Quantitative, chemically specific imaging of selenium transformation in plants

Quantitative, chemically specific images of biological systems would be invaluable in unraveling the bioinorganic chemistry of biological tissues. Here we report the spatial distribution and chemical forms of selenium in Astragalus bisulcatus (two-grooved poison or milk vetch), a plant capable of accumulating up to 0.65% of its shoot dry biomass as Se in its natural habitat. By selectively tuning incident x-ray energies close to the Se K-absorption edge, we have collected quantitative, 100-μm-resolution images of the spatial distribution, concentration, and chemical form of Se in intact root and shoot tissues. To our knowledge, this is the first report of quantitative concentration-imaging of specific chemical forms. Plants exposed to 5 μM selenate for 28 days contained predominantly selenate in the mature leaf tissue at a concentration of 0.3–0.6 mM, whereas the young leaves and the roots contained organoselenium almost exclusively, indicating that the ability to biotransform selenate is either inducible or developmentally specific. While the concentration of organoselenium in the majority of the root tissue was much lower than that of the youngest leaves (0.2–0.3 compared with 3–4 mM), isolated areas on the extremities of the roots contained concentrations of organoselenium an order of magnitude greater than the rest of the root. These imaging results were corroborated by spatially resolved x-ray absorption near-edge spectra collected from selected 100 × 100 μm2 regions of the same tissues.

Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity

Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens.

The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene

The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (Citrullus vulgaris) cotyledons two hsps with molecular masses of 70 and 72 kDa. Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of glyoxysomes and plastids. Affinity purification and partial amino acid determination revealed the 70-kDa protein to share high sequence identity with cytosolic hsp70s from a number of plant species, while the 72 kDa protein was very similar to plastid hsp70s from pea and cucumber. A full-length cDNA clone encoding the 72-kDa hsp70 was isolated and identified two start methionines in frame within the N-terminal presequence leading either to an N-terminal extension of 67 amino acids or to a shorter one of 47 amino acids. The longer presequence was necessary and sufficient to target a reporter protein into watermelon proplastids in vitro. The shorter extension starting from the second methionine within the long version harbored a consensus peroxisomal targeting signal (RT-X5-KL) that directed in vivo a reporter protein into peroxisomes of the yeast Hansenula polymorpha. Peroxisomal targeting was however prevented, when the 67-residue presequence was fused to the reporter protein, indicating that the peroxisomal targeting signal 2 information is hidden in this context. We propose that the 72-kDa hsp70 is encoded by a single gene, but targeted alternatively into two organelles by the modulated use of its presequence.

Telomerase activity: A biomarker of cell proliferation, not malignant transformation

Telomerase activity is readily detected in most cancer biopsies, but not in premalignant lesions or in normal tissue samples with a few exceptions that include germ cells and hemopoietic stem cells. Telomerase activity may, therefore, be a useful biomarker for diagnosis of malignancies and a target for inactivation in chemotherapy or gene therapy. These observations have led to the hypothesis that activation of telomerase may be an important step in tumorigenesis. To test this hypothesis, we studied telomerase activity in isogeneic samples of uncultured and cultured specimens of normal human uroepithelial cells (HUCs) and in uncultured and cultured biopsies of superficial and myoinvasive transitional cell carcinoma (TCC) of the bladder. Our results demonstrated that four of four TCC biopsies, representing both superficial and myoinvasive TCCs, were positive for telomerase activity, but all samples of uncultured HUC were telomerase negative. However, when the same normal HUC samples were established as proliferating cultures in vitro, telomerase activity was readily detected but usually at lower levels than in TCCs. Consistent with the above observation of the telomerase activity in HUCs, telomeres did not shorten during the HUC in vitro lifespan. Demonstration of telomerase in proliferating human epithelial cells in vitro was not restricted to HUCs, because it was also present in prostate and mammary cell cultures. Notably, telomerase activity was relatively low or undetectable in nonproliferating HUC cultures. These data do not support a model in which telomerase is inactive in normal cells and activated during tumorigenic transformation. Rather, these data support a model in which the detection of telomerase in TCC biopsies, but not uncultured HUC samples, reflects differences in proliferation between tumor and normal cells in vivo.

A point mutation in the MET oncogene abrogates metastasis without affecting transformation

The MET oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF), known to stimulate invasive growth of epithelial cells. MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. To investigate whether this oncogene is directly responsible for the acquisition of the metastatic phenotype, we exploited a single-hit oncogenic version of MET, able to transform and to confer invasive and metastatic properties to nontumorigenic cells, both in vitro and in nude mice. We mutagenized the signal transducer docking site of Met (Y1349VHVX3Y1356VNV), which has the uncommon property of binding and activating multiple src homology region 2 (SH2)-containing intracellular effectors. Notably, a point mutation (H1351 → N) increased the transforming ability of the oncogene but abolished its metastatic potential. This mutation duplicates the Grb2 binding site, super-activating the Ras pathway and preventing the binding of the other intracellular transducers. Complementation in trans with another nonmetastatic mutant (N1358 → H), recruiting all the transducers downstream to Met except Grb2, rescued the invasive–metastatic phenotype. It is concluded that the metastatic potential of the MET oncogene relies on the properties of its multifunctional docking site, and that a single point mutation affecting signal transduction can dissociate neoplastic transformation from metastasis.

Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets

5′-End fragments of two genes encoding plastid-localized acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) of wheat (Triticum aestivum) were cloned and sequenced. The sequences of the two genes, Acc-1,1 and Acc-1,2, are 89% identical. Their exon sequences are 98% identical. The amino acid sequence of the biotin carboxylase domain encoded by Acc-1,1 and Acc-1,2 is 93% identical with the maize plastid ACCase but only 80–84% identical with the cytosolic ACCases from other plants and from wheat. Four overlapping fragments of cDNA covering the entire coding region were cloned by PCR and sequenced. The wheat plastid ACCase ORF contains 2,311 amino acids with a predicted molecular mass of 255 kDa. A putative transit peptide is present at the N terminus. Comparison of the genomic and cDNA sequences revealed introns at conserved sites found in the genes of other plant multifunctional ACCases, including two introns absent from the wheat cytosolic ACCase genes. Transcription start sites of the plastid ACCase genes were estimated from the longest cDNA clones obtained by 5′-RACE (rapid amplification of cDNA ends). The untranslated leader sequence encoded by the Acc-1 genes is at least 130–170 nucleotides long and is interrupted by an intron. Southern analysis indicates the presence of only one copy of the gene in each ancestral chromosome set. The gene maps near the telomere on the short arm of chromosomes 2A, 2B, and 2D. Identification of three different cDNAs, two corresponding to genes Acc-1,1 and Acc-1,2, indicates that all three genes are transcriptionally active.

Genetic transformation of HeLa cells by Agrobacterium

Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. In nature, however, Agrobacterium also may encounter organisms belonging to other kingdoms such as insects and animals that feed on the infected plants. Can Agrobacterium, then, also infect animal cells? Here, we report that Agrobacterium attaches to and genetically transforms several types of human cells. In stably transformed HeLa cells, the integration event occurred at the right border of the tumor-inducing plasmid's transferred-DNA (T-DNA), suggesting bona fide T-DNA transfer and lending support to the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells. Collectively, our results suggest that Agrobacterium can transport its T-DNA to human cells and integrate it into their genome.

Plastid redox state and sugars: Interactive regulators of nuclear-encoded photosynthetic gene expression

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway.

Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine–Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5′-untranslated region (5′-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5′-UTR and DB region promotes accumulation of NPTII up to ∼16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield (∼23% of TSP) was obtained from a construct in which the T7g10 5′-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.

Capabilities of liposomes for topological transformation

Dynamic behaviors of liposomes caused by interactions between liposomal membranes and surfactant were studied by direct real-time observation by using high-intensity dark-field microscopy. Solubilization of liposomes by surfactants is thought to be a catastrophic event akin to the explosion of soap bubbles in the air; however, the actual process has not been clarified. We studied this process experimentally and found that liposomes exposed to various surfactants exhibited unusual behavior, namely continuous shrinkage accompanied by intermittent quakes, release of encapsulated liposomes, opening up, and inside–out topological inversion.

Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes

The uptake and expression of extracellular DNA has been established as a mechanism for horizontal transfer of genes between bacterial species. Such transfer can support acquisition of advantageous elements, including determinants that affect the interactions between infectious organisms and their hosts. Here we show that erythrocyte-stage Plasmodium falciparum malaria parasites spontaneously take up DNA from the host cell cytoplasm into their nuclei. We have exploited this finding to produce levels of reporter expression in P.falciparum that are substantially improved over those obtained by electroporation protocols currently used to transfect malaria parasites. Parasites were transformed to a drug-resistant state when placed into cell culture with erythrocytes containing a plasmid encoding the human dihydrofolate reductase sequence. The findings reported here suggest that the malaria genome may be continually exposed to exogenous DNA from residual nuclear material in host erythrocytes.

Role of the cytoplasmic tyrosines of β1A integrins in transformation by v-src

GD25 cells lacking β1 integrins or expressing β1A with mutations of conserved cytoplasmic tyrosines (Y783, Y795) to phenylalanine have poor directed migration to platelet-derived growth factor or lysophosphatidic acid when compared with GD25 cells expressing wild-type β1A. We studied the effects of v-src on these cells. Transformation with v-src caused tyrosine and serine phosphorylation of wild-type β1A but not of Y783/795F doubly mutated β1A. v-src-transformed cells had rounded and/or fusiform morphology and poor assembly of fibronectin matrix. Adhesion to fibronectin or laminin and coupling of focal contacts to actin-containing cytoskeleton were preserved in transformed Y783/795F cells but lost on transformation when β1A was wild type. Transformed Y783/795F cells also retained ability, albeit limited, to migrate across filters, whereas transformed cells with wild-type β1A were unable to transverse filters. Studies of single tyrosine mutants showed that the more important tyrosine for retaining ability to adhere, assemble focal contacts, and migrate is Y783. These results suggest that overactive phosphorylation of cytoplasmic residues of β1A, particularly Y783, accounts in part for the phenotype of v-src-transformed cells.

Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for ≈25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.