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.     

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.     

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.     

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.     

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.     

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.     

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.     

Hematology and serum chemistry values in the beluga (Delphinapterus leucas)

Normal values and ranges for 31 clinical hematology and serum chemistry tests are reported for the beluga or white whale (Delphinapterus leucas). The values were collected over a 6-yr period from eight belugas maintained for display at Sea World (San Diego, California, USA) facilities and represent long-term evaluations for each animal in a controlled environment. They represent the first report for a number of serum chemistry values for the beluga. Normal values such as these provide an important data base from which to detect diagnostically important changes in health status for belugas in a zoological setting. They also establish a baseline from which to evaluate differences in normal values in free-ranging belugas and from which to diagnose disease problems in wild populations.     

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.     

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.     

Possible genomic imprinting of three human obesity-related genetic loci

To detect potentially imprinted, obesity-related genetic loci, we performed genomewide parent-of-origin linkage analyses under an allele-sharing model for discrete traits and under a family regression model for obesity-related quantitative traits, using a European American sample of 1,297 individuals from 260 families, with 391 microsatellite markers. We also used two smaller, independent samples for replication (a sample of 370 German individuals from 89 families and a sample of 277 African American individuals from 52 families). For discrete-trait analysis, we found evidence for a maternal effect in chromosome region 10p12 across the three samples, with LOD scores of 5.69 (single-point) and 4.52 (multipoint) for the pooled sample. For quantitative-trait analysis, we found the strongest evidence for a maternal effect (single-point LOD of 2.85; multipoint LOD of 4.01 for body mass index [BMI] and 3.69 for waist circumference) in region 12q24 and for a paternal effect (single-point LOD of 4.79; multipoint LOD of 3.72 for BMI) in region 13q32, in the European American sample. The results suggest that parent-of-origin effects, perhaps including genomic imprinting, may play a role in human obesity.     

Screening for aneuploidy in twin pregnancies: maternal age- and race-specific risk assessment between 9-14 weeks.

The aim of this study was to calculate the risk for aneuploidy in twin pregnancies between 9-14 weeks utilizing maternal age, race and dizygotic twinning rates. Using previously published risks for aneuploidy in singletons and twins at the time of amniocentesis and at term, we calculated new risk estimates for twins at 9-14 weeks gestation or at the time of chorionic villus sampling. Using these tables, the risk for trisomy 21 in at least one fetus of a twin gestation in a 32-year-old at 9-14 weeks is 1/285 for Whites and for African-Americans. This is equivalent to the risk for trisomy 21 (1/265) in a 35-year-old woman with a singleton at the same gestational age. The risks for trisomies 18 and 13 also follow similar trends. In counseling women with twin pregnancies at the time of first trimester nuchal translucency screening or chorionic villus sampling, it should be noted that the maternal age-related risk for aneuploidy for a 32-year-old is equivalent to that of a 35-year-old woman with a singleton gestation.     

Repetitive mating and female fitness in Dysdercus cardinalis (Hemiptera: Pyrrhocoridae)

Using three mating-treatment groups–pairing with a male for life, pairing with a male from adult emergence to first oviposition, and no pairing (virgin)–of Dysdercus cardinalis females in a laboratory study, the following results were obtained. Most of the virgin females did not lay eggs; those that did oviposited for the first time at a considerably older age than females in the other two groups. In a lifetime, females pairing with a male for life and those pairing with a male up to first oviposition produced essentially the same number of eggs, and this was more than 3.5 times the number of eggs laid by a virgin female. Virgin females produced an average of 1.3 clutches in a lifetime, compared with about 4.5 clutches produced by females in the other two groups. Reproductive span was significantly shorter, and fecundity per day of reproductive span significantly greater, for females pairing with males for life than for those pairing with a male up to first oviposition. Age at death was significantly different amongst females in different mating-treatment groups. Virgin females had the longest life, followed by females pairing with a male up to first oviposition and females pairing with a male for life, in that order. There was a positive correlation between lifetime gross reproduction and age at death for females pairing with a male for life. There was no relationship between these two characters for females pairing with a male up to first oviposition. Both females pairing with a male for life and those pairing with a male to first oviposition exhibited a significant inverse relationship between fecundity per day and age at death. The results obtained indicate that (1) mating is a prerequisite for normal gonadal activity in Dysdercus cardinalis , and (2) repetitive mating increases the rate of reproduction. This would reduce the mean age of parenthood which is inversely related to the intrinsic rate of increase.     

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.     

Rapid assessment of female preference for male size predicts subsequent choice of spawning partner in a socially monogamous cichlid fish

Although size-assortative mating in convict cichlids, Amatitliana nigrofasciata, is supposed to result from mutual mating preference for larger individuals, female choice in relation to male size remains ambiguous. We revisited the evidence for directional preference for larger males in female convict cichlids using a classical two-way choice apparatus in which each female could decide to spend time in front of a small male or a large one. We found evidence for female preference for large males, as assessed from association preference during a 4-hour period following encounter. Furthermore, females decided to spawn in front of the initially preferred male more often than expected by chance. Our results thus confirm the existence of a directional preference for large males in female convict cichlids, and indicate that association preference measured over a short period of time can provide a quick and reliable proxy for reproductive preference in this species.     

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.