High-affinity binding of Ca2+ to bovine alpha-lactalbumin in the absence and presence of EGTA.

Literature values for the Kd for Ca2+ in bovine alpha-lactalbumin range over 3 orders of magnitude. There is a difference between two results obtained with EGTA as a metal-ion buffer, partly because different values for the Kd of Ca2+-EGTA were used in the calculations, and a much wider difference between results obtained in the presence and absence of EGTA, which has been attributed to an interaction between EGTA and the protein. Titrations in a flow-dialysis cell showed that Mn2+ competed with Ca2+ for the high-affinity site on the protein, and the results, combined with a Kd for Mn2+ of 2.1 +/- 0.1 microM, which was determined fluorimetrically, gave a Kd for Ca2+ of 1.3 +/- 0.1 nM. When alpha-lactalbumin containing 45Ca2+ was titrated with EGTA in a flow-dialysis cell, and widely accepted metal-chelation data for EGTA were used in the calculations, a Kd for Ca2+ of 1.10 +/- 0.03 nM was obtained. The results from the two methods are so similar as to indicate that the affinity for Ca2+ was unaffected by the presence of EGTA.     

The Efficacy of an Immunoisolating Membrane System for Islet Xenotransplantation in Minipigs

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.     

The role of phadiatop in the screening of respiratory allergy

Phadiatop is a new in vitro screening test for respiratory allergy. This test, based on the RAST procedure, detects in serum, the presence of specific IgE to a mixture of common inhalent allergens. Among 70 patients (26 children and 44 adults) consulting for respiratory syndrome, Phadiatop was positive in 31 cases. There were a good correlation between this new test and skin tests (59% for adults and 92% for children), total IgE (70% for adults and 65% for children) and RAST (93% for adults and 96% for children). Phadiatop, with a specificity of 100%, a sensitivity of 82% (76% for adults and 92% for children) and an efficiency of 90% (86% for adults and 96% for children), is a more accurate test than total IgE and could be an excellent in vitro screening test for respiratory allergy.     

Inter-plot competition in yield trials of potatoes (Solanum tuberosum L.) with single-drill plots

An experiment was carried out to assess the significance of inter-plot competition in a yield trial of potato cultivars. Seventeen cultivars were deliberately chosen and assessed for yield in single-drill and four-drill plots. Inter-plot competition for fresh-weight yield was a significant factor in the single-drill plots. It was modelled using a common competition coefficient with a covariate based on neighbour fresh-weight yields. In contrast, there was no statistically significant inter-plot competition for specific gravity. After adjustment for inter-plot competition, varietal ranking in estimated monoculture yield differed little from that based on unadjusted means. However, there was a reduction in the range of yield estimates, and a closer agreement with the observed pure-stand yields from the inner two drills of the four-drill plots. The adjustment for monoculture performance was most pronounced for the higher and lower yielding varieties, as expected from the assumption that the performance of high yielding varieties was enhanced in a competitive environment at the expense of low yielding ones. A general and flexible method of estimating competition coefficients in variety trials, together with a suitable algorithm, was developed and is explained in an appendix. It was used to check for inter-plot competition in a number of potato trials with single-drill plots and a large number of entries. Competition was found in some trials but not in others. Thus, where potato tubers are grown in single-drill plots for assessment of fresh-weight yield, adjustment should be made for inter-plot competition when evidence of inter-drill competition is found.     

Site-directed mutants of charged residues in the active site of tyrosine hydroxylase

