High-dose ascorbic acid (vitamin C) therapy (66 mg/kg per hour) attenuates postburn lipid peroxidation, resuscitation fluid volume requirements, and edema generation in severely burned patients. Study Design and Setting:A prospective, randomized study at a university trauma and critical care center in Japan.Subjects and Methods: Thirty-seven patients with burns over more than 30% of their total body surface area (TBSA) hospitalized within 2 hours after injury were randomly divided into ascorbic acid and control groups. Fluid resuscitation was performed using Ringer lactate solution to maintain stable hemodynamic measurements and adequate urine output (0.5-1.0 mL/kg per hour). In the ascorbic acid group (n = 19; mean burn size, 63% ± 26% TBSA; mean burn index, 57 ± 26; inhalation injury, 15/ 19), ascorbic acid was infused during the initial 24hour study period. In the control group (n = 18; mean burn size, 53% ± 17% TBSA; mean burn index, 47 ± 13; inhalation injury, 12/18), no ascorbic acid was infused. We compared hemodynamic and respiratory measurements, lipid peroxidation, and fluid balance for 96 hours after injury. Two-way analysis of variance and Tukey test were used to analyze the data.

As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.

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The WRKY proteins comprise a major family of transcription factors that are essential in pathogen and salicylic acid responses of higher plants as well as a variety of plant-specific reactions. They share a DNA binding domain, designated as the WRKY domain, which contains an invariant WRKYGQK sequence and a CX 4-5 CX 22-23 HXH zinc binding motif. Herein, we report the NMR solution structure of the C-terminal WRKY domain of the Arabidopsis thaliana WRKY4 protein. The structure consists of a four-stranded b-sheet, with a zinc binding pocket formed by the conserved Cys/His residues located at one end of the b-sheet, revealing a novel zinc and DNA binding structure. The WRKYGQK residues correspond to the most N-terminal b-strand, kinked in the middle of the sequence by the Gly residue, which enables extensive hydrophobic interactions involving the Trp residue and contributes to the structural stability of the b-sheet. Based on a profile of NMR chemical shift perturbations, we propose that the same strand enters the DNA groove and forms contacts with the DNA bases.

The B3 DNA binding domain is shared amongst various plant-specific transcription factors, including factors involved in auxin-regulated and abscisic acid-regulated transcription. Herein, we report the NMR solution structure of the B3 domain of the Arabidopsis thaliana cold-responsive transcription factor RAV1. The structure consists of a seven-stranded open b-barrel and two a-helices located at the ends of the barrel and is significantly similar to the structure of the noncatalytic DNA binding domain of the restriction enzyme EcoRII. An NMR titration experiment revealed a DNA recognition interface that enabled us to propose a structural model of the protein-DNA complex. The locations of the DNA-contacting residues are also likely to be similar to those of the EcoRII DNA binding domain.