New Insights into Syndecan-2 Expression and Tumourigenic Activity in Colon Carcinoma Cells

Adhesion receptors play crucial roles in the neoplastic transformation of normal cells through induction of cancer-specific cellular behaviour and morphology. This implies that cancer cells likely express and utilize a distinct set of adhesion receptors during carcinogenesis. Colon cancer is an excellent model system for the study of this process, since both molecular genetic and morphological changes have been well established for this disease. We recently reported increased expression of the cell surface adhesion receptor, syndecan-2, in several colon carcinoma cell lines. Indeed, increased syndecan-2 expression was necessary for tumourigenic activity, suggesting that syndecan-2 might have value as both a new diagnostic marker and a possible therapeutic target. Here, we review recent advances in understanding the role of syndecan-2 in the carcinogenesis of colon cells, and discuss a leading role for this molecule in a new era for colon cancer treatment.     

Coordinated Regulation of the Orosomucoid-like Gene Family Expression Controls de Novo Ceramide Synthesis in Mammalian Cells

The orosomucoid-like (ORMDL) protein family is involved in the regulation of de novo sphingolipid synthesis, calcium homeostasis, and unfolded protein response. Single nucleotide polymorphisms (SNPs) that increase ORMDL3 expression have been associated with various immune/inflammatory diseases, although the pathophysiological mechanisms underlying this association are poorly understood. ORMDL proteins are claimed to be inhibitors of the serine palmitoyltransferase (SPT). However, it is not clear whether individual ORMDL expression levels have an impact on ceramide synthesis. The present study addressed the interaction with and regulation of SPT activity by ORMDLs to clarify their pathophysiological relevance. We have measured ceramide production in HEK293 cells incubated with palmitate as a direct substrate for SPT reaction. Our results showed that a coordinated overexpression of the three isoforms inhibits the enzyme completely, whereas individual ORMDLs are not as effective. Immunoprecipitation and fluorescence resonance energy transfer (FRET) studies showed that mammalian ORMDLs form oligomeric complexes that change conformation depending on cellular sphingolipid levels. Finally, using macrophages as a model, we demonstrate that mammalian cells modify ORMDL genes expression levels coordinately to regulate the de novo ceramide synthesis pathway. In conclusion, we have shown a physiological modulation of SPT activity by general ORMDL expression level regulation. Moreover, because single ORMDL3 protein alteration produces an incomplete inhibition of SPT activity, this work argues against the idea that ORMDL3 pathophysiology could be explained by a simple on/off mechanism on SPT activity.     

Regulation of the Nitrogen Transfer Pathway in the Arbuscular Mycorrhizal Symbiosis: Gene Characterization and the Coordination of Expression with Nitrogen Flux

