Light regulation of nuclear photosynthetic genes in higher plants

Light can modulate development and differentiation of the vital photosynthetic organelle, chloroplast, by photomorphogenetic mechanisms involved in regulating transcription of various photosynthetic genes encoded by nuclear genome. The nuclear genes encode majority of proteins involved in photosynthesis. After perception, light signal is transduced via signaling intermediates, which have been identified using various approaches. These signaling components can either directly influence the binding of light regulatory trans-acting factors to cis-acting elements present in a photosynthetic gene promoter or modulate their activity by various means to facilitate transcription in response to light. Some cis-acting elements show a high degree of conservation among photo-responsive nuclear genes in plants and have been designated as light regulatory elements (LREs). The gene regulation seems to involve interplay of several cis-acting elements and regulatory factors. Additional information available about the post-transcriptional mechanisms responsible for light-regulated expression of photosynthetic genes suggests the importance of these levels in regulation biology

Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from arabidopsis thaliana and regulation of its expression

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected

Binding of 4-methylumbelliferyl β-D-galactopyranoside to Momordica charantia lectin fluorescence-quenching studies

The binding of 4-methylumbelliferyl β-D-galactopyranoside (MeUmb-Galp), to Mormordica charantia lectin was studied by equilibrium dialysis and quenching of ligand fluorescence. The fluorescence of MeUmb-Galp decreases as a function of solvent polarity. On binding to M. charantia lectin, its fluorescence was nearly 100% quenched, showing that the binding of the glycoside takes place in a hydrophobic environment. The binding of the fluorescent sugar was saccharide-specific as evidenced by reversal of MeUmb-Galp fluorescence quenching by lactose. The association constant is independent of the experimental method used and at 25°C the value is (1.96±0.05)×10<SUP>4</SUP> M<SUP>−1</SUP>. The number of binding sites as determined by equilibrium dialysis and fluorescence quenching agree very well with each other; n being equal to 1.98±0.02. The k<SUB>a</SUB> value for the glycoside was also determined by competition studies employing reversal of fluorescence quenching of MeUmb-Galp by lactose. The value of K<SUB>a</SUB> obtained for lactose is 1.21×10<SUP>4</SUP> M<SUP>−1</SUP> at 30°C. The internal consistency of the association constant and number of binding site values at low and high saturation indicates the absence of additional subsite on M. charantia lectin. The thermodynamic parameters do not differ greatly with change in temperature: the values of −ΔH° and −ΔS° are equal to 30±0.63 kJ mol<SUP>−1</SUP> and 21±0.3 J mol<SUP>−1</SUP> K<SUP>−1</SUP> respectively in the range of 15-35°C indicating that the binding of M. charantia lectin to saccharide is exothermic in nature

MicroRNA control of bone formation and homeostasis

MicroRNAs (miRNAs) repress cellular protein levels to provide a sophisticated parameter of gene regulation that coordinates a broad spectrum of biological processes. Bone organogenesis is a complex process involving the differentiation and crosstalk of multiple cell types for formation and remodeling of the skeleton. Inhibition of mRNA translation by miRNAs has emerged as an important regulator of developmental osteogenic signaling pathways, osteoblast growth and differentiation, osteoclast-mediated bone resorption activity and bone homeostasis in the adult skeleton. miRNAs control multiple layers of gene regulation for bone development and postnatal functions, from the initial response of stem/progenitor cells to the structural and metabolic activity of the mature tissue. This Review brings into focus an emerging concept of bone-regulating miRNAs, the evidence for which has been gathered largely from in vivo mouse models and in vitro studies in human and mouse skeletal cell populations. Characterization of miRNAs that operate through tissue-specific transcription factors in osteoblast and osteoclast lineage cells, as well as intricate feedforward and reverse loops, has provided novel insights into the supervision of signaling pathways and regulatory networks controlling normal bone formation and turnover. The current knowledge of miRNAs characteristic of human pathologic disorders of the skeleton is presented with a future goal towards translational studies

Pristine Graphene–Copper(II) Oxide Nanocatalyst: A Novel and Green Approach in CuAAC Reactions

Pristine graphene, as the name suggests is the closest to graphite structurally among all the forms of graphene that are synthesized using different methods. High electronic conductivity, large surface area, and absence of defects make this perfect two-dimensional arrangement of sp² hybridized carbon atoms a perfect support material for metal or metal oxide nanoparticles. We have introduced a quick and green route to synthesize CuO nanocomposites having pristine graphene as a support material by microwave assisted hydrothermal reaction. The nanocomposite exhibited very high catalytic activity in copper catalyzed azide-alkyne cycloaddition (CuAAC) reactions compared with reduced graphene oxide (RGO)-CuO nanocomposite and CuO nanoparticles. The presence of pristine graphene in the nanocomposite increases the catalytic activity due to its better conductivity and ability to adsorb reactants through π–π interaction than RGO. The pristine graphene-CuO nanocomposite showed very good recyclability with much less leaching of the metal from it. The CuAAC reactions could be completed in a short duration (1 h), at low reaction temperature (30 °C), using water as a “green” solvent with a small amount of the pristine graphene-CuO nanocomposite as catalyst (0.51 mol %) and sodium ascorbate as cocatalyst (1 mol %)

