Effects of the utilization of homeopathic elements in commercial diluent on swine sperm viability

It has been speculated that the homeopathic treatment of sperm cells in order to improve semen quality could be promising. However, few data is available and its use in spermatozoa requires investigation. It is well established that mitochondrial membrane potential is an important viability parameter of spermatozoa and it is intimately related to reproductive efficiency. In this manner, new technologies in order to improve the activity of sperm cells and, finally, the fecundity of swine herds are of extremely importance. Due to the lack of knowledge of homeopathic treatment effect on spermatozoa, the aim of the present study was to verify the effect of three different homeopathic treatments on viability of boar sperm cells. Three homeopathic treatments composed by Pulsatila CH6, Pulsatila and Avena CH6, Avena CH6 and one control treatment (sucrose) were added to diluted boar semen, which were cooled for 24 or 48 h. Interestingly, no positive effect of homeopathic treatments was observed over semen viability. However, it was demonstrated that the 24 h of cooling storage provided more viable sperm cells when compared to the 48-h period. This effect of storage period on sperm viability was assessed by intact plasmatic membrane, intact acrosome and mitochondrial membrane potential evaluation.     

Predicting novel binding modes of agonists to β adrenergic receptors using all-atom molecular dynamics simulations

Understanding the binding mode of agonists to adrenergic receptors is crucial to enabling improved rational design of new therapeutic agents. However, so far the high conformational flexibility of G protein-coupled receptors has been an obstacle to obtaining structural information on agonist binding at atomic resolution. In this study, we report microsecond classical molecular dynamics simulations of β(1) and β(2) adrenergic receptors bound to the full agonist isoprenaline and in their unliganded form. These simulations show a novel agonist binding mode that differs from the one found for antagonists in the crystal structures and from the docking poses reported by in silico docking studies performed on rigid receptors. Internal water molecules contribute to the stabilization of novel interactions between ligand and receptor, both at the interface of helices V and VI with the catechol group of isoprenaline as well as at the interface of helices III and VII with the ethanolamine moiety of the ligand. Despite the fact that the characteristic N-C-C-OH motif is identical in the co-crystallized ligands and in the full agonist isoprenaline, the interaction network between this group and the anchor site formed by Asp(3.32) and Asn(7.39) is substantially different between agonists and inverse agonists/antagonists due to two water molecules that enter the cavity and contribute to the stabilization of a novel network of interactions. These new binding poses, together with observed conformational changes in the extracellular loops, suggest possible determinants of receptor specificity.     

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity

Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.     

G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection

Background

Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.

Methods

Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.

Results

These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.

Conclusions

These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.

General observation

This study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.     

Complications of Elastase-Induced Arterial Saccular Aneurysm in Rabbits: Case Reports and Literature Review

Endoluminal infusion and incubation of elastase with or without collagenase into the rabbit common carotid artery is an established model of arterial saccular aneurysm. The model mimics naturally occurring human cerebral aneurysms in many ways, including histologic and morphologic characteristics, hemodynamic pressures, and shear stresses. However, complications have been associated with the model. Here, we report 2 complications: 1) the first known case of iatrogenic laryngeal hemiplegia in a rabbit; and 2) histopathologically confirmed iatrogenic hippocampal and cerebellar infarcts (stroke). Finally, we present and review data from current literature on the morbidity and mortality associated with this model.Abbreviations: CCA, common carotid artery; RLN, recurrent laryngeal nerveElastase-induced aneurysm in the rabbit common carotid artery (CCA) is an established model of arterial saccular aneurysm^2,6 that provides an alternative to large animal (for example, swine and canine) models of saccular aneurysm. The degradation of the elastic lamina with elastase leads to a cascade of changes in extracellular matrix proteins and progressive inflammatory infiltration as seen in naturally occurring cerebral and aortic aneurysms.¹⁵ These changes lead to thin-walled aneurysms⁹ that are morphometrically²⁸ and histologically¹ similar to naturally occurring human cerebral aneurysms. In addition, the created aneurysms have hemodynamic pressures and shear stresses similar to those of human cerebral aneurysms,²⁴ because the rabbit''s carotid artery is also comparable in size to human middle cerebral arteries.¹ Rabbits also have comparable thrombotic and thrombolytic profiles to those of humans.¹⁶Despite the advantages, this rabbit model has limitations. These include the inability to control the size of aneurysms (potentially corrected by adjusting the ligation site);¹⁰ sacrifice of the arterial access for each angiogram;⁴ absence of clear atherosclerotic changes, thus limiting data extrapolation to the “true human degenerative aneurysm”;²⁷ and small size of rabbits, thus limiting the evaluation of large devices or techniques requiring multiple catheters.¹³ In addition, rare spontaneous ruptures of elastase-induced aneurysms have been reported;^1,6,17,19 these ruptures were postulated to be due to residual collagen in the vessel wall, given that collagen is important to maintain the wall''s tensile strength.³³ Therefore, the natural history of human intracranial aneurysm is not precisely produced in this rabbit model. Because alterations in either the quantity^14,26,30 or quality³² of collagen may be related to the development of cerebral aneurysms in humans, the traditional technique for creating these aneurysms in rabbits was modified to include the infusion and incubation of collagenase into the CCA.Creating the model involves careful dissection of the ventral neck region to isolate the CCA and infuse it with elastase with or without collagenase. Complications arising from this technique have been reported. Here, we present 2 clinical cases based on our experience: the first known case of iatrogenic laryngeal hemiplegia in a rabbit and a histopathologically confirmed case of iatrogenic rabbit stroke. We also review the literature regarding complications associated with creating this model.     

