Sucrose transporter plays a role in phloem loading in CMV-infected melon plants that are defined as symplastic loaders.

Based on the high density of plasmodesmata interconnecting the intermediary cells and their neighboring phloem parenchyma or bundle-sheath cells, and based on the insensitivity to the sucrose transport inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), cucurbits have been concluded to be symplastic loaders. In the present study, we identified and characterized the full-length sequence of sucrose transporter gene (CmSUT1) from melon (Cucumis melo L. cv. Hale's best jumbo). In vitro experiments confirmed that the identified gene product has sucrose transporter activity in baker's yeast. Healthy and cucumber mosaic virus (CMV)-infected melon plants were employed to examine sucrose transporter activity in planta. Pretreatment with PCMBS inhibited loading of newly fixed ¹4CO2 into minor veins of CMV-infected plants. Moreover, CMV infection caused significant increase in CmSUT1 transcripts expression, mainly in vascular bundles of minor veins, which was associated with elevated sucrose content in phloem sap collected from source-leaf petioles. We propose that cucurbit plants contain the machinery for apoplastic phloem loading and that CMV infection causes a quantitative shift in the mode by which photoassimilates are loaded into the sieve tube.

Vorinostat enhances the antimyeloma effects of melphalan and bortezomib.

OBJECTIVES: Examine the antitumor activity of the histone deacetylase inhibitor vorinostat's antitumor activity against multiple myeloma (MM) using cell lines and a murine xenograft model. METHODS: RPMI8226, U266, and MM1S cells were cultured for 48 h in the presence of media, vorinostat, melphalan, or bortezomib alone, or combinations of vorinostat with melphalan or bortezomib. Cell proliferation was measured using the MTS [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfphophenyl)-2H-tetrazolium, inner salt] assay. Severe combined immunodeficient mice bearing LAGkappa-1B tumors were treated with vorinostat [30, 60, or 100 mg/kg daily for five consecutive days per week (qdx5d), 100 or 300 mg/kg daily for 2 d/wk (qdx2d)], melphalan (1, 3, or 10 mg/kg qdx1d), bortezomib (0.25 or 0.5 mg/kg qdx2d), or combinations thereof for 35 d. Tumor growth was determined via measurement of human immunoglobulin G (hIgG) levels and tumor volume. RESULTS AND CONCLUSIONS: Vorinostat enhanced the anti-MM effects of melphalan and bortezomib in vitro. Synergism was observed with vorinostat and melphalan in RPMI8226 and U266 cell lines. Vorinostat 100 mg/kg in combination with melphalan 3 mg/kg resulted in significant inhibition of tumor growth in vivo, compared with control (tumor volume P = 0.0001; hIgG P = 0.0001), single-agent vorinostat (tumor volume P = 0.0025; hIgG P = 0.0137), and single-agent melphalan (tumor volume P = 0.0043; hIgG P = 0.0426). Vorinostat also enhanced the antimyeloma effects of bortezomib in vivo. Vorinostat enhances the anti-MM activity of melphalan and bortezomib in vitro and in vivo. This study provides rationale for further evaluation of vorinostat in combination with chemotherapeutic agents and bortezomib for the treatment of MM.

Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis.

Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)-marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Arabidopsis cryptochrome 2 completes its posttranslational life cycle in the nucleus.

CRY2 is a blue light receptor regulating light inhibition of hypocotyl elongation and photoperiodic flowering in Arabidopsis thaliana. The CRY2 protein is found primarily in the nucleus, and it is known to undergo blue light-dependent phosphorylation and degradation. However, the subcellular location where CRY2 exerts its function or undergoes blue light-dependent phosphorylation and degradation remains unclear. In this study, we analyzed the function and regulation of conditionally nuclear-localized CRY2. Our results show that CRY2 mediates blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation in the nucleus. Consistent with this result and a hypothesis that blue light-dependent phosphorylation is associated with CRY2 function, we demonstrate that CRY2 undergoes blue light-dependent phosphorylation in the nucleus. CRY2 phosphorylation is required for blue light-dependent CRY2 degradation, but only a limited quantity of CRY2 is phosphorylated at any given moment in seedlings exposed to blue light, which explains why continuous blue light illumination is required for CRY2 degradation. Finally, we showed that CRY2 is ubiquitinated in response to blue light and that ubiquitinated CRY2 is degraded by the 26S proteasome in the nucleus. These findings demonstrate that a photoreceptor can complete its posttranslational life cycle (from protein modification, to function, to degradation) inside the nucleus.