The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom. Within the cleft are several charged residues which are conserved across the family of pterin-dependent hydroxylases. We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues. Replacement of arginine 316 with lysine results in a protein with a Ktyr value that is at least 400-fold greater and a V/Ktyr value that is 4000-fold lower than those found in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme. Arginine 316 is therefore critical for the binding of tyrosine. Replacement of glutamate 326 with alanine has no effect on the KM value for tyrosine and results in a 2-fold increase in the KM value for tetrahydropterin. The Vmax for DOPA production is reduced 9-fold, and the Vmax for dihydropterin formation is reduced 4-fold. These data suggest that glutamate 326 is not directly involved in catalysis. Replacement of aspartate 328 with serine results in a 26-fold higher KM value for tyrosine, a 8-fold lower Vmax for dihydropterin formation, and a 13-fold lower Vmax for DOPA formation. These data suggest that aspartate 328 has a role in tyrosine binding. Replacement of glutamate 332 with alanine results in a 10-fold higher KM value for 6-methyltetrahydropterin with no change in the KM value for tyrosine, a 125-fold lower Vmax for DOPA formation, and an only 3.3-fold lower Vmax for tetrahydropterin oxidation. These data suggest that glutamate 332 is required for productive tetrahydropterin binding.     

Mass Ligand Binding Screening for Receptor Antagonists: Prototype New Drugs and Blind Alleys

Abstract

The ligand binding assay is a powerful tool in the search for antagonists for novel receptors, and for identification of novel classes of antagonists for well-known receptors. Ligand binding mass screening can be adapted for very high throughput. In order for mass screening to be useful, it is necessary to strictly define the binding characteristics for a compound to be considered a putative receptor antagonist. In practice, we have found that synthetic pursuit of a compound with a K_i of ± 1 uM is likely to lead down a blind alley unless very good evidence for specificity is available. Even potent competitors for binding should be thoroughly evaluated in assays of biological activity before a synthetic program is initiated in earnest.     

Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system

The large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters. We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli. A high level of recombinant protein expression is a requirement for purification of protein for structural determination and other purposes. In other applications, successful complementation of a missing enzyme activity in E. coli, as well as directed evolution studies and metabolic engineering, often require a much lower level of protein expression. In this report we describe the construction of a number of new pHOST vectors that can be screened for both low- and high-level expression. We isolated a mutant vector for MBP fusions that exhibited a more optimal level of expression for complementation of aerobic respiration in hemA(-) E. coli, our functional assay for the alternative oxidase. We then demonstrated the use of our system to rapidly screen for both optimal functional expression and optimal overexpression of the alternative oxidase as well as two other members of a family of membrane-bound diiron carboxylate proteins, the plastid terminal oxidase and 5-demethoxyquinone hydroxylase.     

The effect of exposure to black background and to α-MSH on black-white background preference in the amphibian Xenopus laevis (Daudin)

Young frogs, Xenopus laevis, raised on a white background showed a slight preference for the black background of a black-white preference testing apparatus. Keeping frogs for either 24 hr or 20 days on a black background resulted in a further increase in their preference for black. Injecting 1 μg of synthetic α-MSH into white-adapted frogs had no effect on their black-white preference. On the other hand, Xenopus laevis tadpoles, at an early stage of metamorphosis, raised on a white background showed a significant preference for white. Keeping these tadpoles for 24 hr on a black background resulted in a shift from white to black preference. Injecting synthetic α-MSH into white-adapted tadpoles resulted in an increase in their preference for black. A behavioral feedback mechanism appears to exist between the release of endogenous MSH when a tadpole is exposed to a black background and its subsequent preference for background color.     

Kinetic analyses and structure-activity relationship studies of synthetic lysine acetylation catalysts

Lysine acylation of proteins is a crucial chemical reaction, both as a post-translational modification and as a method for bioconjugation. We previously developed a chemical catalyst, DSH, which activates a chemically stable thioester including acyl-CoA, allowing the site-selective lysine acylation of histones under physiological conditions. However, a more active catalyst is required for efficient lysine acylation in more complex biological milieu, such as in living cells, but there are no rational guidelines for developing efficient lysine acylation catalysts for use under physiological conditions as opposed to in organic solvents. We, herein, conducted a kinetic analysis of the ability of DSH and several derivatives to mediate lysine acetylation to better understand the structural elements essential for high acetylation activity under physiological conditions. Interestingly, the obtained trend in reactivity was different from that observed in organic solvents, suggesting that a different principle is necessary for designing chemical catalysts specifically for use under physiological conditions compared to catalysts for use in organic solvents. Based on the obtained information, we identified a new catalyst scaffold with high activity and structural flexibility for further modification to improve this catalyst system.     

