Overexpression of a GmCnx1 gene enhanced activity of nitrate reductase and aldehyde oxidase, and boosted mosaic virus resistance in soybean.

Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 µg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.

Combination of all-trans retinoic acid and lithium chloride surmounts a retinoid differentiation block induced by expression of Scl and Rbtn2 transcription factors in myeloid leukemia cells.

We have previously shown that forced expression of the transcription factor Scl in WEHI-3B D(+) cells prevents ATRA-induced cell differentiation. We now find that the overexpression of Rbtn2 also interferes with induction of differentiation by ATRA. Addition of LiCl to ATRA treatment restored the capacity of both Scl- and Rbtn2-expressing cells to respond to the retinoid in a synergistic manner. Similar results were obtained with Scl-transfected HL60 cells where its expression diminished responsiveness to ATRA. These findings suggest that if Scl and/or Rbtn2 are involved in the non-responsiveness of AML patients to ATRA-induced differentiation, addition of LiCl may reverse insensitivity.

Anti-membrane-bound transferrin-like protein antibodies induce cell-shape change and chondrocyte differentiation in the presence or absence of concanavalin A.

Membrane-bound transferrin-like protein (MTf), a glycosylphosphatidylinositol-anchored protein, is expressed at high levels in many tumors and in several fetal and adult tissues including cartilage and the intestine, as well as in the amyloid plaques of Alzheimer's disease, although its role remains unknown. MTf is one of the major concanavalin A-binding proteins of the cell surface. In this study, we examined the effects of anti-MTf antibodies and concanavalin A on cell shape and gene expression, using cultures of chondrocytes and MTf-overexpressing ATDC5 and C3H10T1/2 cells. In cultures expressing MTf at high levels, concanavalin A induced cell-shape changes from fibroblastic to spherical cells, whereas no cell-shape changes were observed with wild-type ATDC5 or C3H10T1/2 cells expressing MTf at very low levels. The cell-shape changes were associated with enhanced proteoglycan synthesis and expression of cartilage-characteristic genes, including aggrecan and type II collagen. Some anti-MTf antibodies mimicked this action of concanavalin A, whereas other antibodies blocked the lectin action. The findings suggest that the crosslinking of MTf changes the cell shape and induces chondrogenic differentiation. MTf represents the first identification of a plant lectin receptor involved in cell-shape changes and the differentiation of animal cells.

Effects of zinc occupancy on human O6-alkylguanine-DNA alkyltransferase.

A recent crystallographic study of recombinant human O(6)-alkylguanine-DNA alkyltransferase (hAGT) revealed a previously unknown zinc atom [Daniels et al., (2000) EMBO J. 19, 1719-1730]. The effects of zinc on the properties of hAGT are reported here. In bacterial expression systems, recombinant hAGT was produced in increasingly larger quantities when growth media are supplemented with up to 0.1 mM ZnCl(2). Metal-enriched hAGT samples had a 5-fold increase in repair rate constant over conventionally purified protein samples and a 60-fold increase over metal-stripped hAGT. In addition, mutants of the zinc-binding residues had decreases in zinc occupancy that correlated with reductions in repair rate. Zinc modulation did not abolish the repair capacity of a fraction of the hAGT population, as evidenced by the stoichiometric reaction with an oligodeoxyribonucleotide substrate. Zinc occupancy had a similar effect on the rate of reaction with O(6)-benzylguanine, a free base substrate, as on the repair of methylated DNA. Differentially zinc-treated hAGTs showed the same affinity for binding to native DNA and substrate oligodeoxyribonucleotides. Metal content manipulations had little effect upon the CD spectrum of hAGT, but fluorescence studies revealed a small conformational change based upon metal binding, and zinc occupancy correlated with enhanced hAGT stability as evidenced by resistance to the denaturing effects of urea. These results indicate that the presence of zinc confers a mechanistic enhancement to repair activity that does not result from an increase in substrate binding affinity. Zinc also provides conformational stability to hAGT that may influence its regulation.

The effect of intracellular molybdenum in Hydrogenophaga pseudoflava on the crystallographic structure of the seleno-molybdo-iron-sulfur flavoenzyme carbon monoxide dehydrogenase.

