Investigating Models of Protein Function and Allostery With a Widespread Mutational Analysis of a Light-Activated Protein

To investigate the relationship between a protein’s sequence and its biophysical properties, we studied the effects of more than 100 mutations in Avena sativa light-oxygen-voltage domain 2, a model protein of the Per-Arnt-Sim family. The A. sativa light–oxygen–voltage domain 2 undergoes a photocycle with a conformational change involving the unfolding of the terminal helices. Whereas selection studies typically search for winners in a large population and fail to characterize many sites, we characterized the biophysical consequences of mutations throughout the protein using NMR, circular dichroism, and ultraviolet/visible spectroscopy. Despite our intention to introduce highly disruptive substitutions, most had modest or no effect on function, and many could even be considered to be more photoactive. Substitutions at evolutionarily conserved sites can have minimal effect, whereas those at nonconserved positions can have large effects, contrary to the view that the effects of mutations, especially at conserved positions, are predictable. Using predictive models, we found that the effects of mutations on biophysical function and allostery reflect a complex mixture of multiple characteristics including location, character, electrostatics, and chemistry.     

Tolerance of low oxygen stress in insecticide-resistant and susceptible populations of mosquitofish (Gambusia affinis)

The tolerance of low oxygen stress in insecticide-resistant and susceptible populations of mosquitofish ($\text{Gambusia}$$\text{affinis}$) was investigated. Dissolved oxygen was reduced chemically with formaldehyde and sodium sulfite treatment. Available oxygen was reduced physically through barriers, both by preventing oxygen exchange at the air-water interface and by preventing the fish from contacting the surface of the water. In all cases, the tolerance to low oxygen stress was reduced in the insecticide-resistant population compared to the susceptible population. The results indicate that although tolerance of pesticides in the resistant population has been dramatically increased by environmental selective pressures, tolerance to other stresses has not been concomitantly increased.     

Mirex-induced liver enlargement in rats is dependent upon an intact pituitary-adrenalcortical axis

The effect of prior hypophysectomy upon mirex-induced liver hypertrophy in male Sprague-Dawley rats was examined. Mirex had no effect upon adrenal weight, liver weight, plasma glucose or plasma corticosterone in hypophysectomized rats. However, daily corticosterone supplements (20 mg/kg body weight, sc) given to mirex-treated hypophysectomized animals yielded a 52% increase in liver weight to body weight ratios over those observed in mirex-treated hypophysectomized animals not receiving supplement. In intact rats, both liver weight to body weight ratios and plasma ACTH were significantly increased over controls 2 days after mirex treatment. These results indicate that mirex-induced liver enlargement not only requires corticosterone, but that the response is dependent upon an intact pituitary-adrenalcortical axis.     

Physiological effects of crude oil exposure in the striped mullet, Mugil cephalus

Juvenile mullet ($\text{Mugil cephalus}$) were exposed to a surface slick of Empire Mix crude oil for a three week period in a simulated estuarine ecosystem. Liver weight to body weight ratios were increased in the mullet from the oil-treated ponds when compared to those from the control ponds. Activities of alkaline phosphatase, which were elevated in gill and muscle of oil-treated mullet, and β-glucuronidase, which was elevated in the muscle of oil-treated mullet, may be related to the degree of stress the animals were experiencing. Malic dehydrogenase, which was depressed in the livers and elevated in the muscle of oil-treated organisms, indicate changes in aerobic metabolism in response to the stress of crude oil exposure. Muscle acetylcholinesterase was not affected by oil exposure.     

Kinetics of interaction of 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate with bovine brain tubulin

The binding of the guanine nucleotide analogue 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (S6-GTP) to tubulin from which the associated proteins and exchangeably bound nucleotide have been removed produces about a 15% decrease in intrinsic tubulin fluorescence. Using a fluorescence stopped-flow technique, we have examined the kinetics and mechanism of this process. Analysis of the data reveals that the binding is complex, involving at least one conformational change subsequent to nucleotide binding. The bimolecular association rate constant for binding of S6-GTP to tubulin is approximately 6 X 10(5) M-1 s-1, suggesting that the orientation requirements are stringent. The kinetic parameters for dissociation of GDP, S6-GTP, and S6-GDP from the exchangeable nucleotide binding site have also been determined. S6-GDP and GDP were found to have comparable rates of dissociation; S6-GTP dissociated approximately twice as slowly as either GDP or S6-GDP. Glycerol produces a significant decrease in the rates of nucleotide dissociation. The mechanism whereby glycerol produces such an effect is not known; however, it may involve slight changes in the conformation of the tubulin protomer.     

Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface suggested that only four residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent residues in DR TrxR (M84, K137, F148, and F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. When M84 and K137 were changed to match equivalent EC TrxR residues (K137R and M84F), the DR TrxR substrate specificity was altered from its own Trx to that of EC Trx. The results suggest that a small subset of the TrxR-Trx interface residues is responsible for the majority of Trx binding affinity and species-specific recognition.     

The Open Microscopy Environment (OME) Data Model and XML file: open tools for informatics and quantitative analysis in biological imaging

The Open Microscopy Environment (OME) defines a data model and a software implementation to serve as an informatics framework for imaging in biological microscopy experiments, including representation of acquisition parameters, annotations and image analysis results. OME is designed to support high-content cell-based screening as well as traditional image analysis applications. The OME Data Model, expressed in Extensible Markup Language (XML) and realized in a traditional database, is both extensible and self-describing, allowing it to meet emerging imaging and analysis needs.     

Amphiphilic conjugates of human brain natriuretic peptide designed for oral delivery: in vitro activity screening

Congestive heart failure (CHF) is a complex syndrome involving altered neurohormonal levels and impaired cardiac and renal function. In recent years, intravenous administration of exogenous human brain-type natriuretic peptide (hBNP) has become an important therapy in treating patients with acutely decompensated CHF. However, reports during the past year suggest that hBNP could play a prominent role in the chronic treatment of CHF patients as well. We are currently developing conjugates of hBNP suitable for oral delivery to provide a patient-friendly treatment option for chronic heart failure patients. In this report, we present in vitro activity results obtained from hBNP conjugates featuring a variety of rationally designed amphiphilic oligomers. Mapping studies revealed that the hydrophobic/hydrophilic balance of the oligomer impacted the regioselectivity of conjugation. Additionally, the regiochemistry and extent of conjugation had a significant impact on activity. Many monoconjugates retained activity comparable to native peptide and are currently under evaluation in subsequent in vivo screens.     

Deaths of detainees in the custody of US forces in Iraq and Afghanistan from 2002 to 2005

In light of the large number of detainees who continue to be taken and held in US custody in settings with limited judicial or public oversight, deaths of detainees warrant scrutiny. We have undertaken the task of reviewing all known detainee deaths between 2002 and early 2005 based on reports available in the public domain. Using documents obtained from the Department of Defense through a Freedom of Information Act request, combined with a review of anecdotal published press accounts, 112 cases of death of detainees in United States custody (105 in Iraq, 7 in Afghanistan) during the period from 2002 to early 2005 were identified. Homicide accounted for the largest number of deaths (43) followed by enemy mortar attacks against the detention facility (36). Deaths attributed to natural causes numbered 20. Nine were listed as unknown cause of death, and 4 were reported as accidental or natural. A clustering of 8 deaths ascribed to natural causes in Iraq in August 2003 raises questions about the adequacy and availability of medical care, as well as other conditions of confinement that may have had an impact on the mortality rate.