The Sam domain of the lipid phosphatase Ship2 adopts a common model to interact with Arap3-Sam and EphA2-Sam

Background  

Sterile alpha motif (Sam) domains are small protein modules that can be involved in homotypic or heterotypic associations and exhibit different functions. Previous studies have demonstrated that the Sam domain of the lipid phosphatase Ship2 can hetero-dimerize with the Sam domain of the PI3K effector protein Arap3.     

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish)

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT–PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.     

Peptides targeting chemokine receptor CXCR4: structural behavior and biological binding studies

CXCR4 is a G‐protein‐coupled receptor involved in a number of physiological processes in the hematopoietic and immune systems. CXCL12/CXCR4 axis plays a central role in diseases, such as HIV, cancer, WHIM syndrome, rheumatoid arthritis, pulmonary fibrosis, and lupus and, hence, indicated as putative therapeutic target. Although multiple CXCR4 antagonists have been developed, there is only one marketed drug, plerixafor, indicated for stem cell mobilization in poor mobilizer patients. In this work, we have designed and synthesized two peptides, six and seven residues long, using as template the N‐terminal region of CXCL12; analyzed their conformations by CD, NMR, and molecular dynamics simulations; simulated their complexes with CXCR4 by docking methods; and validated these data by in vitro studies. The results showed that the two peptides are rather flexible in aqueous solution lacking ordered secondary structure elements and present a promising affinity for CXCR4. This affinity is not revealed for CXCR7, indicating a specificity for CXCR4. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Autologous immune responses to the major oncornavirus polypeptides in unmanipulated AKR/J mice.

The autologous immune response of AKR/J mice to the structural proteins of murine leukemia virus (MuLV) was examined. Immunoglobulins from the renal glomeruli were chemically eluted, separated from antigens, recovered, and tested for immunological reactivity against MuLV structural proteins. Analyzing immune precipitates obtained after mixing radiolabeled Tween-disrupted MuLV preparations with eluates from AKR/J mice on sodium dodecyl sulfategel electrophoresis, we found evidence of antibodies to the major classes of MuLV structural components: gp70, gp45, p30, and one or more proteins in the 10,000- to 15,000-dalton class. Using rate zonal centrifugation we confirmed that the eluates from AKR/J glomeruli contained antibody(s) that bound specifically to p30. These results indicate that AKR/J mice spontaneously mount immune responses against the major oncornavirus polypeptide antigens.     

Endothelin-1 induces proliferation of human lung fibroblasts and IL-11 secretion through an ET(A) receptor-dependent activation of MAP kinases

Endothelin-1 (ET-1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET-1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET-1 in the presence or absence of an overnight pre-treatment with either ET(A) or ET(B) selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen-activated protein kinases (MAPK). ET-1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL-6/IL-11 release into cell culture supernatants. All these effects were inhibited by the selective ET(A) antagonist BQ-123, but not by the specific ET(B) antagonist BQ-788. The stimulatory influence of ET-1 on IL-11, but not on IL-6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET-1 exerts a profibrogenic action via an ET(A) receptor-dependent, MAPK-mediated induction of IL-11 release and cell proliferation.