Crystal structures of carbon monoxide dehydrogenase (CODH), a seleno-molybdo-iron-sulfur flavoprotein from the aerobic carbon monoxide utilizing carboxidotrophic eubacterium Hydrogenophaga pseudoflava, have been determined from the enzyme synthesized at high (Mo(plus) CODH) and low intracellular molybdenum content (Mo(minus) CODH) at 2.25 A and 2.35 A resolution, respectively. The structures were solved by Patterson search methods utilizing the enzyme from Oligotropha carboxidovorans as the initial model. The CODHs from both sources are structurally very much conserved and show the same overall fold, architecture and arrangements of the molybdopterin-cytosine dinucleotide-type of molybdenum cofactor, the type I and type II [2Fe-2S] clusters and the flavin-adenine dinucleotide. Unlike the CODH from O. carboxidovorans, the enzyme from H. pseudoflava reveals a unique post-translationally modified C(gamma)-hydroxy-Arg384 residue which precedes the catalytically essential S-selanyl-Cys385 in the active-site loop. In addition, the Trp193 which shields the isoalloxazine ring of the flavin-adenine dinucleotide in the M subunit of the H. pseudoflava CODH is a Tyr193 in the O. carboxidovorans CODH. The hydrogen bonding interaction pattern of the molybdenum cofactor involves 27 hydrogen bonds with the surrounding protein. Of these, eight are with the cytosine moiety, eight with the pyrophosphate, six with the pyranopterin, and five with the ligands of the Mo ion. The structure of the catalytically inactive Mo(minus) CODH indicates that an intracellular Mo-deficiency affects exclusively the active site of the enzyme as an incomplete non-functional molybdenum cofactor was synthesized. The 5'-CDP residue was present in Mo(minus) CODH, whereas the Mo-pyranopterin moiety was absent. In Mo(plus) CODH the selenium faces the Mo ion and flips away from the Mo site in Mo(minus) CODH. The different side-chain conformations of the active-site residues S-selanyl-Cys385 and Glu757 in Mo(plus) and Mo(minus) CODH indicate a side-chain flexibility and a function of the Mo ion in the proper orientation of both residues.

Azuki bean cells are hypersensitive to cadmium and do not synthesize phytochelatins.

Suspension-cultured cells of azuki bean (Vigna angularis) as well as the original root tissues were hypersensitive to Cd (<10 microM). Repeated subculturings with a sublethal level of Cd (1-10 microM) did not affect the subsequent response of cells to inhibitory levels of Cd (10-100 microM). The azuki bean cells challenged to Cd did not contain phytochelatin (PC) peptides, unlike tomato (Lycopersicon esculentum) cells that have a substantial tolerance to Cd (>100 microM). Both of the cell suspensions contained a similar level of reduced glutathione (GSH) when grown in the absence of Cd. Externally applied GSH to azuki bean cells recovered neither Cd tolerance nor PC synthesis of the cells. Furthermore, enzyme assays in vitro revealed that the protein extracts of azuki bean cells had no activity converting GSH to PCs, unlike tomato. These results suggest that azuki bean cells are lacking in the PC synthase activity per se, hence being Cd hypersensitive. We concluded that the PC synthase has an important role in Cd tolerance of suspension-cultured cells.

Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus.

Pleurotus ostreatus is a white rot basidiomycete that produces several extracellular laccase isoenzymes, including phenol oxidase A1b (POXA1b), POXA2, and POXC. POXC was the most abundant isoenzyme produced under all of the growth conditions examined in this study. Copper was the most efficient inducer of laccase activity among the putative inducers tested. The amounts of all of the previously described laccase isoenzymes increased substantially in copper-supplemented cultures. Under these conditions expression of POX isoenzymes was regulated at the level of gene transcription. It is worth noting that poxa1b mRNA was the most abundant induced transcript at all of the growth times analyzed, and the amount of this mRNA increased until day 7. The discrepancy between the poxa1b transcript and protein amounts can be explained by the presence of a high level of the protein in P. ostreatus cellular extract, which indicated that the POXA1b isoenzyme could be inefficiently secreted and/or that its physiological activity could occur inside the cell or on the cell wall. Moreover, the POXA1b isoenzyme behaved uniquely, as its activity was maximal on the second day of growth and then decreased. An analysis performed with protease inhibitors revealed that the loss of extracellular POXA1b activity could have been due to the presence of specific proteases secreted into the copper-containing culture medium that affected the extracellular POXA1b isoenzyme.

Molecular cloning of the cDNA coding for mouse aldehyde oxidase: tissue distribution and regulation in vivo by testosterone.

