Structure of aldose reductase from Giardia lamblia.

Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP(+)-binding site located within the eight ß-strands of the interior.

The Seattle Structural Genomics Center for Infectious Disease (SSGCID).

The NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium established to apply structural genomics approaches to potential drug targets from NIAID priority organisms for biodefense and emerging and re-emerging diseases. The mission of the SSGCID is to determine approximately 400 protein structures over the next five years. In order to maximize biomedical impact, ligand-based drug-lead discovery campaigns will be pursued for a small number of high-impact targets. Here we review the center's target selection processes, which include pro-active engagement of the infectious disease research and drug therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. This is followed by a brief overview of the SSGCID structure determination pipeline and ligand screening methodology. Finally, specifics of our resources available to the scientific community are presented. Physical materials and data produced by SSGCID will be made available to the scientific community, with the aim that they will provide essential groundwork benefiting future research and drug discovery.

RNA methylation under heat shock control.

Structural, biochemical, and genetic techniques were applied to investigate the function of FtsJ, a recently identified heat shock protein. FtsJ is well conserved, from bacteria to humans. The 1.5 A crystal structure of FtsJ in complex with its cofactor S-adenosylmethionine revealed that FtsJ has a methyltransferase fold. The molecular surface of FtsJ exposes a putative nucleic acid binding groove composed of highly conserved, positively charged residues. Substrate analysis showed that FtsJ methylates 23S rRNA within 50S ribosomal subunits in vitro and in vivo. Null mutations in ftsJ show a dramatically altered ribosome profile, a severe growth disadvantage, and a temperature-sensitive phenotype. Our results reveal an unexpected link between the heat shock response and RNA metabolism.

Structure of Hsp15 reveals a novel RNA-binding motif.

We have solved the crystal structure of the heat shock protein Hsp15, a newly isolated and very highly inducible heat shock protein that binds the ribosome. Comparison of its structure with those of two RNA-binding proteins, ribosomal protein S4 and threonyl-tRNA synthetase, reveals a novel RNA-binding motif. This newly recognized motif is remarkably common, present in at least eight different protein families that bind RNA. The motif's surface is populated by conserved, charged residues that define a likely RNA-binding site. An intriguing pattern emerges: stress proteins, ribosomal proteins and tRNA synthetases repeatedly share a conserved motif. This may imply a hitherto unrecognized functional similarity between these three protein classes.

Effect of pentoxifylline on radiation-induced lung and skin toxicity in rats.

PURPOSE: There is currently substantial clinical interest in pentoxifylline as an inhibitor of radiation-related normal tissue injury. To further assess this drug's potential toxicity-sparing effects, pentoxifylline was studied in rats using a radiation-induced lung injury model. METHODS AND MATERIALS: Adult male rats were exposed to either sham irradiation or a single fraction of 21 Gy delivered to the left hemithorax. Four study groups were defined: those that received neither radiation nor pentoxifylline, those that received pentoxifylline (500 mg/L in drinking water) but no irradiation, those that underwent irradiation without pentoxifylline, and those that received both pentoxifylline and radiation. Lung injury was measured by changes in relative left:right lung perfusion ratios derived from quantitative gamma camera imaging of 99mTechnetium-macroaggregated albumin uptake in the pulmonary circulation. Serial scans were done over a 40-week period following radiation. Skin toxicity was also assessed. After 40 weeks, the animals were killed, and lung tissue was assayed for angiotensin converting enzyme activity as a marker for endothelial cell damage. RESULTS: Both groups of radiated (with or without pentoxifylline) rats showed equivalent acute sharp decreases in left:right lung perfusion ratios compared to the nonirradiated groups, reaching a mean nadir value of 0.29 at week 4. Irradiated lung perfusion in subsequent weeks in the radiation-only group showed minimal recovery, with a plateau mean ratio of 0.37 (0.36-0.39). However, there was apparent later recovery of lung perfusion in the radiation with pentoxifylline group from weeks 14 through 40, to a mean ratio of 0.47 (0.43-0.52) (p < 0.01 compared to the radiation-only group). Angiotensin converting enzyme activity correlated closely with lung perfusion data. No effect of pentoxifylline on acute or late skin toxicity was detected. CONCLUSIONS: This study suggests that pentoxifylline does not have any measurable effect on acute lung injury following hemithoracic irradiation in rats, but does result in sparing of later lung toxicity.

Effect of bicarbonate on stability of the gallium-transferrin complex.

When gallium citrate is injected intravenously the gallium is rapidly transchelated to transferrin. If the stability of the gallium-transferrin association is sufficiently strong it should be a good macromolecular tracer suitable for quantitative measurements of vascular permeability. Studies of the stability of the gallium transferrin complex in human plasma and serum were done using ultrafiltration and dialysis. It was found that the stability was sensitive to bicarbonate level. Above 13 mM of bicarbonate, 97%-99% of the gallium is bound to molecules greater than 10,000 MW by ultrafiltration. However, at 10 mM binding is 95% and at 7 mM 83%. Similar results were obtained with dialysis. This suggests the gallium-transferrin complex may not be sufficiently stable for quantitative measures of vascular permeability, particularly if the bicarbonate concentration is low.