Capsicum annuum tobacco mosaic virus-induced clone 1 expression perturbation alters the plant's response to ethylene and interferes with the redox homeostasis

Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.     

Cysteine repeat domains and adjacent sequences determine distinct bone morphogenetic protein modulatory activities of the Drosophila Sog protein

The Drosophila short gastrulation gene (sog) encodes a large extracellular protein (Sog) that inhibits signaling by BMP-related ligands. Sog and its vertebrate counterpart Chordin contain four copies of a cysteine repeat (CR) motif defined by 10 cysteine residues spaced in a fixed pattern and a tryptophan residue situated between the first two cysteines. Here we present a structure-function analysis of the CR repeats in Sog, using a series of deletion and point mutation constructs, as well as constructs in which CR domains have been swapped. This analysis indicates that the CR domains are individually dispensable for Sog function but that they are not interchangeable. These studies reveal three different types of Sog activity: intact Sog, which inhibits signaling mediated by the ligand Glass bottom boat (Gbb), a more broadly active class of BMP antagonist referred to as Supersog, and a newly identified activity, which may promote rather than inhibit BMP signaling. Analysis of the activities of CR swap constructs indicates that the CR domains are required for full activity of the various forms of Sog but that the type of Sog activity is determined primarily by surrounding protein sequences. Cumulatively, our analysis suggests that CR domains interact physically with adjacent protein sequences to create forms of Sog with distinct BMP modulatory activities.     

Lysophosphatidic acid induces exocytic trafficking of Na(+)/H(+) exchanger 3 by E3KARP-dependent activation of phospholipase C

Lysophosphatidic acid (LPA) stimulates Na(+)/H(+) exchanger 3 (NHE3) activity in opossum kidney proximal tubule (OK) cells by increasing the apical membrane amount of NHE3. This occurs by stimulation of exocytic trafficking of NHE3 to the apical plasma membrane by an E3KARP-dependent mechanism. However, it is still unclear how E3KARP leads to the LPA-induced exocytosis of NHE3. In the current study, we demonstrate that stable expression of exogenous E3KARP increases LPA-induced phospholipase C (PLC) activation and subsequent elevation of intracellular Ca(2+) in opossum kidney proximal tubule (OK) cells. Pretreatment with U73122, a PLC inhibitor, prevented the LPA-induced NHE3 activation and the exocytic trafficking of NHE3. To understand how the elevation of intracellular Ca(2+) leads to the stimulation of NHE3, we pretreated OK cells with BAPTA-AM, an intracellular Ca(2+) chelator. BAPTA-AM completely blocked the LPA-induced increase of NHE3 activity and surface NHE3 amount by decreasing the LPA-induced exocytic trafficking of NHE3. Pretreatment with GF109203X, a PKC inhibitor, did not affect the percent of LPA-induced NHE3 activation and increase of surface NHE3 amount. From these results, we suggest that E3KARP plays a necessary role in LPA-induced PLC activation, and that PLC-dependent elevation of intracellular Ca(2+) but not PKC activation is necessary for the LPA-induced increase of NHE3 exocytosis.     

Single-molecule three-color FRET

Fluorescence resonance energy transfer (FRET) measured at the single-molecule level can reveal conformational changes of biomolecules and intermolecular interactions in physiologically relevant conditions. Thus far single-molecule FRET has been measured only between two fluorophores. However, for many complex systems, the ability to observe changes in more than one distance is desired and FRET measured between three spectrally distinct fluorophores can provide a more complete picture. We have extended the single-molecule FRET technique to three colors, using the DNA four-way (Holliday) junction as a model system that undergoes two-state conformational fluctuations. By labeling three arms of the junction with Cy3 (donor), Cy5 (acceptor 1), and Cy5.5 (acceptor 2), distance changes between the donor and acceptor 1, and between the donor and acceptor 2, can be measured simultaneously. Thus we are able to show that the acceptor 1 arm moves away from the donor arm at the same time as the acceptor 2 arm approaches the donor arm, and vice versa, marking the first example of observing correlated movements of two different segments of a single molecule. Our data further suggest that Holliday junction does not spend measurable time with any of the helices unstacked, and that the parallel conformations are not populated to a detectable degree.     

Changes in parvalbumin immunoreactivity in the parietofrontal cortex after transient forebrain ischemia in the Mongolian gerbil

We investigated the changes in parvalbumin (PV)-immunoreactive (IR) neurons in the parietofrontal cortex after transient forebrain ischemia. In the sham-operated group, PV-IR neurons were present in all layers of the parietofrontal cortex except layer I. Shortly after ischemia the number of PV-IR neurons in layer II/III first increased, and then declined dramatically 12 h after ischemic insult, followed by a second increase after 2 days. At this time the PV immunoreactivity was very weak and only present in the peripheral neuronal cytoplasm. The reversible increase in the number of PV-IR neurons and in the level of their immunoreactivity could result from a transient ischemia-induced increase in intracellular calcium. This pattern of expression was particularly pronounced in layer II/III of the parietofrontal cortex, suggesting that these neurons are especially\susceptible to ischemic insult.     