The cDNA coding for mouse aldehyde oxidase (AO), a molybdoflavoprotein, has been isolated and characterized. The cDNA is 4347 nt long and consists of an open reading frame predicting a polypeptide of 1333 amino acid residues, with 5' and 3' untranslated regions of 13 and 335 nt respectively. The apparent molecular mass of the translation product in vitro derived from the corresponding cRNA is consistent with that of the monomeric subunit of the AO holoenzyme. The cDNA codes for a catalytically active form of AO, as demonstrated by transient transfection experiments conducted in the HC11 mouse mammary epithelial cell line. The deduced primary structure of the AO protein contains consensus sequences for two distinct 2Fe-2S redox centres and a molybdopterin-binding site. The amino acid sequence of the mouse AO has a high degree of similarity with the human and bovine counterparts, and a significant degree of relatedness to AO proteins of plant origin. Northern blot and in situ hybridization analyses demonstrate that hepatocytes, cardiocytes, lung endothelial or epithelial cells and oesophagus epithelial cells express high levels of AO mRNA. In the various tissues and organs considered, the level of AO mRNA expression is not strictly correlated with the amount of the corresponding protein, suggesting that the synthesis of the AO enzyme is under translational or post-translational control. In addition, we observed sex-related regulation of AO protein synthesis. In the liver of male animals, despite similar amounts of AO mRNA, the levels of the AO enzyme and corresponding polypeptide are significantly higher than those in female animals. Treatment of female mice with testosterone increases the amounts of AO mRNA and of the relative translation product to levels similar to those in male animals.

A zyxin-related protein whose synthesis is reduced in virally transformed fibroblasts.

We have cloned the gene for a novel LIM-domain protein from human fibroblasts whose expression is substantially decreased in simian-virus-40-(SV40)-transformed cells. This protein has a calculated molecular mass of 61 kDa and comprises a proline-rich domain followed by three LIM motifs. It appears to be identical to the focal adhesion protein p83 that has recently been isolated and characterized from porcine and human platelets. Hybridization experiments demonstrate a very low degree of evolutionary conservation of its sequence between mammals and birds. It is therefore possible that the novel protein represents the human equivalent of the chicken protein zyxin as the two proteins display a very similar overall structure, although their amino acid sequences diverge markedly from each other. The repression of this zyxin-related protein in virally transformed fibroblasts may explain, at least in part, the dramatic morphological changes that occur at the cell surface and in the cytoskeleton of transformed cells.

Genetic modification of plants: significant issues and hurdles to success.

Transformation and regeneration is routine for many crop plants. A genetically engineered tomato with a longer shelf life at full ripeness was introduced in the United States in 1994, and other soon-to-be-released products, both foods and fibers, incorporate genes for resistance to pests, diseases, and environmentally benign herbicides. Other possibilities are altered plant fats and oils, methionine- and lysine-enhanced grain and legume proteins, plant foods that can deliver immunizing antigens, and other ways of controlling fruit ripening. Food safety concerns include the inadvertent production of toxicants and allergens. Foreign DNA can be introduced into plants by bacterial vectors, direct uptake by protoplasts, and mechanical introduction on metal particles or other materials. Limitations include little or no control of copy number or site of integration of the introduced DNA, dependence on selectable markers for recovery of traits, and inadequate knowledge of how to control key metabolic steps to maximize desirable traits. Directed genetic change still requires conventional crop breeding to deliver benefits to farmers and consumers.

Metallopanstimulin gene product produced in a baculovirus expression system is a nuclear phosphoprotein that binds to DNA.

The protein product of the human Metallopanstimulin (MPS-1) gene was produced in the insect cell line Spodoptera frugiperda (Sf9) using the baculovirus expression vector system Autographa californica nuclear polyhedrosis virus (AcNPV). When a cloned MPS-1 complementary DNA sequence was inserted into the AcNPV viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately 10,000 was observed in extracts of infected Sf9 cells. This protein was not detected in Sf9 cells infected with AcNPV-MPS-1-Del, a vector in which the MPS-1 gene was deleted. The MPS-1 protein was produced at high levels in this host-vector system (congruent to 12% of total labeled soluble protein). Characterization of the MPS-1 protein extracted from Sf9 infected cells showed that it: binds zinc ions specifically; is phosphorylated; accumulates in the nucleus; is tightly bound to the nucleus; and binds to calf thymus DNA-cellulose. The MPS-1 protein constitutes one of the major proteins in the nuclear fraction of Sf9 cells and can be purified from this source to near homogeneity by a two-step procedure composed of high-performance liquid chromatography and gel electrophoresis. Antibodies were raised against selected peptide sequences of the MPS-1 protein and used to detect MPS-1 in insect cells and in various cultured mammalian cell types. Western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunological properties equivalent to those of MPS-1 spontaneously expressed in various human mammalian cell lines. Finally, recombinant MPS-1 generated a specific protein-DNA complex with a duplex oligomer containing a cyclic AMP-responsive element, as assessed by gel mobility shift assays. These results support the hypothesis that the MPS-1 protein may act, at least in part, by interacting with genes possessing the cyclic AMP-responsive element sequence.