Aspects éthiques des prélèvements d’organes à coeur arrêté

Medical progress in organ preservation during cardiopulmonary resuscitation allow to obtain organ for transplantation in patients suffering from a cardiac arrest. The different theoretical situations have been summarized in an international classification (Maastricht classification). In France, the fact that patients deceased after a phase of withholding/withdrawing active therapies in intensive care (Maastricht class III) are specifically excluded from this discussion represent an ethical safeguard for the public and the health-care providers. Ethically, in cardiac arrest patients, there is a will of maintaining a clear distinction between the care of the patients for therapeutic purposes for the patient (that cannot lead to an organ withdrawal except for the specific case of a secondary evolution towards brain death) and for a pursuit of therapeutic maneuvers in order to obtain organs for transplantation.     

Modeling the Hydration Layer around Proteins: HyPred

Protein hydration plays an integral role in determining protein function and stability. We develop a simple method with atomic level precision for predicting the solvent density near the surface of a protein. A set of proximal radial distribution functions are defined and calculated for a series of different atom types in proteins using all-atom, explicit solvent molecular dynamic simulations for three globular proteins. A major improvement in predicting the hydration layer is found when the protein is held immobile during the simulations. The distribution functions are used to develop a model for predicting the hydration layer with sub-1-Ångstrom resolution without the need for additional simulations. The model and the distribution functions for a given protein are tested in their ability to reproduce the hydration layer from the simulations for that protein, as well as those for other proteins and for simulations in which the protein atoms are mobile. Predictions for the density of water in the hydration shells are then compared with high occupancy sites observed in crystal structures. The accuracy of both tests demonstrates that the solvation model provides a basis for quantitatively understanding protein solvation and thereby predicting the hydration layer without additional simulations.     

Molecular modeling and dynamics simulations of PNP from Streptococcus agalactiae

This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.     

Affinity of corticosteroids for mineralocorticoid and glucocorticoid receptors of the rabbit kidney: effect of steroid substitution

Corticosteroid derivatives coupled in the C3, C7 or C17 position with a long aliphatic chain were synthesized in order to select a suitable ligand for the preparation of a biospecific affinity adsorbent for mineralocorticoid receptor purification. The affinity of these derivatives for mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) was explored in rabbit kidney cytosol. In this model, aldosterone bound to a single class of receptors with high affinity (Kd 1 nM) and mineralocorticoid specificity. RU26988, a highly specific ligand for GR, did not compete for these sites. The C7 and C17 positions were found to be of crucial importance in the steroid's interaction with the mineralocorticoid receptors, since the linkage of a long side chain in these positions induced complete loss of affinity. Hence, deoxycorticosterone no longer bound to MR after 17 beta substitution with a 9-carbon aliphatic chain. This loss of affinity was not observed for glucocorticoids. The 17 beta nonylamide derivative of dexamethasone still competed for GR. Increasing the length of the C7 side of the spirolactone SC26304 suppressed its affinity for MR. Finally, C3 was an appropriate position for steroid substitution. The 3-nonylamide of carboxymethyloxime deoxycorticosterone bound to MR but not to GR, and therefore constitutes a suitable ligand for the preparation of a mineralocorticoid adsorbent.     

Re-entry programs, from an anti-unconventional ex-offender

The purpose of this article seeks to question the relationship of higher levels of educational attainment and the ability for successful reintegration into mainstream society for the formerly incarcerated. Specifically this article highlights societal negligence for individual success of the formerly incarcerated, opting for a corporatized exploitation of those in the reentry process premised on the socially constructed label “ex-con.” Research surrounding labeling and reentry suggests once individuals are released from prison the social stigma experienced in society is a recipe for failure and reoffending. One way this emerges is in the form of hiring policies in corporate occupations, prohibiting the hiring of individual s convicted of a felony. Critical criminology suggests the class structure is in place for those in power to stay in power and keep those who are inferior in inferior social positions. Using both points of view as a framework, with the idea of corporate monopoly as a lens, allows for an untraditional critical perspective of the reentry process as a capitalist, for-profit industry. Questioning the role and intentions of reentry initiatives provides a general discussion for increasing success rates of those in the reentry process and reducing capital costs spent for incarceration. The specific focus of this article presents my experience of the reentry process.     

Estimates of time and propensity for dental treatment among institutionalised elders

The oral health and concerns of elderly residents were surveyed in a stratified random selection of 41 long-term care facilities in Vancouver. The residents who could participate were examined and interviewed to determine their oral health and concerns about dental treatment. The need and time required for treatment were estimated in six groups to reflect the propensity for treatment in dentate and edentate subjects. The propensity for treatment was high in about one-third of the participants whereas it was unlikely that one-tenth of those examined would ever use a dental service. In general, there was a widespread need for a diagnostic service because so few had been examined by a dentist in the previous year. Prosthodontics accounted for most of the treatment in all of the propensity groups, with substantially more time required by the dentate residents. There was also a substantial need for management of mucosal pathoses and oral hygiene and, to a lesser extent, for dental restorations and endodontics. Overall, our estimates indicate a substantial need for dental treatment among residents of long-term care facilities, although the need is reduced by half if the propensity for treatment is considered.     

Experimental tests for an evolutionary trade-off between growth rate and yield in E. coli

Theoretical studies have predicted a trade-off between growth rate and yield in heterotrophic organisms. Here we test for the existence of this trade-off by analyzing the growth characteristics of 12 E. coli B populations that evolved for 20,000 generations under a constant selection regime. We performed three different tests. First, we analyzed changes in growth rate and yield over evolutionary time for each population. Second, we tested for a negative correlation between rate and yield across the 12 populations. Finally, we isolated clones from four selected populations and tested for a negative correlation between rate and yield within these populations. We did not find evidence for a trade-off based on the first two tests. However, we did observe a trade-off based on the within-population correlation of yield and rate. Our results indicate that, at least for the populations studied here, an analysis of the within-population diversity might be the most sensitive test for the existence of a trade-off. The observation of a trade-off within, but not between, populations suggests that the populations evolved different genetic solutions for growth in the selective environment, which in turn led to different physiological constraints.     

Relationship between thermodynamic driving force and one-way fluxes in reversible processes

Chemical reaction systems operating in nonequilibrium open-system states arise in a great number of contexts, including the study of living organisms, in which chemical reactions, in general, are far from equilibrium. Here we introduce a theorem that relates forward and reverse fluxes and free energy for any chemical process operating in a steady state. This relationship, which is a generalization of equilibrium conditions to the case of a chemical process occurring in a nonequilibrium steady state in dilute solution, provides a novel equivalent definition for chemical reaction free energy. In addition, it is shown that previously unrelated theories introduced by Ussing and Hodgkin and Huxley for transport of ions across membranes, Hill for catalytic cycle fluxes, and Crooks for entropy production in microscopically reversible systems, are united in a common framework based on this relationship.     

A complete graphical criterion for the adjustment formula in mediation analysis

Various assumptions have been used in the literature to identify natural direct and indirect effects in mediation analysis. These effects are of interest because they allow for effect decomposition of a total effect into a direct and indirect effect even in the presence of interactions or non-linear models. In this paper, we consider the relation and interpretation of various identification assumptions in terms of causal diagrams interpreted as a set of non-parametric structural equations. We show that for such causal diagrams, two sets of assumptions for identification that have been described in the literature are in fact equivalent in the sense that if either set of assumptions holds for all models inducing a particular causal diagram, then the other set of assumptions will also hold for all models inducing that diagram. We moreover build on prior work concerning a complete graphical identification criterion for covariate adjustment for total effects to provide a complete graphical criterion for using covariate adjustment to identify natural direct and indirect effects. Finally, we show that this criterion is equivalent to the two sets of independence assumptions used previously for mediation analysis.