletion analyses of upstream sequences indicated the contribution of additional cis-acting elements with either positive or negative effects on transgene expression. These results show differences in the transcriptional activation through the heat shock element of small heat shock protein gene promoters in seeds compared with the heat shock response. In addition, they suggest that heat shock transcription factors and other distinct trans-acting factors cooperate in the regulation of Ha hsp17.6 G1 during seed desiccation. (+info)
Lymphokine-induced production and release of lysosomal enzymes by macrophages.
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MACROPHAGES ARE ASSOCIATED WITH MOST CHRONIC INFLAMMATORY LESIONS, AND THESE CELLS CONTAIN ENZYMES THAT ARE ABLE TO DESTROY CONNECTIVE TISSUE CONSTITUENTS. Normal lymphoid cells responding to a mitogen, phytohemagglutinin-P, release factor(s) that cause a marked increase in the size and enzyme content for mononuclear phagocytes maintained in culture. The stimulated macrophages, which by several criteria remain otherwise viable and healthy, selectively release large quantities of hydrolytic enzymes to the culture medium. (+info)
Biotransformation of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, in mice, rats, rabbits, dogs, monkeys, and chimpanzees.
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The study objectives were to characterize the metabolism of nevirapine (NVP) in mouse, rat, rabbit, dog, monkey, and chimpanzee after oral administration of carbon-14-labeled or -unlabeled NVP. Liquid scintillation counting quantitated radioactivity and bile, plasma, urine, and feces were profiled by HPLC/UV diode array and radioactivity detection. Metabolite structures were confirmed by UV spectral and chromatographic retention time comparisons with synthetic metabolite standards, by beta-glucuronidase incubations, and in one case, by direct probe electron impact ionization/mass spectroscopy, chemical ionization/mass spectroscopy, and NMR. NVP was completely absorbed in both sexes of all species except male and female dogs. Parent compound accounted for <6% of total urinary radioactivity and <5.1% of total fecal radioactivity, except in dogs where 41 to 46% of the radioactivity was excreted as parent compound. The drug was extensively metabolized in both sexes of all animal species studied. Oxidation to hydroxylated metabolites occurred before glucuronide conjugation and excretion in urine and feces. Hydroxylated metabolites were 2-, 3-, 8-, and 12-hydroxynevirapine (2-, 3-, 8-, and 12-OHNVP). 4-carboxynevirapine, formed by secondary oxidation of 12-OHNVP, was a major urinary metabolite in all species except the female rat. Glucuronides of the hydroxylated metabolites were major or minor metabolites, depending on the species. Rat plasma profiles differed from urinary profiles with NVP and 12-OHNVP accounting for the majority of the total radioactivity. Dog plasma profiles, however, were similar to the urinary profiles with 12-OHNVP, its glucuronide conjugate, 4-carboxynevirapine, and 3-OHNVP glucuronide being the major metabolites. Overall, the same metabolites are formed in animals as are formed in humans. (+info)
Effects of 60 Hz magnetic field exposure on polymorphonuclear leukocyte activation.
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We have investigated the effects of a sinusoidal 60 Hz magnetic field on free radical (superoxide anion) production, degranulation (beta-glucuronidase and lysozyme release) and viability in human neutrophils (PMNs). Experiments were performed blindly in very controlled conditions to examine the effects of a magnetic field in resting PMNs and in PMNs stimulated with a tumor promoter: phorbol 12-myristate 13-acetate (PMA). Exposure of unstimulated human PMNs to a 60 Hz magnetic field did not affect the functions examined. In contrast, exposure of PMNs to a 22 milliTesla (mT), 60 Hz magnetic field induced significant increases in superoxide anion (O2-) production (26.5%) and in beta-glucuronidase release (53%) when the cells were incubated with a suboptimal stimulating dose of PMA. Release of lysozyme and lactate dehydrogenase was unchanged by the magnetic field, whether the cells were stimulated or not. A 60 Hz magnetic field did not have any effect on O2- generation by a cell-free system xanthine/xanthine oxidase, suggesting that a magnetic field could upregulate common cellular events (signal transduction) leading to O2- generation and beta-glucuronidase release. In conclusion, exposure of PMNs to a 22 mT, 60 Hz magnetic field potentiates the effect of PMA on O2- generation and beta-glucuronidase release. This effect could be the result of an alteration in the intracellular signaling. (+info)
Heat shock response: presence and effects in burn patient neutrophils.
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Heat shock proteins (HSPs) are present in neutrophils (PMNs) from critically ill patients. We investigated whether HSPs were present in PMNs from burn patients and whether heat shock contributed to the functional defects observed in burn PMNs. Using both flow cytometry and Western blot techniques it was observed that inducible HSP72 (iHSP72) was present in PMNs and leukocytes from burn patients, especially in patients with inhalation injury. Similar to burn PMNs, and in contrast to normal cells, heat shocked PMNs (43 degrees C incubation) expressed iHSP72 and were unable to increase the expression of CD11b/CD18 in response to pro-inflammatory stimuli. Degranulation after pro-inflammatory stimuli was decreased for both burn- and heat-shocked PMNs when compared to normal controls. In burn PMNs these functional abnormalities were mainly due to decreased quantities of proteins (CD11b, albumin, B12 binding protein, beta-glucuronidase) present within cytoplasmic granules. However, in heat-shocked PMNs the abnormalities were primarily related to abnormal exocytosis. In conclusion, our data show that decreased quantities of cytoplasmic granule proteins and, to a smaller degree, defective exocytosis are involved in the functional abnormalities observed in burn PMNs. (+info)