Effect of selenium deficiency on type I 5'-deiodinase.

The type I iodothyronine 5'-deiodinase (5'-DI) present in rat liver and kidney has recently been demonstrated to be a selenoprotein. The goal of the present study was to examine in detail the effect of selenium (Se) deficiency on 5'-DI at the protein and mRNA levels. In weanling rats fed a selenium-deficient (Se(-)) diet for 6 weeks, 5'-DI activity was decreased 91 and 69% relative to control activities in liver and kidney, respectively. Administration of 3,5,3'-triiodothyronine resulted in a 2-fold increase in 5'-DI activity in control animals, but had little or no effect on 5'-DI activity in Se(-) animals. Western analysis using a specific antiserum directed against a bacterial fusion protein containing the carboxyl-terminal half of the 5'-DI protein demonstrated that this decrease in 5'-DI activity in Se(-) animals was explained by a marked decrease in 5'-DI protein. Administration of Se to Se(-) animals resulted in parallel increases in 5'-DI protein and activity over a 72-h time period. It was also shown that selenium deficiency was accompanied by a 40% decrease in 5'-DI mRNA levels in the kidney, but not in the liver. In both tissues, the administration of 3,5,3'-triiodothyronine resulted in increased 5'-DI mRNA levels which were not altered by selenium status. These studies indicate that selenium deficiency decreases 5'-DI activity by decreasing the amount of 5'-DI protein. The mechanism of this impairment in enzyme synthesis appears to be a defect in translation, presumably due to a block in the UGA-directed selenocysteine incorporation in selenium deficiency.

Increased metallothionein content in rat liver induced by X irradiation and exposure to high oxygen tension.

X irradiation and exposure to high oxygen tension are known to induce lipid peroxidation. The effects of these stresses on hepatic content of metallothionein, which may be involved in the regulation of zinc and copper metabolism, have been studied. The amount of metallothionein in rat liver was increased 11-fold by a high dose of X irradiation (1000 R). Increased metallothionein content (about 15 times) was also observed in liver of rats exposed to high oxygen tension for 3 days.

Inducible protective proteins: a potentially novel approach to chemotherapy.

A number of toxic chemical and physical agents elicit the induction of a series of protein species, some of which react with the agents and render them nontoxic. A few of the induced species (such as metallothionein) are rich in thiol groups that might be expected to react with alkylating agents and render them nontoxic. If a safe means could be found for selectively enhancing the synthesis of alkylating-agent-reactive species in normal but not tumor cells, such a procedure would have ramifications in the area of cancer chemotherapy. In this report, we have utilized a variety of trace elements (Zn, Se, Cu, As) as inducers of synthesis of protective species in line CHO Chinese hamster cells and in a number of derived variants to determine whether this type of approach can be utilized to increase resistance to alkylating-agent toxicity. Our results indicate that Zn, Se and Cu elicit a protective response (increased survival, monitored by colony-forming ability) against the toxic effects of iodoacetate or melphalan, and, at least in the case of zinc, at levels that are physiologically reasonable. Arsenite appears to be a marginally effective inducer in the CHO cell and an ineffective inducer in the Cdr20F4 variant cell. The increased survival is not attributable to metallothionein inducibility, decreased availability of the alkylating agent in the medium, or decreased uptake of the drug into the trace-element-pretreated cells. The protective responses induced by zinc or selenite alone are additive in cells receiving both trace elements prior to exposure to alkylating agent, which suggests that different domains of response are elicited by the two metals. In view of reported differences in inducibility of protective proteins between normal and tumor cells, a possibility is raised for a novel approach to alkylating-agent chemotherapy that is somewhat analogous to the protocol utilized in high-dose methotrexate therapy.

Regulation of intestinal metallothionein biosynthesis in rats by dietary zinc.