Antimyeloma effects of arsenic trioxide are enhanced by melphalan, bortezomib and ascorbic acid.

Arsenic trioxide (ATO) induces apoptosis of malignant plasma cells through multiple mechanisms, including inhibition of DNA binding by nuclear factor kappa-B, a key player in the development of chemoresistance in multiple myeloma (MM). This activity suggests that ATO may be synergistic when combined with other active antimyeloma drugs. To evaluate this, we examined the antimyeloma effects of ATO alone and in combination with bortezomib, melphalan and ascorbic acid (AA) both in vitro and in vivo using a severe combined immunodeficient (SCID)-hu murine myeloma model. Marked synergistic antimyeloma effects were demonstrated when human MM Los Angeles xenograft IgG lambda light chain (LAGlambda-1) cells were treated in vitro with ATO and any one of these agents. SCID mice bearing human MM LAGlambda-1 tumours were treated with single-agent ATO, bortezomib, melphalan, or AA, or combinations of ATO with either bortezomib or melphalan and AA. Animals treated with any of these drugs alone showed tumour growth and increases in paraprotein levels similar to control mice, whereas animals treated with ATO-containing combinations showed markedly suppressed tumour growth and significantly reduced serum paraprotein levels. These in vitro and in vivo results suggest that addition of ATO to other antimyeloma agents may result in improved outcomes for patients with relapsed or refractory MM.

Derepression of the NC80 motif is critical for the photoactivation of Arabidopsis CRY2.

Cryptochromes are blue light receptors that regulate photomorphogenesis in plants and the circadian clock in animals and plants. Arabidopsis cryptochrome 2 (CRY2) mediates blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation. CRY2 undergoes blue light-induced phosphorylation, which was hypothesized to be associated with CRY2 photoactivation. To further investigate how light activates CRY2, we analyzed the physiological activities and phosphorylation of various CRY2 fusion proteins in transgenic plants. Our results showed that an 80-residue motif, referred to as NC80, was sufficient to confer the physiological function of CRY2. The GUS-NC80 fusion protein expressed in transgenic plants is constitutively active but unphosphorylated, suggesting that the blue light-induced CRY2 phosphorylation causes a conformational change to derepress the NC80 motif. Consistent with this hypothesis, the CRY2 C-terminal tail was found to be required for the blue light-induced CRY2 phosphorylation but not for the CRY2 activity. We propose that the PHR domain and the C-terminal tail of the unphosphorylated CRY2 form a "closed" conformation to suppress the NC80 motif in the absence of light. In response to blue light, the C-terminal tail of CRY2 is phosphorylated and electrostatically repelled from the surface of the PHR domain to form an "open" conformation, resulting in derepression of the NC80 motif and signal transduction to trigger photomorphogenic responses.

Pleiotrophin is highly expressed by myeloma cells and promotes myeloma tumor growth.

Pleiotrophin (PTN) is an important developmental cytokine that is highly expressed during embryogenesis but shows very limited expression in adult tissues, where it is largely restricted to the brain. High PTN serum levels are associated with a variety of solid tumors. We recently showed that patients with multiple myeloma (MM) also have elevated serum levels of this protein and the amount of PTN correlated with the patients' disease status and response to treatment. In this study, we demonstrate that MM cell lines and the malignant cells from MM patients' bone marrow produced PTN and secreted PTN protein into the supernatants during short-term culture. Moreover, Ptn gene expression correlated with the patients' disease status. Inhibition of PTN with a polyclonal anti-PTN antibody reduced growth and enhanced apoptosis of MM cell lines and freshly isolated bone marrow tumor cells from MM patients in vitro. Importantly, this antibody also markedly suppressed the growth of MM in vivo using a severe combined immunodeficiency (SCID)-hu murine model. This represents the first study showing the importance of PTN in the growth of any hematological disorder. Because the expression of this protein is very limited in normal adult tissues, PTN may represent a new target for the treatment of MM.

Regulation of flowering time in Arabidopsis by K homology domain proteins.

