Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase

Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5′-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T_m values of 46 and 67?°C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer–dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T_m values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.     

Biological rhythms in birds — development, insights and perspectives

The aim of this review is to show that probably the internal clock of precocial birds is imprinted in the prenatal period by exogenous factors (zeitgeber). The activity of organ functions occurs early during embryonic development, before this function is ultimately necessary to ensure the survival of the embryo. Prenatal activation of some functional systems may have a training effect on the postnatal efficiency.The development of physiological control systems is influenced by endogenous and exogenous factors during the late prenatal and early postnatal period: epigenetic adaptation processes play an important role in the development of animals; they have acquired characteristics which are innated but not genetically fixed. As a rule, the actual value during the determination period has a very strong influence on the set-point of the system. This will be explained using the example of thermoregulation.It is shown in detail that it seems to be possible to imprint the prenatal development of circadian rhythms by periodic changes of the light-dark cycle but not by rhythmic influence of acoustic signals.Altogether, there are more questions open than solved concerning the perinatal genesis of circadian rhythms in birds. Topics are given for the future research.     

Differences between horse selection based on two forms of osteochondrosis in fetlock

Horses lose potential opportunities because of health problems. Available breeding strategies are not effective enough, probably also because of the different definition used and its genetic usefulness. The aim of the study was to compare the genetic background estimated by the genome-wide association study (GWAS) for osteochondrosis using two different scaling osteochondrosis (OC)/healthy and osteochondrosis dissecans (OCD)/healthy systems for evaluating the disease status of investigated fetlock joints. Two hundred one Warmblood horses trained for performance tests (87 stallions and 114 mares) were phenotyped and genotyped. Four fetlock x-ray images per horse were collected using the RTG Girth HF 80 and Vet Scan ray 3600. The DNA of each horse was genotyped using the BeadChip 70K. To identify SNPs that significantly affect the probability of osteochondrosis, two different methods were applied: the Cochran-Armitage test based on an additive mode of inheritance and logistic regression. The genetic background for osteochondrosis, expressed in the number of SNPs found with significant associations with osteochondrosis, was higher by evaluation in the scale of OCD/healthy horses (16 SNPs on several chromosomes mainly on the ECA1 and ECA10) than OC/healthy (2 SNPs on the ECA15 and one SNP on the ECA10). Detailed definition of osteochondrosis is needed in breeding and in veterinary practice. The genetic background for osteochondrosis and osteochondrosis dissecans seems not the same. Suggestive SNPs could be the candidate markers for osteochondrosis but should be checked on a larger population before usage.     

Statistical Analysis of Sleep Spindle Occurrences

Spindles - a hallmark of stage II sleep - are a transient oscillatory phenomenon in the EEG believed to reflect thalamocortical activity contributing to unresponsiveness during sleep. Currently spindles are often classified into two classes: fast spindles, with a frequency of around 14 Hz, occurring in the centro-parietal region; and slow spindles, with a frequency of around 12 Hz, prevalent in the frontal region. Here we aim to establish whether the spindle generation process also exhibits spatial heterogeneity. Electroencephalographic recordings from 20 subjects were automatically scanned to detect spindles and the time occurrences of spindles were used for statistical analysis. Gamma distribution parameters were fit to each inter-spindle interval distribution, and a modified Wald-Wolfowitz lag-1 correlation test was applied. Results indicate that not all spindles are generated by the same statistical process, but this dissociation is not spindle-type specific. Although this dissociation is not topographically specific, a single generator for all spindle types appears unlikely.     

Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

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.     

Walnut diet reduces accumulation of polyubiquitinated proteins and inflammation in the brain of aged rats

An increase in the aggregation of misfolded/damaged polyubiquitinated proteins has been the hallmark of many age-related neurodegenerative diseases. The accumulation of these potentially toxic proteins in brain increases with age, in part due to increased oxidative and inflammatory stresses. Walnuts, rich in omega fatty acids, have been shown to improve memory, cognition and neuronal effects related to oxidative stress (OS) and inflammation (INF) in animals and human trials. The current study found that feeding 19-month-old rats with a 6% or 9% walnut diet significantly reduced the aggregation of polyubiquitinated proteins and activated autophagy, a neuronal housekeeping function, in the striatum and hippocampus. Walnut-fed animals exhibited up-regulation of autophagy through inhibiting phosphorylation of mTOR, up-regulating ATG7 and Beclin 1, and turnover of MAP1BLC3 proteins. The clearance of polyubiquitinated protein aggregates such as p62/SQSTM1 was more profound in hippocampus, a critical region in the brain involved in memory and cognitive performance, than striatum. The clearance of ubiquitinated aggregates was in tandem with significant reductions in OS/INF, as indicated by the levels of P38-MAP kinase and phosphorylations of nuclear factor kappa B and cyclic AMP response element binding protein. The results demonstrate the effectiveness of a walnut-supplemented diet in activating the autophagy function in brain beyond its traditionally known antioxidant and anti-inflammatory benefits.