Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mm nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 x atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mm nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.     

Sorgoleone

Sorgoleone, a major component of the hydrophobic root exudate of sorghum [Sorghum bicolor (L.) Moench], is one of the most studied allelochemicals. The exudate also contains an equivalent amount of a lipid resorcinol analog as well as a number of minor sorgoleone congeners. Synthesis of sorgoleone is constitutive and compartmentalized within root hairs, which can accumulate up to 20 μg of exudate/mg root dry weight. The biosynthesis pathway involves unique fatty acid desaturases which produce an atypical 16:3 fatty acyl-CoA starter unit for an alkylresorcinol synthase that catalyzes the formation of a pentadecatrienylresorcinol intermediate. This intermediate is then methylated by SAM-dependent O-methyltransferases and dihydroxylated by cytochrome P450 monooxygenases. An EST data set derived from a S. bicolor root hair-specific cDNA library contained all the candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Sorgoleone interferes with several molecular target sites, including inhibition of photosynthesis in germinating seedlings. Sorgoleone is not translocated acropetally in older plants, but can be absorbed through the hypocotyl and cotyledonary tissues. Therefore, the mode of action of sorgoleone may be the result of inhibition of photosynthesis in young seedlings in concert with inhibition of its other molecular target sites in older plants. Due to its hydrophobic nature, sorgoleone is strongly sorbed in soil which increases its persistence, but experiments show that it is mineralized by microorganisms over time.     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

Spatial and temporal control of expression of therapeutic genes using heat shock protein promoters

Heat-shock protein promoters, particularly hsp70, have been used for gene therapy strategies because of their efficiency and the possibility of induction by external heat. This review describes some of the characteristics of hsp70 promoters that make them attractive for use in gene therapy. The human hsp70B promoter is especially promising because of its dose response effect with regard to temperature. Spatial and temporal control of transgene expression using hsp70 promoters necessitates non-invasive methods of local heat deposition and accurate local control of temperature. Special emphasis is given to Focused Ultrasound heating guided by Magnetic Resonance temperature mapping.     

Characterization of single chain antibody targets through yeast two hybrid

Background  

Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.     

Assay and inhibition of diacylglycerol lipase activity

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.     

Altered processing of sensorimotor feedback in schizophrenia

Schizophrenia is characterized by an altered sense of the reality, associated with hallucinations and delusions. Some theories suggest that schizophrenia is related to a deficiency of the system that generates information about the sensory consequences of the actions realized by the subject. This system monitors the reafferent information resulting from an action and allows its anticipation. In the present study, we examined visual-event-related potentials (ERPs) generated by a sensorimotor task in 15 patients with schizophrenia and 15 normal controls. The visual feedback from hand movements performed by the subjects was experimentally distorted. Behavioral results showed that patients were impaired in recognizing their own movements. The ERP signal in patients also differed from those of control subjects. In patients, the ERP waveform was affected during the early part of the response (200 ms). This early effect in schizophrenic patients reveals a modified processing of the visual consequence of their actions.