The transition from vegetative growth to reproductive development in Arabidopsis is regulated by multiple floral induction pathways, including the photoperiodic, the autonomous, the vernalization, and the hormonal pathways. These pathways converge to regulate the expression of a small set of genes critical for floral initiation and different signal transduction pathways can interact to govern the time to flower. One important regulator of floral initiation is the MADS-box transcription factor FLC, which acts as a negative regulator of flowering in response to both endogenous and environmental signals. In this report, we describe a study of the flowering-time gene, FLK [flowering locus K homology (KH) domain] that encodes a putative RNA-binding protein with three KH domains. The flk mutations cause delayed flowering without a significant effect on the photoperiodic or vernalization responses. FLK functions primarily as a repressor of FLC expression, although it also modestly affects expression of genes associated with the photoperiodic pathway. In addition to FLK, the expression of two other KH domain genes are modestly affected by the flk mutation, suggesting a possible involvement of more than one KH domain protein in the regulation of flowering time in Arabidopsis.

Manipulating volatile emission in tobacco leaves by expressing Aspergillus nigerbeta-glucosidase in different subcellular compartments.

Expression of the Aspergillus nigerbeta-glucosidase gene, BGL1, in Nicotiana tabacum plants (cv. Xanthi) had a profound effect on the volatile emissions of intact and crushed leaves. BGL1 was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and targeted to the cytoplasm, cell wall, lytic vacuole (LV), chloroplast or endoplasmic reticulum (ER). Subcellular localization was confirmed by gold immunolabelling, followed by transmission electron microscopy (TEM). Significant beta-glucosidase activity was observed in transgenic plants expressing BGL1 in the cell wall, LV and ER. Compared with controls, all intact transgenic leaves were found to emit increased levels of 2-ethylhexanol, as determined by gas chromatography-mass spectrometry (GC-MS) analysis of the headspace volatiles. Plants expressing BGL1 in the cell wall (Tcw) emitted more trans-caryophyllene than did non-transgenic controls, whereas plants expressing BGL1 in the ER (Ter) and LV (Tvc) emitted more cembrene than did non-transgenic controls. Volatiles released from crushed transgenic leaves and collected with solid-phase microextraction (SPME) polydimethylsiloxane fibre were distinctly enhanced. Significant increases in linalool, nerol, furanoid cis-linalool oxide, 4-methyl-1-pentanol, 6-methyl-hept-5-en-2-ol and 2-ethylhexanol were detected in transgenic plants when compared with wild-type controls. 3-Hydroxyl-beta-ionone levels were increased in crushed Tcw and Ter leaves, but were undetectable in Tvc leaves. The addition of glucoimidazole, a beta-glucosidase inhibitor, abolished the increased emission of these volatiles. These results indicate that the expression of a fungal beta-glucosidase gene in different subcellular compartments has the potential to affect the emission of plant volatiles, and thereby to modify plant-environment communication and aroma of agricultural products.

Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1.

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light-dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light-dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light-induced phosphorylation. These results demonstrate that blue light-dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.

Cryptochrome structure and signal transduction.

Cryptochromes are photosensory receptors mediating light regulation of growth and development in plants. Since the isolation of the Arabidopsis CRY1 gene in 1993, cryptochromes have been found in every multicellular eukaryote examined. Most plant cryptochromes have a chromophore-binding domain that shares similar structure with DNA photolyase, and a carboxyl terminal extension that contains a DQXVP-acidic-STAES (DAS) domain conserved from moss, to fern, to angiosperm. In Arabidopsis, cryptochromes are nuclear proteins that mediate light control of stem elongation, leaf expansion, photoperiodic flowering, and the circadian clock. Cryptochromes may act by interacting with proteins such as phytochromes, COP1, and clock proteins, or/and chromatin and DNA. Recent studies suggest that cryptochromes undergo a blue light-dependent phosphorylation that affects the conformation, intermolecular interactions, physiological activities, and protein abundance of the photoreceptors.

Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent phosphorylation.

Cryptochromes are blue/ultraviolet-A light receptors that mediate various light responses in plants and animals. But the initial photochemical reaction of cryptochrome is still unclear. For example, although most photoreceptors are known to undergo light-dependent protein modification such as phosphorylation, no blue-light dependent phosphorylation has been reported for a cryptochrome. Arabidopsis cryptochrome 2 (cry2) mediates light regulation of seedling development and photoperiodic flowering. The physiological activity and cellular level of cry2 protein are light-dependent, and protein protein interactions are important for cry2 function. Here we report that cry2 undergoes a blue-light-dependent phosphorylation, and that cry2 phosphorylation is associated with its function and regulation. Our results suggest that, in the absence of light, cry2 remains unphosphorylated, inactive and stable; absorption of blue light induces the phosphorylation of cry2, triggering photomorphogenic responses and eventually degradation of the photoreceptor.