The arbuscular mycorrhiza (AM) brings together the roots of over 80% of land plant species and fungi of the phylum Glomeromycota and greatly benefits plants through improved uptake of mineral nutrients. AM fungi can take up both nitrate and ammonium from the soil and transfer nitrogen (N) to host roots in nutritionally substantial quantities. The current model of N handling in the AM symbiosis includes the synthesis of arginine in the extraradical mycelium and the transfer of arginine to the intraradical mycelium, where it is broken down to release N for transfer to the host plant. To understand the mechanisms and regulation of N transfer from the fungus to the plant, 11 fungal genes putatively involved in the pathway were identified from Glomus intraradices, and for six of them the full-length coding sequence was functionally characterized by yeast complementation. Two glutamine synthetase isoforms were found to have different substrate affinities and expression patterns, suggesting different roles in N assimilation. The spatial and temporal expression of plant and fungal N metabolism genes were followed after nitrate was added to the extraradical mycelium under N-limited growth conditions using hairy root cultures. In parallel experiments with ¹⁵N, the levels and labeling of free amino acids were measured to follow transport and metabolism. The gene expression pattern and profiling of metabolites involved in the N pathway support the idea that the rapid uptake, translocation, and transfer of N by the fungus successively trigger metabolic gene expression responses in the extraradical mycelium, intraradical mycelium, and host plant.The arbuscular mycorrhizal (AM) symbiosis brings together the roots of the majority of land plant species and fungi of the phylum Glomeromycota to great mutual advantage (Smith and Read, 2008). AM fungi improve the acquisition of phosphate, nitrogen (N), sulfur, and trace elements such as copper and zinc (Clark and Zeto, 2000; Allen and Shachar-Hill, 2008) and increase the biotic and abiotic stress resistance of their host (Smith et al., 2010). In return, the host transfers up to 20% of its photosynthetically fixed carbon to the AM fungus (Jakobsen and Rosendahl, 1990), which depends on its host plant for its carbon supply (Bago et al., 2000).N is the nutrient whose availability most commonly limits plant growth in natural ecosystems. AM fungi can take up NO₃⁻NH₄⁺ and can also increase access to organic N sources from the soil (Ames et al., 1983; Johansen et al., 1993; Bago et al., 1996; Hodge et al., 2001). The translocation by the fungus can represent a significant route for N uptake by the plant (Johansen and Jensen, 1996). For example, Toussaint et al. (2004) showed that in an in vitro mycorrhiza at least 21% of the total N uptake in the roots came from the fungal extraradical mycelium (ERM); for other mycorrhizal systems, even larger proportions have been described (more than 30% and 50%; Govindarajulu et al., 2005; Jin et al., 2005). Tanaka and Yano (2005) reported that 75% of the N in leaves of mycorrhizal maize (Zea mays) was taken up by the ERM of Glomus aggregatum.A mechanism of N transfer from the fungus to the plant has been proposed (Bago et al., 2001) that involves the operation of a novel metabolic route in which N was translocated from the ERM to the intraradical mycelium (IRM) as Arg but transferred to the plant without carbon as inorganic N. This mechanism has been supported by labeling experiments (Johansen et al., 1996; Govindarajulu et al., 2005; Jin et al., 2005), enzyme activity analysis (Cruz et al., 2007), and limited gene expression data (Govindarajulu et al., 2005; Gomez et al., 2009; Guether et al., 2009). Nevertheless, our molecular knowledge of the metabolic and transport pathways involved and how they are regulated is still rudimentary. A better understanding of the mechanism and regulation of N uptake assimilation, translocation, and transfer to the host is important for potential applications of AM fungi as biofertilizers, bioprotectors, and bioregulators in sustainable agriculture and restoration as well as for understanding the role of AM fungi in natural ecosystems (Bruns et al., 2008).In this study, we postulate that the uptake, translocation, and transfer of N by the fungus triggers the metabolic gene expression responses successively in the ERM, IRM, and host plant, which will result in the synthesis and accumulation of Arg in the ERM, the turnover of Arg to release ammonium in the IRM, and the assimilation of ammonium by the host plant via the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway inside the root (Fig. 1). To test these predictions, 11 genes involved in the N primary assimilation and metabolism were cloned and verified from Glomus intraradices; six enzymes with full-length coding sequences (CDSs) were functionally characterized by yeast knockout mutant complementation. Two GS proteins were found to have different substrate affinities and expression patterns, suggesting that they have different roles in N assimilation. The time courses of gene expression and N movement in fungal and host tissues were analyzed following nitrate supply to the ERM of a mycorrhiza grown under N-limited conditions. The results substantially increase our knowledge of the identity and regulation of most of the metabolic and transport genes involved in N movement through the AM symbiosis.Open in a separate windowFigure 1.Working model of N transport and metabolism in the symbiosis between plant roots and arbuscular mycorrhizal fungi. N moves (black arrows) from the soil into the fungal ERM, through a series of metabolic conversion reactions into Arg, which is transported into the IRM within the root (host) and there is broken down; N is transferred to and assimilated by the host as ammonia. Red circles refer to the sites of action of the genes identified and analyzed in this study. Blue arrows indicate mechanisms hypothesized to regulate gene expression by N metabolites involved in the pathway.     

