Using model proteins to quantify the effects of pathogenic mutations in Ig-like proteins

It has proved impossible to purify some proteins implicated in disease in sufficient quantities to allow a biophysical characterization of the effect of pathogenic mutations. To overcome this problem we have analyzed 37 different disease-causing mutations located in the L1 and IL2Rgamma proteins in well characterized related model proteins in which mutations that are identical or equivalent to pathogenic mutations were introduced. We show that data from these models are consistent and that changes in stability observed can be correlated to severity of disease, to correct trafficking within the cell and to in vitro ligand binding studies. Interestingly, we find that any mutations that cause a loss of stability of more than 2 kcal/mol are severely debilitating, even though some model proteins with these mutations can be easily expressed and analyzed. Furthermore we show that the severity of mutation can be predicted by a DeltaDeltaG(evolution) scale, a measure of conservation. Our results demonstrate that model proteins can be used to analyze disease-causing mutations when wild-type proteins are not stable enough to carry mutations for biophysical analysis.     

Exploring the pharmacokinetic properties of phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2)

We report the preparation and structure–activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.     

Functional genomics of the yeast DNA-damage response

High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage.     

Normalization of ground reaction forces

The appropriateness of normalizing data, as one method to reduce the effects of a covariate on a dependent variable, should be evaluated. Using ratio, 0.67-nonlinear, and fitted normalizations, the aim of this study was to investigate the relationship between ground reaction force variables and body mass (BM). Ground reaction forces were recorded for 40 female subjects running at 3.7 +/- 0.18 m x s(-1) (mass = 58 +/- 6 kg). The explained variance for mass to forces (peak-impact-vertical = 70%; propulsive-vertical = 27%; braking = 40%) was reduced to <0.1% for mass to ratio normalized forces (i.e., forces/BM1) with statistically significantly different power exponents (p < 0.05). The smaller covariate effect of mass on loading rate variables of 2-16% was better removed through fitted normalization (e.g., vertical-instantaneous-loading rate/ BM(0.69+/-0.93); +/-95% CI) with nonlinear power exponents ranging from 0.51 to 1.13. Generally, these were similar to 0.67 as predicted through dimensionality theory, but, owing to the large confidence intervals, these power exponents were not statistically significantly different from absolute or ratio normalized data (p > 0.05). Further work is warranted to identify the appropriate method to normalize loading rates either to mass or to another covariate. Ratio normalization of forces to mass, as predicted through Newtonian mechanics, is recommended for comparing subjects of different masses.     

The receptor for branch-site docking within a group II intron active site

The distinguishing feature of group II introns, and the property that links them with spliceosomal catalysis, is their ability to undergo splicing through branching. In this reaction, the 2'-hydroxyl group of a specific adenosine within intron domain 6 serves as the nucleophile for attack on the 5' splice site. We know less about branching than any other feature of group II intron catalysis, largely because the receptor structure for activating the branch site is unknown. Here, we identify the intronic region that binds the branch site of a group IIB intron. Located in domain 1, close to receptors for intron domain 5 and both splice sites, we demonstrate that the branch-site receptor is a functional element required for transesterification. Furthermore, we show that crosslinked branch sites can carry out both steps of splicing, suggesting that the conformational state of the intron core is set early and that it persists throughout the entire splicing process.     

The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9

The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9. We investigated whether the genomic Cf-9 gene functions in potato and tobacco. Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection. Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco. A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent. However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed. Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings. Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9. Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.