Age-related changes in the primary motor cortex of newborn to adult domestic pig sus scrofa domesticus

The pig has been increasingly used as a suitable animal model in translational neuroscience. However, several features of the fast-growing, immediately motor-competent cerebral cortex of this species have been adequately described. This study analyzes the cytoarchitecture of the primary motor cortex (M1) of newborn, young and adult pigs (Sus scrofa domesticus). Moreover, we investigated the distribution of the neural cells expressing the calcium-binding proteins (CaBPs) (calretinin, CR; parvalbumin, PV) throughout M1. The primary motor cortex of newborn piglets was characterized by a dense neuronal arrangement that made the discrimination of the cell layers difficult, except for layer one. The absence of a clearly recognizable layer four, typical of the agranular cortex, was noted in young and adult pigs. The morphometric and immunohistochemical analy-ses revealed age-associated changes characterized by (1) thickness increase and neuronal density (number of cells/mm2 of M1) reduction during the first year of life; (2) morphological changes of CR-immunoreactive neurons in the first months of life; (3) higher density of CR-and PV-immunopositive neurons in newborns when compared to young and adult pigs. Since most of the present findings match with those of the human M1, this study strengthens the growing evidence that the brain of the pig can be used as a potentially valuable translational animal model during growth and development

Osteopontin expression in healing wounds of horses and in human keloids

REASONS FOR PERFORMING STUDY: Convincing evidence shows that persistent or excessive expression of osteopontin (OPN) is linked to fibroproliferation of various organs in laboratory animals and in man, such that its downregulation is a logical therapeutic objective. OBJECTIVES: To investigate OPN expression in an equine model of wound healing and in clinical specimens of equine exuberant granulation tissue and human keloids in an effort to better understand the contribution of this protein to inflammation-associated skin fibrosis. STUDY DESIGN: Description of gene and protein expression in an experimental equine model of wound healing and clinical specimens in horse and man. METHODS: Osteopontin gene expression was evaluated by quantitative PCR, while protein expression was investigated by means of immunohistochemical staining. RESULTS: Quantitative PCR showed that the OPN gene is expressed in normal intact skin of horses and continues to be expressed during the wound-healing process. An increase in gene expression was observed throughout the phases of wound healing, with a final decrease at wound closure. The protein was not detected in normal skin. Keratinocytes in wound-edge samples did not express the protein, whereas dermal immunoreactivity was confined to inflammatory cells. Healed wounds were devoid of staining. Equine exuberant granulation tissue showed immunoreactivity of the surrounding epidermis, infiltrating neutrophils, mononuclear cells, endothelial cells and fibroblasts. Human keloids showed OPN immunoreactivity throughout the epidermis as well as in mononuclear cells and scattered fibroblasts. CONCLUSIONS: Immunohistochemical data show a different pattern of expression between normally healing and fibrotic wounds (exuberant granulation tissue and keloids), thus suggesting a role in fibroproliferation in horses and man

Investigating the properties of TBA variants with twin thrombin binding domains

In this paper, we report studies concerning thrombin binding aptamer (TBA) dimeric derivatives in which the 3'-ends of two TBA sequences have been joined by means of linkers containing adenosine or thymidine residues and/or a glycerol moiety. CD and electrophoretic investigations indicate that all modified aptamers are able to form G-quadruplex domains resembling that of the parent TBA structure. However, isothermal titration calorimetry measurements of the aptamer/thrombin interaction point to different affinities to the target protein, depending on the type of linker. Consistently, the best ligands for thrombin show anticoagulant activities higher than TBA. Interestingly, two dimeric aptamers with the most promising properties also show far higher resistances in biological environment than TBA

Folding mechanisms steer the amyloid fibril formation propensity of highly homologous proteins

Significant advances in the understanding of the molecular determinants of fibrillogenesis can be expected from comparative studies of the aggregation propensities of proteins with highly homologous structures but different folding pathways. Here, we fully characterize, by means of stopped-flow, T-jump, CD and DSC experiments, the unfolding mechanisms of three highly homologous proteins, zinc binding Ros87 and Ml153-149 and zinc-lacking Ml452-151. The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml153-149 appear to be much more stable to guanidine denaturation and are characterized by folding mechanisms including the presence of an intermediate. On the other hand, metal lacking Ml452-151 folds according to a classic two-state model. Successively, we have monitored the capabilities of Ros87, Ml452-151 and Ml153-149 to form amyloid fibrils under native conditions. Particularly, we show, by CD, fluorescence, DLS, TEM and SEM experiments, that after 168 hours, amyloid formation of Ros87 has started, while Ml153-149 has formed only amorphous aggregates and Ml452-151 is still monomeric in solution. This study shows how metal binding can influence protein folding pathways and thereby control conformational accessibility to aggregation-prone states, which in turn changes aggregation kinetics, shedding light on the role of metal ions in the development of protein deposition diseases

The Thioredoxin-Regulated α-Amylase 3 of Arabidopsis thaliana Is a Target of S-Glutathionylation

Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member, AtAMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that AtAMY3 activity was sensitive to ROS, such as H2O2. Treatments with H2O2 inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized AtAMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated AtAMY3 and performing oxidation experiments of Cys-to-Ser variants of AtAMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the pKa values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (pKa1 = 5.70 ± 0.28; pKa2 = 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of AtAMY3 is proposed

Design, Synthesis, and Anticancer Activity of a Selenium‐Containing Galectin‐3 and Galectin‐9N Inhibitor

Galectins are soluble β‐D‐galactoside‐binding proteins whose implication in cancer progression and disease outcome makes them prominent targets for therapeutic intervention. In this frame, the development of small inhibitors that block selectively the activity of galectins represents an important strategy for cancer therapy which is, however, still relatively underdeveloped. To this end, we designed here a rationally and efficiently novel diglycosylated compound, characterized by a selenoglycoside bond and the presence of a lipophilic benzyl group at both saccharide residues. The relatively high binding affinity of the new compound to the carbohydrate recognition domain of two galectins, galectin 3 and galectin 9, its good antiproliferative and anti‐migration activity towards melanoma cells, as well as its anti‐angiogenesis properties, pave the way for its further development as an anticancer agent

Awareness, treatment, and control of hypertension in the elderly in a general practice experience

In 3858 ambulatory elderly people (age greater than or equal to 65 years) prevalence of hypertension was 67.8%. The hypertensive status was unknown to both the doctor and the patient in 21.4% of cases. More than 90% of known hypertensives were treated, but hypertension could be considered as controlled in less than 30% of them

The laminar organization of the motor cortex in monodactylous mammals: a comparative assessment based on horse, chimpanzee, and macaque

The architecture of the neocortex classically consists of six layers, based on cytological criteria and on the layout of intra/interlaminar connections. Yet, the comparison of cortical cytoarchitectonic features across different species proves overwhelmingly difficult, due to the lack of a reliable model to analyze the connection patterns of neuronal ensembles forming the different layers. We first defined a set of suitable morphometric cell features, obtained in digitized Nissl-stained sections of the motor cortex of the horse, chimpanzee, and crab-eating macaque. We then modeled them using a quite general non-parametric data representation model, showing that the assessment of neuronal cell complexity (i.e., how a given cell differs from its neighbors) can be performed using a suitable measure of statistical dispersion such as the mean absolute deviation\u2014mean absolute deviation (MAD). Along with the non-parametric combination and permutation methodology, application of MAD allowed not only to estimate, but also to compare and rank the motor cortical complexity across different species. As to the instances presented in this paper, we show that the pyramidal layers of the motor cortex of the horse are far more irregular than those of primates. This feature could be related to the different organizations of the motor system in monodactylous mammals