Developmental Gene Expression in Amphioxus: New Insights into the Evolutionary Origin of Vertebrate Brain Regions, Neural Crest, and Rostrocaudal Segmentation

SYNOPSIS. Amphioxus is widely held to be the closest invertebraterelative of the vertebrates and the best available stand-infor the proximate ancestor of the vertebrates. The spatiotemporalexpression patterns of developmental genes can help suggestbody part homologies between vertebrates and amphioxus. Thisapproach is illustrated using five homeobox genes (AmphiHoxl,AmphiHox2, AmphiOtx, AmphiDll, and AmphiEri) to provide insightsinto the evolutionary origins of three important vertebratefeatures: the major brain regions, the neural crest, and rostrocaudalsegmentation. During amphioxus development, the neural expressionpatterns of these genes are consistent with the presence ofa forebrain (detailed neuroanatomy indicates that the forebrainis all diencephalon without any telencephalon) and an extensivehindbrain; the possible presence of a midbrain requires additionalstudy. Further, during neurulation, the expression pattern ofAmphiDll as well as migratory cell behavior suggest that theepidermal cells bordering the neural plate may represent a phylogeneticprecursor of the vertebrate neural crest. Finally, when theparaxial mesoderm begins to segment, the earliest expressionof AmphiEn is detected in the posterior part of each nascentand newly formed somite. This pattern recalls the expressionof the segment-polarity gene engrailed during establishmentof the segments of metameric protostomes. Thus, during animalevolution, the role of engrailed in establishing and maintainingmetameric body plans may have arisen in a common segmented ancestorof both the protostomes and deuterostomes.     

Stress-Induced Cytokinin Synthesis Increases Drought Tolerance through the Coordinated Regulation of Carbon and Nitrogen Assimilation in Rice

The effects of water deficit on carbon and nitrogen metabolism were investigated in flag leaves of wild-type and transgenic rice (Oryza sativa japonica ‘Kitaake’) plants expressing ISOPENTENYLTRANSFERASE (IPT; encoding the enzyme that mediates the rate-limiting step in cytokinin synthesis) under the control of P_SARK, a maturation- and stress-induced promoter. While the wild-type plants displayed inhibition of photosynthesis and nitrogen assimilation during water stress, neither carbon nor nitrogen assimilation was affected by stress in the transgenic P_SARK::IPT plants. In the transgenic plants, photosynthesis was maintained at control levels during stress and the flag leaf showed increased sucrose (Suc) phosphate synthase activity and reduced Suc synthase and invertase activities, leading to increased Suc contents. The sustained carbon assimilation in the transgenic P_SARK::IPT plants was well correlated with enhanced nitrate content, higher nitrate reductase activity, and sustained ammonium contents, indicating that the stress-induced cytokinin synthesis in the transgenic plants played a role in maintaining nitrate acquisition. Protein contents decreased and free amino acids increased in wild-type plants during stress, while protein content was preserved in the transgenic plants. Our results indicate that the stress-induced cytokinin synthesis in the transgenic plants promoted sink strengthening through a cytokinin-dependent coordinated regulation of carbon and nitrogen metabolism that facilitates an enhanced tolerance of the transgenic plants to water deficit.Plant hormones control many aspects of plant growth and development and the responses of plants to abiotic and biotic stresses. Cytokinins (CKs) have been shown to regulate plant cell differentiation, leaf senescence, and other key developmental processes (Sakakibara, 2006). It has also been shown that CKs regulate assimilate partitioning (Ronzhina and Mokronosov, 1994), sink strength (Kuiper, 1993), and source/sink relationships (Roitsch, 1999). The localized expression in tobacco (Nicotiana tabacum) of a promoterless ISOPENTENYLTRANSFERASE (IPT), a gene encoding the enzyme that catalyzes the rate-limiting step in CK synthesis, enhanced the local sink strength and quickly mobilized nutrients to the tissues with elevated CK (Guivarc’h et al., 2002). Changes in sink/source relationships were also observed in CK-deficient tobacco shoots and roots (Werner et al., 2008). Elevated CK levels enhanced the survival of plants under water-stress conditions (Rivero et al., 2007). The overexpression of IPT under the control of SENESCENCE-ASSOCIATED RECEPTOR KINASE (SARK; a maturation- and stress-induced promoter) improved the drought tolerance of both eudicots (Rivero et al., 2007; Qin et al., 2011) and monocots (Peleg et al., 2011). After a water-stress episode during the reproductive stages (pre and post anthesis), transgenic P_SARK::IPT rice (Oryza sativa) plants displayed higher grain yield than the wild type (Peleg et al., 2011). The transgenic P_SARK::IPT rice exhibited a differential expression of genes encoding enzymes associated with hormone synthesis and hormone-regulated pathways. These results suggested that changes in hormone homeostasis induced the modification of source/sink relationships in the transgenic plants, resulting in higher grain yields under stress conditions (Peleg et al., 2011).The maintenance of carbon (C) and nitrogen (N) assimilation is of paramount importance to ensure sink strength and improve stress tolerance without yield penalties. The interactions between C and N metabolism are vital for plant growth and development, and complex mechanisms operate in the plant to coordinate C assimilation with N metabolism (Nunes-Nesi et al., 2010). Thus, plants respond to changes in C and N metabolites through the regulation of translation and posttranslational modification mechanisms. C and N metabolites activate signaling pathways that regulate enzyme and transporter activities that control C and N fluxes, optimizing the plant response to developmental and environmental cues changing source/sink relationships (Coruzzi and Zhou, 2001).Plant hormones affect, either directly or indirectly, these pathways and can act antagonistically or synergistically when responding to environmental stress (Wilkinson et al., 2012). The exposure of plants to water-limiting conditions results in abscisic acid (ABA) synthesis that induces ABA-dependent gene expression (Yamaguchi-Shinozaki and Shinozaki, 2006), triggering the closure of stomata and reducing water loss during drought (Wilkinson and Davies, 2010). Other hormones, in particular CK, salicylic acid, ethylene, and jasmonic acid, also play direct or indirect roles in the plant responses to abiotic stress (Peleg and Blumwald, 2011). Under drought stress, plant CK content decreases, and the reduction in CK increases the plant responses to increasing ABA (Davies and Zhang, 1991), inducing stomata closure and inhibiting photosynthesis (Rivero et al., 2010). Our previous results suggested that the stress-induced CK synthesis, driven by a stress-induced promoter, protected against the deleterious effects of water deficit on the photosynthetic apparatus, allowing higher photosynthetic rates and higher yields after water deficit in tobacco (Rivero et al., 2009) and cotton (Gossypium hirsutum; Kuppu et al., 2013) plants grown in the greenhouse and peanut (Arachis hypogaea) plants grown under field conditions (Qin et al., 2011).Here, we analyzed gene expression profiles, metabolites, and enzymatic and photosynthetic activities of flag leaves of wild-type and transgenic rice expressing P_SARK::IPT exposed to water deficit during the reproductive stage and identified metabolic processes associated with the enhanced tolerance of the transgenic plants to water deficit. Our results indicate that the stress-induced CK synthesis in the transgenic plants promoted sink strengthening through the maintenance and coordination of N and C assimilation during water stress.     