Activated liver X receptors stimulate adipocyte differentiation through induction of peroxisome proliferator-activated receptor gamma expression

Liver X receptors (LXRs) are nuclear hormone receptors that regulate cholesterol and fatty acid metabolism in liver tissue and in macrophages. Although LXR activation enhances lipogenesis, it is not well understood whether LXRs are involved in adipocyte differentiation. Here, we show that LXR activation stimulated the execution of adipogenesis, as determined by lipid droplet accumulation and adipocyte-specific gene expression in vivo and in vitro. In adipocytes, LXR activation with T0901317 primarily enhanced the expression of lipogenic genes such as the ADD1/SREBP1c and FAS genes and substantially increased the expression of the adipocyte-specific genes encoding PPARγ (peroxisome proliferator-activated receptor γ) and aP2. Administration of the LXR agonist T0901317 to lean mice promoted the expression of most lipogenic and adipogenic genes in fat and liver tissues. It is of interest that the PPARγ gene is a novel target gene of LXR, since the PPARγ promoter contains the conserved binding site of LXR and was transactivated by the expression of LXRα. Moreover, activated LXRα exhibited an increase of DNA binding to its target gene promoters, such as ADD1/SREBP1c and PPARγ, which appeared to be closely associated with hyperacetylation of histone H3 in the promoter regions of those genes. Furthermore, the suppression of LXRα by small interfering RNA attenuated adipocyte differentiation. Taken together, these results suggest that LXR plays a role in the execution of adipocyte differentiation by regulation of lipogenesis and adipocyte-specific gene expression.     

Site-directed mutagenesis of human brain GABA transaminase: lysine-357 is involved in cofactor binding at the active site

gamma-Aminobutyrate transaminase (GABA-T), a key enzyme of the GABA shunt, converts the major inhibitory neurotransmitter, GABA, to succinic semialdehyde. Although GABA-T is a pivotal factor implicated in the pathogenesis of various neurological disorders, its function remains to be elucidated. In an effort to clarify the structural and functional roles of specific lysyl residue in human brain GABA-T, we constructed human brain GABA-T mutants, in which the lysyl residue at position 357 was mutated to various amino acids including asparagine (K357N). The purified mutant GABA-T enzymes displayed neither catalytic activity nor absorption bands at 330 and 415 nm that are characteristic of pyridoxal-5'-phosphate (PLP) covalently linked to the protein. The wild type apoenzyme reconstituted with exogenous PLP had catalytic activity, while the mutant apoenzymes did not. These results indicate that lysine 357 is essential for catalytic function, and is involved in binding PLP at the active site.     

Controlled alternate partial root-zone irrigation: its physiological consequences and impact on water use efficiency

Controlled alternate partial root-zone irrigation (CAPRI), also called partial root-zone drying (PRD) in other literature, is a new irrigation technique and may improve the water use efficiency of crop production without significant yield reduction. It involves part of the root system being exposed to drying soil while the remaining part is irrigated normally. The wetted and dried sides of the root system are alternated with a frequency according to soil drying rate and crop water requirement. The irrigation system is developed on the basis of two theoretical backgrounds. (i) Fully irrigated plants usually have widely opened stomata. A small narrowing of the stomatal opening may reduce water loss substantially with little effect on photosynthesis. (ii) Part of the root system in drying soil can respond to the drying by sending a root-sourced signal to the shoots where stomata may be inhibited so that water loss is reduced. In the field, however, the prediction that reduced stomatal opening may reduce water consumption may not materialize because stomatal control only constitutes part of the total transpirational resistance. The boundary resistance from the leaf surface to the outside of the canopy may be so substantial that reduction in stomatal conductance is small and may be partially compensated by the increase in leaf temperature. It is likely that densely populated field crops, such as wheat and maize, may have a different stomatal control over transpiration from that of fruit trees which are more sparsely separated. It was discussed how long the stomata can keep 'partially' closed when a prolonged and repeated 'partial' soil drying is applied and what role the rewatering-stimulated new root growth may play in sensing the repeated soil drying. The physiological and morphological alternation of plants under partial root-zone irrigation may bring more benefits to crops than improved water use efficiency where carbon redistribution among organs is crucial to the determination of the quantity and quality of the products.     

Cysteine-321 of human brain GABA transaminase is involved in intersubunit cross-linking

Gamma-aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5 cent-dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T.