Getting the most out of X-ray home sources.

The structures of a 14 kDa phospholipase, an 18 kDa proteinase inhibitor and a novel glycoside hydrolase with molecular weight 60 kDa were solved using the SAD technique and the effects of the amount of anomalous signal, completeness and redundancy of data on heavy-atom substructure determination, phasing and model building were analyzed. All diffraction data sets were collected on a Cu-anode X-ray home source. The structure of the phospholipase was obtained using the anomalous scattering contribution from its 16 S atoms. Three-dimensional models for the other two macromolecules were obtained using the anomalous contribution of I atoms rapidly incorporated into the crystal through the quick cryo-soaking method of derivatization. These results were used to discuss the application of sulfur- and iodine-SAD approaches in combination with X-ray home sources for high-throughput protein crystal structure solution. The estimates of the anomalous signal from S atoms in the gene products of four genomes are given and the prospects for increasing the anomalous contribution using longer wavelengths (e.g. from a chromium home source) and quick cryo-soaking derivatization are discussed. The possibility of rapidly preparing tangible home-source isomorphous derivatives suggests that this approach might become a valuable tool in the future of post-genomic projects.

Anti-adhesion molecule strategies as potential neuroprotective agents in cerebral ischemia: a critical review of the literature.

Despite recent advances in the understanding of the pathophysiology of cerebral ischemia, current approaches attempting to prevent ischemic brain damage after an acute stroke remain quite inadequate. Today, ischemic stroke remains the third leading cause of death in industrialized nations, and the leading cause of disability requiring long term institutional care in the U.S and other industrialized nations. While one treatment, tissue plasminogen activator, has shown efficacy in clinical trials, safety concerns limit its role in clinical practice to a narrow time window of use. Acute cerebral ischemia has been shown to evoke a profound and deleterious upregulation of the inflammatory response, initiated within the cerebral microvasculature. Recently, research efforts have focused on targeting individual components of the inflammatory cascade, such as leukocyte activation and adhesion, in an attempt to develop potential neuroprotective agents. While these strategies have shown promise preclinically, clinical trials have yet to show clear benefit. Here, we review the current understanding of the pathophysiologic consequences of acute cerebral ischemic injury. Additionally, we discuss the role of the inflammatory cascade, with specific attention given to the deleterious role played by leukocyte activation and adhesion in stroke. Finally, relevant efforts to translate these basic science observations into clinical efficacy in acute stroke trials are critically reviewed.

Laser Doppler flowmetry in non-human primate stroke studies: model refinement for pre-clinical development of cerebroprotective strategies.

BACKGROUND: Safety, feasibility, and efficacy trials in non-human primate stroke models are essential to the evaluation of experimental therapies and their translation to humans. Although Laser Doppler Flowmetry has been successfully employed in rodent stroke to continuously monitor cerebral blood flow, it has not been applied in primate studies. This investigation examined the utility of Laser Doppler Flowmetry in refining an existing baboon model of cerebral ischemia/reperfusion. METHOD: Continuous Laser Doppler Flowmetry monitoring was used, in non-human primates, to document local cerebral blood flow before, during, and after middle cerebral artery territory occlusion. In each baboon (n = 7) a single Doppler probe was placed into the left frontal cortex through a precoronal burr hole. Correlations between Laser Doppler Flowmetry values and latencies to Motor Evoked Potential dropout were compared using a linear regression model. FINDINGS: Placement of the Laser Doppler probe was easily accomplished in all animals. Laser Doppler Flowmetry tracings accurately documented blood flow changes that occurred with each technical manipulation during the procedure. Laser Doppler confirmed decreased perfusion that coincided both regionally and temporally with vessel occlusion. Depth of ischemia as measured by Laser Doppler Flowmetry was associated with Motor Evoked Potential dropout latencies for individual animals. CONCLUSIONS: Continuous, single probe Laser Doppler Flowmetry is a reliable method of documenting perfusion changes following middle cerebral artery territory occlusion in a baboon model of reperfused stroke. This advanced intraoperative monitoring technique may lead to more accurate evaluation of acute stroke therapies in pre-clinical trials.

The role of the complement cascade in ischemia/reperfusion injury: implications for neuroprotection.

BACKGROUND: The complement cascade plays a deleterious role in multiple models of ischemia/reperfusion (I/R) injury, including stroke. Investigation of the complement cascade may provide a critical approach to identifying neuroprotective strategies that can be effective at clinically relevant time points in cerebral ischemia. This review of the literature describes the deleterious effects of complement activation in systemic I/R models and previous attempts at therapeutic complement inhibition, with a focus on the potential role of complement inhibition in ischemic neuroprotection. Translation of these concepts into ischemic stroke models and exploration of related neuroprotective strategies are also reviewed. SUMMARY OF REVIEW: We performed a MEDLINE search to identify any studies published between 1966 and 2001 dealing with complement activation in the setting of I/R injury. We also searched for studies demonstrating up-regulation of any complement components within the central nervous system during inflammation and/or ischemia. CONCLUSIONS: The temporal and mechanistic overlap of the complement cascade with other biochemical events occurring in cerebral I/R injury is quite complex and is only beginning to be understood. However, there is compelling evidence that complement is quite active in the setting of acute stroke, suggesting that anticomplement strategies should be further investigated through genetic analysis, nonhuman primate models, and clinical investigations.

Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance.

Galectins, beta-galactoside-binding lectins, are extensively distributed in the animal kingdom and share some basic molecular properties. Galectin-3, a member of this family, is generally associated with differentiation, morphogenesis, and metastasis. In this study, galectin-3 was isolated from ovine placental cotyledons round the middle of the gestation period by lactose extraction followed by affinity chromatography on lactosyl-agarose, and separated from galectin-1 by size exclusion chromatography on a Superose 12 column. Under native conditions this lectin behaved as a monomer with an apparent molecular weight of approximately 29,000 and an isoelectric point of 9.0. The partial amino acid sequence of the peptides obtained by tryptic digestion of this protein followed by HPLC separation showed striking homology with other members of the galectin-3 subfamily. Furthermore, ovine placental galectin-3 exhibited specific mitogenic activity toward rat spleen mononuclear cells. Besides, this protein strongly reacted with a rabbit antiserum raised against a chicken galectin. Results obtained by Western blot analysis showed that its expression was greatly decreased in term placenta with respect to the middle of the gestation period, suggesting a regulated expression throughout development.

The heparin-binding lectin from ovine placenta: purification and identification as histone H4.

The heparin-binding lectin complex from ovine placental cotyledons was purified by affinity chromatography on heparin-agarose column. It showed three protein bands, which had molecular weights of 13000, 15000 and 17000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of DNA by agarose gel electrophoresis. The protein components of the complex were separated by reverse-phase HPLC. The minimum inhibitory concentrations of glycosaminoglycans were significantly different for the lectin complex and the separated proteins, suggesting affinity changes upon DNA binding. The haemagglutinating activity specificity allowed the characterization of the fraction with a molecular weight of 13000 as the heparin-binding lectin. This protein was identified as histone H4 by internal sequencing, thus showing that this is the histone responsible for the heparin-binding property of the complex. The accompanying proteins were tentatively identified as histones H2A and H2B.