Milk Protein Synthesis, Gene Expression, and Hormonal Responsiveness in Primary Cultures of Mammary Cells from Lactating Sheep

A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation, With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lecturing sheep. α- and β-casein protein synthesis, β-lactoglobulin synthesis, and α-casein, β-casein, and β-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of α-S₁-casein, β-lactoglobulin, and α-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled α-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro.     

Development of Melatonin Synthesis in Chicken Retina: Regulation of Serotonin N-Acetyltransferase Activity by Light, Circadian Oscillators, and Cyclic AMP

In chicken retinas, melatonin levels and the activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme of melatonin biosynthesis, are expressed as circadian rhythms with peaks of levels and activity occurring at night. In the present study, NAT activity was examined in retinas of embryonic and posthatch chicks to assess the ontogenic development of regulation of the enzyme by light, circadian oscillators, and the second messenger cyclic AMP. During embryonic development, NAT activity was consistently detectable by embryonic day 6 (E6). Significant light-dark differences were first observed on E20, and increased to a maximum amplitude of sixfold by posthatch day 3 (PH3). Circadian rhythmicity of NAT activity appears to develop at or prior to hatching, as evidenced by day-night differences of activity in constant darkness observed in PH1 chicks that had been exposed to a light-dark cycle in ovo only. NAT activity is regulated by a cyclic AMP-dependent mechanism. Activity was significantly increased by incubating retinas with forskolin or dibutyryl cyclic AMP as early as E7, and seven- to ninefold increases were observed following treatment with these agents on E14. Thus, development of the cyclic AMP-dependent mechanism for increasing NAT activity significantly precedes that of rhythmicity, suggesting that the onset of rhythmicity may be related to the onset of photoreception or development of the circadian oscillator in chick retina.     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.