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.     

Efficient screening of process variables inPenicillium janthinellum fermentations

Summary Several potentially important process variables involved in xylanase production byPenicillium janthinellum in large lab-scale fermentors were efficiently screened by employing a Plackett-Burman statistical design. Results illustrate the design's general applicability to experimentation with fermentation processes.     

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.     

The legacy of forest logging on organic matter inputs and storage in tropical streams

Riparian forests play an important role in stream ecosystems, as they support biodiversity, reduce water erosion, and provide litter that fuels aquatic biota. However, they are affected by great array of anthropogenic threats (e.g., fire, logging, and organic pollution), which alter species composition and their physical structure. Although forest recovery after disturbance such as logging can take decades, the legacy of forest clear-cut logging on key processes in tropical riparian ecosystems is mostly unknown. Here, we investigated how litter inputs (leaves, twigs, and reproductive parts) and storage, key processes for carbon and nutrient recycling and for forest and stream biota, are influenced by riparian vegetation undergoing succession (after 28 years from logging) through the comparison of reference and logged forest sites in the Cerrado biome. Litterfall was overall similar between forest types, but litterfall of twigs was twofold higher at logged than reference sites. Similarly, litter inputs from the bank to the stream (i.e., lateral inputs) and streambed storage were 50–60% higher at logged than reference sites. The higher litterfall observed in logged forests could be related to higher proportion of tree species that are characteristic of primary and secondary successional stages, including fast-growing and liana species, which often are more productive and common in anthropogenic areas. Our results showed that the legacy impact of clear-cut logging, even if residual woody vegetation is maintained in riparian buffers, can shift the type, quantity, and seasonality of litter subsidies to tropical streams. This knowledge should be considered within the context of management and conservation of communities and ecosystem processes in the forest-stream interfaces.     

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.     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

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.