Expression of secreted frizzled related protein 1, a Wnt antagonist, in brain, kidney, and skeleton is dispensable for normal embryonic development

Secreted frizzled related protein-1 (sFRP1), an antagonist of Wnt signaling, regulates cell proliferation, differentiation and apoptosis and negatively regulates bone formation. The spatial and temporal pattern of endogenous sFRP1 expression and loss-of-function were examined in the sFRP1-LacZ knock-in mouse (sFRP1-/-) during embryonic development and post-natal growth. beta-gal activity representing sFRP1 expression is robust in brain, skeleton, kidney, eye, spleen, abdomen, heart and somites in early embryos, but sFRP1 gene inactivation in these tissues did not compromise normal embryonic and post-natal development. Kidney histology revealed increased numbers of glomeruli in KO mice, observed after 5 years of breeding. In the skeleton, we show sFRP1 expression is found in relation to the mineralizing front of bone tissue during skeletal development from E15.5 to birth. Trabecular bone volume and bone mineral density in the sFRP1-/- mouse compared to WT was slightly increased during post-natal growth. Calvarial osteoblasts from newborn sFRP1-/- mice exhibited a 20% increase in cell proliferation and differentiation at the early stages of osteoblast maturation. sFRP1 expression was observed in osteoclasts, but this did not affect osteoclast number or activity. These findings have identified functions for sFRP1 in kidney and bone that are not redundant with other sFRPs. In summary, the absence of major organ abnormalities, the enhanced bone formation and a normal life span with no detection of spontaneous tumors suggests that targeting sFRP1 can be used as a therapeutic strategy for increasing bone mass in metabolic bone disorders or promoting fracture healing by modulating Wnt signaling

Enhanced fracture repair by leukotriene antagonism is characterized by increased chondrocyte proliferation and early bone formation: a novel role of the cysteinyl LT-1 receptor

Inflammatory mediators and drugs which affect inflammation can influence the healing of injured tissues. Leukotrienes are potent inflammatory mediators, and similar to prostaglandins, are metabolites of arachidonic acid which can have positive or negative effects on bone and cartilage tissues. Here we tested the hypothesis that blocking the negative regulation of leukotrienes, would lead to enhanced endochondral bone formation during fracture repair. A closed femoral fracture was created in mice. Animals were divided into three groups for treatment with either montelukast sodium, a cysteinyl leukotriene type 1 receptor antagonist (trade name Singulair), zileuton, a 5-lipoxygenase enzyme inhibitor (trade name Zyflo), or carrier alone. The fractures were analyzed using radiographs, quantitative gene expression, histology and histomorphometry, and immunohistochemistry. Both the montelukast sodium group and the zileuton group exhibited enhanced fracture repair when compared with controls. Both treatment groups exhibited increased callous size and earlier bone formation when compared to controls as early as day 7. Gene expression analysis of treatment groups showed increased markers of chondrocyte proliferation and differentiation, and increased early bone formation markers when compared with controls. Treatment with montelukast sodium directly targeted the cysteinyl leukotriene type 1 receptor, leading to increased chondrocyte proliferation at early time points. These novel findings suggests a potential mechanism by which the cysteinyl leukotriene type 1 receptor acts as a negative regulator of chondrocyte proliferation, with important and previously unrecognized implications for both fracture repair, and in a broader context, systemic chondrocyte growth and differentiation

Networks and hubs for the transcriptional control of osteoblastogenesis

We present an overview of the concepts of tissue-specific transcriptional control mechanisms essential for development of the bone cell phenotype. BMP2 induced transcription factors constitute a network of activities and molecular switches for bone development and osteoblast differentiation. Among these regulators are Runx2 (Cbfa1/AML3), the principal osteogenic master gene for bone formation, as well as homeodomain proteins and osterix. Runx2 has multiple regulatory activities, including activation or repression of gene expression, and integration of biological signals from developmental cues, such as BMP/TGFbeta, Wnt and Src signaling pathways. Runx2 provides a new paradigm for transcriptional control by functioning as a principal scaffolding protein in nuclear microenvironments to control gene expression in response to physiologic signals (growth factors, cytokines and hormones). The protein serves as a hub for the coordination of activities essential for the expansion and differentiation of osteogenic lineage cells through the formation of co-regulatory protein complexes organized in subnuclear domains. Mechanisms by which Runx2 supports commitment to osteogenesis and determines cell fate involve its retention on mitotic chromosomes. Disruption of a unique protein module, the subnuclear targeting signal of Runx2, has profound effects on osteoblast differentiation and metastasis of cancer cells in the bone microenvironment. Runx2 target genes include regulators of cell growth control, components of the bone extracellular matrix, angiogenesis, and signaling proteins for development of the osteoblast phenotype and bone turnover. The specificity of Runx2 regulatory activities provides a basis for novel therapeutic strategies to correct bone disorders

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults