Allele-specific Characterization of Alanine: Glyoxylate Aminotransferase Variants Associated with Primary Hyperoxaluria

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.     

The initial fertilizing capacity of longerm-stored liquid boar semen following pre- and postovulatory insemination

In pigs, high variation is seen in the duration of estrus and in the time of ovulation. This is one of a wide range of factors not related to semen quality, which possibly influences the results of field insemination trials. Experiment 1 (n=81 gilts) was performed to determine the influence of the time of ovulation on the fertilizing capacity of liquid boar semen stored up to 118 h. The objective of Experiment 2 (n=102 gilts) was to study the fertilizing potential of semen stored up to 120 h in 2 different extenders, Androhep and Beltsville Thawing Solution (BTS), by means of postovulatory AI. Inseminations were performed 0 to 4 h after ovulation in order to standardize the trial conditions. Fertilization rates based on Day-2 to Day-4 embryos, and the number of accessory spermatozoa per zona pellucida did not differ between semen stored for 0 to 48 and 48 to 87 h in gilts ovulating within 12 after insemination (Experiment 1). Gilts with an interval of 12 to 24 h between AI and ovulation had lower fertility results using semen stored for more than 48 h. A further decrease was observed when semen storage exceeded 87 h in those gilts ovulating later than 24 h after insemination. The time of ovulation has to be considered as being a major factor of variation in the fertility results of AI trials. In Experiment 2, fertilization rates and numbers of accessory spermatozoa decreased between semen stored for 0 to 24 and 24 to 48 h in BTS, and between semen stored for 0 to 24 and 48 to 72 h in Androhep. Significant differences in fertility between diluents were seen only when using semen stored for more than 96 h, with semen extended with Androhep giving the higher results. The results indicate that the decrease in fertilizing capacity due to in vitro aging of spermatozoa cannot be prevented even during the first days of storage.     

Equilibria of polyoxometalates in aqueous solution

In most aqueous polyoxometalate systems, numerous, often highly negatively charged species, are formed. To establish the speciation in such complex polyanion systems, many experimental methods and techniques must be used. Moreover, it is of vital importance that the experimental data are of the highest accuracy and that the data collected from different methods are treated simultaneously with an appropriate computer program. In systems containing one or more sensitive NMR nuclei, a combined EMF-NMR method has been shown to be extremely powerful.This article firstly gives some general comments on equilibria in aqueous polyoxometalate systems including ionic medium effects, and then describes an equilibrium EMF-NMR (³¹P and⁵¹V) study of the five component system H⁺-Mo(VI)-V(V)-P(V)-e^–. The study has been focused on so-called Keggin ratio solutions, Mo+V):P=12:1, since these are commonly used in selective oxidation processes. Special attention has been given to Mo₁₀V₂P and Mo₉V₃P solutions, where positional isomers occur. We have been able to identify and characterize all the five possible isomers of -Mo₁₀V₂PO ₄₀ ^5– at 25 and 90 °C. Besides the results from the five component system, some interesting findings from the binary, ternary and quarternary sybsystems are also reported.     

Artifacts arising from sampling interval in dive depth studies of marine endotherms

Most depth recorders used to study the diving behaviour of polar marine endotherms record depth data at specific time intervals. The length of recording interval can have potentially profound implications for the interpretation of the data. We used data acquired on the diving behaviour of king penguins, Aptenodytes patagonicus, to examine the validity of various analyses routinely conducted on depth data. In our experiments, increasing the sampling interval led to an underestimation of the number of dives performed, an overestimation in mean dive duration and substantial changes in the form of the dive profile. Our analysis indicates that depth data should be recorded at a minimum rate corresponding to 10% of the total dive duration and that conventional dive profile categorization may be inappropriate. Alternatives that are less subjective, and based on curve fits of dive depth versus time, are proposed.     

Mitochondrial DNA sequence evolution in sharks: rates, patterns, and phylogenetic inferences

Abundant representation of sharks in the fossil record makes this group a superb system in which to investigate rates and patterns of molecular evolution and to explore the strengths and weaknesses of phylogenetic inferences from molecular data. In this report, the molecular evolution of the cytochrome b gene in sharks is described and the information related to results from phylogenetic analysis of the data evaluated in the light of a phylogeny derived independently of the molecular data. Across divergent lineages of sharks there is evidence for significant substitution rate variation, departure from compositional equilibrium, and substantial homoplasy; nevertheless, the signal of evolutionary history is evident in patterns of shared transversions and amino acid replacements.      

Metabolic rate and directional nucleotide substitution in animal mitochondrial DNA

There is marked heterogeneity of nucleotide composition in mitochondrial DNA across divergent animals. Differences in nucleotide composition presumably reflect differences in directional nucleotide substitution for A+T or G+C nucleotides. In mitochondrial DNA, there is A+T directional nucleotide substitution in most (if not all) animals surveyed, and the magnitude of directional A+T nucleotide substitution differs greatly within and among groups. Differences in directional nucleotide substitution among lineages of mammals can be explained by changes in metabolic physiology. This relationship is thought to be mediated by the effect of oxygen radicals because these toxic compounds are by-products of aerobic metabolism and are known mutagens. Association between metabolism and nucleotide composition provides additional evidence in favor of the hypothesis that rates and patterns of nucleotide substitution in mitochondrial DNA can be influenced by factors that impinge on rates of endogenous DNA damage.      

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Acetylenic 2-phenylethylamides and new isobutylamides from Acmella oleracea (L.) R. K. Jansen,a Brazilian spice with larvicidal activity on Aedes aegypti

Ethanol extract obtained from dried leaves of Acmella oleracea afforded after a liquid/liquid partition procedure a larvicidal hexane fraction (LC₅₀ = 145.6 ppm) and a non larvicidal dichloromethane one. From the inactive fraction, three amides were identified, two new structures, named deca-6,9-dihydroxy-(2E,7E)-dienoic acid isobutylamide (1), deca-8,9-dihydroxy-(2E,6Z)-dienoic acid isobutylamide (2) and the known nona-2,3-dihydroxy-6,8-diynoic acid 2-phenylethylamide (3). Bioassay-guided chromatographic fractionation of the hexane partition led to the identification of an amide mixture, nona-(2Z)-en-6,8-diynoic acid 2-phenylethylamide (4) and deca-(2Z)-en-6,8-diynoic acid 2-phenylethlylamide (5). This mixture was active against Aedes aegypti larvae at LC₅₀ = 7.6 ppm. Low toxicity of crude extracts and derived fractions on Artemia salina nauplies showed the possibility of using them to control the A. aegypti mosquito larvae. This is the first report on larvicidal activity of acetylenic 2-phenylethylamides and their identification in A. oleracea leaves.