Response of intestinal metallothionein and zinc to dietary zinc intake was investigated in zinc-depleted rats, after a single feeding by stomach tube of a purified diet supplemented with either 125 ppm zinc or less than 1 ppm zinc. Serum zinc concentration was maximal 3 hours post-feeding, then stabilized at lower levels returning to baseline concentration by 18 hours. Mucosal zinc concentration rose to a maximum by 6 hours after feeding and plateaued at slightly lower levels thereafter, due to association of this zinc with metallothionein. The rate of metallothionein synthesis rose 4-fold, 6-9 hours after feeding, coinciding with the maximal contest of translatable metallothionein mRNA concentration. Together, these findings provide strong evidence for a regulatory role of metallothionein in zinc absorption and homeostasis, where dietary zinc influences subcellular events in mucosa affecting regulation of the metallothionein gene. These events include the induction of de novo metallothionein synthesis, preceded by an increase in translatable mRNA specific for this metalloprotein, and followed by a reduction in zinc absorption simultaneous with the binding of absorbed zinc with newly synthesized thionein polypeptides.

Synthesis of a metallothionein-like protein in cultured human skin fibroblasts: relation to abnormal copper distribution in Menkes' disease.

A metallothionein-like protein (MTP) is synthesized in normal diploid human skin fibroblasts cultured in Zn- or Cu-supplemented medium. Synthesis of MTP is not detected in cells cultured without metal supplementation of complete tissue-culture medium. Cultured fibroblasts from patients with Menkes' disease accumulate excess Cu which chromatographs both with high-molecular-weight protein(s) and with a Cu-MTP. Under normal culture conditions, the Menkes' MTP incorporates [35S]-cystine, but not appreciable amounts of 65Zn. However, Menkes fibroblasts retain the ability to incorporate 65Zn into MTP in response to Zn supplementation of the medium. The results do not support the idea that Menkes' disease results from a failure of Cu to bind to MTP, but rather that an elevated intracellular Cu concentration in Menkes' disease fibroblasts leads to association of excess Cu with high-molecular-weight protein, stimulating synthesis of a Cu-binding MTP.

The production in culture of metalloproteinases and an inhibitor by joint tissues from normal rabbits, and from rabbits with a model arthritis. I. Synovium.

During 5 days of culture, explants of normal rabbit synovium produced no active collagenase, negligible latent collagenase, but significant levels of free collagenase inhibitor. Synovium from joints exhibiting a proliferative arthritis produced greatly elevated levels of collagenase; the appearance of active enzyme in the medium during the second day of culture was associated with the disappearance of free inhibitor. Enzyme levels in the media correlated well with the arthritic status of joints, when explants were prepared up to 10 weeks after the induction of the model arthritis. Synovium from the contralateral joints of rabbits with unilaterally induced arthritis produced no active collagenase, but approximately one-third as much latent collagenase as found with arthritic joints. Enzymatic activities against gelatin and cartilage proteoglycan substrates were demonstrated in synovial culture media in addition to collagenolytic activity. Gel filtration showed that these activities were not due to a single enzyme, and further characterisation confirmed that the enzymes were metalloproteinases. The results are considered in the light of published data, and the involvement of metalloproteinases and their specific inhibitor in the development of arthritic lesions is discussed.

The production in culture of metalloproteinases and an inhibitor by joint tissues from normal rabbits, and from rabbits with a model arthritis. II. Articular cartilage.

During the development of proliferative arthritis in the knee joints of rabbits, there was a large increase in the ability of articular cartilage explants to produce latent collagenase in culture. In parallel, the normally high levels of collagenase inhibitor produced by cartilage in culture fell, but active collagenase was never detectable. Characterisation of the collagenase and other proteinase activities produced by rabbit articular cartilage in culture showed that two activities could be separated by gel filtration, one with activities on gelatin and cartilage proteoglycan and the other degrading collage. Under the conditions employed in this paper no resolution of the gelatin and proteoglycan activities could be achieved. All the activities were in a latent form, activated by 4-aminophenylmercuric acetate (APMA), and inhibited by 1,10-phenanthroline or EDTA, but not by di-isopropylfluorophosphate (DFP), indicating that they are metalloproteinases. Characterization of the collagenase inhibitor showed a single peak of activity of apparent molecular weight of 28,000 on gel filtration. The inhibitor was sensitive to APMA and also inhibited other rabbit metalloproteinases, analogous to the system described for rabbit bone. The physiological significance of the synthesis by articular cartilage of proteinases that destroy connective tissue macromolecules and the presence of an enzyme-inhibitor control system is discussed.