Cathepsin X cleaves the C-terminal dipeptide of alpha- and gamma-enolase and impairs survival and neuritogenesis of neuronal cells

The cysteine carboxypeptidase cathepsin X has been recognized as an important player in degenerative processes during normal aging and in pathological conditions. In this study we identify isozymes alpha- and gamma-enolases as targets for cathepsin X. Cathepsin X sequentially cleaves C-terminal amino acids of both isozymes, abolishing their neurotrophic activity. Neuronal cell survival and neuritogenesis are, in this way, regulated, as shown on pheochromocytoma cell line PC12. Inhibition of cathepsin X activity increases generation of plasmin, essential for neuronal differentiation and changes the length distribution of neurites, especially in the early phase of neurite outgrowth. Moreover, cathepsin X inhibition increases neuronal survival and reduces serum deprivation induced apoptosis, particularly in the absence of nerve growth factor. On the other hand, the proliferation of cells is decreased, indicating induction of differentiation. Our study reveals enolase isozymes as crucial neurotrophic factors that are regulated by the proteolytic activity of cathepsin X.     

Crossing the Hands Increases Illusory Self-Touch

Manipulation of hand posture, such as crossing the hands, has been frequently used to study how the body and its immediately surrounding space are represented in the brain. Abundant data show that crossed arms posture impairs remapping of tactile stimuli from somatotopic to external space reference frame and deteriorates performance on several tactile processing tasks. Here we investigated how impaired tactile remapping affects the illusory self-touch, induced by the non-visual variant of the rubber hand illusion (RHI) paradigm. In this paradigm blindfolded participants (Experiment 1) had their hands either uncrossed or crossed over the body midline. The strength of illusory self-touch was measured with questionnaire ratings and proprioceptive drift. Our results showed that, during synchronous tactile stimulation, the strength of illusory self-touch increased when hands were crossed compared to the uncrossed posture. Follow-up experiments showed that the increase in illusion strength was not related to unfamiliar hand position (Experiment 2) and that it was equally strengthened regardless of where in the peripersonal space the hands were crossed (Experiment 3). However, while the boosting effect of crossing the hands was evident from subjective ratings, the proprioceptive drift was not modulated by crossed posture. Finally, in contrast to the illusion increase in the non-visual RHI, the crossed hand postures did not alter illusory ownership or proprioceptive drift in the classical, visuo-tactile version of RHI (Experiment 4). We argue that the increase in illusory self-touch is related to misalignment of somatotopic and external reference frames and consequently inadequate tactile-proprioceptive integration, leading to re-weighting of the tactile and proprioceptive signals.The present study not only shows that illusory self-touch can be induced by crossing the hands, but importantly, that this posture is associated with a stronger illusion.     

The influence of β-cyclodextrin on the kinetics of progesterone transformation by Rhizopus nigricans

The process of progesterone 11α-hydroxylation by the pelleted growth form of the filamentous fungus Rhizopus nigricans has been described with a mathematical model, based on Michaelis-Menten enzyme kinetics and the rate of substrate dissolution. It was confirmed that the low water solubility of steroids is the limiting step of this process at high steroid concentrations. In order to overcome this problem, β-cyclodextrin, which is known to form inclusion complexes with these organic compounds, was added to the production medium. The phase solubility of the steroid-β-cyclodextrin system was investigated and the effect of β-cyclodextrin addition on progesterone biotransformation evaluated. Enhancement of steroid solubility was demonstrated and nearly two-fold increase in reaction rate was found in the presence of β-cyclodextrin.     

Metabolic fingerprints of human primary endothelial and fibroblast cells

Introduction

Human primary cells originating from different locations within the body could differ greatly in their metabolic phenotypes, influencing both how they act during physiological/pathological processes and how susceptible/resistant they are to a variety of disease risk factors. A novel way to monitor cellular metabolism is through cell energetics assays, so we explored this approach with human primary cell types, as models of sclerotic disorders.

Objectives

In order to better understand pathophysiological processes at the cellular level, our goals were to measure metabolic pathway activities of endothelial cells and fibroblasts, and determine their metabolic phenotype profiles.

Methods

Biolog Phenotype MicroArray? technology was used for the first time to characterize metabolic phenotypes of diverse primary cells. These colorimetric assays enable detection of utilization of 367 specific biochemical substrates by human endothelial cells from the coronary artery (HCAEC), umbilical vein (HUVEC) and normal, healthy lung fibroblasts (NHLF).

Results

Adenosine, inosine, d-mannose and dextrin were strongly utilized by all three cell types, comparable to glucose. Substrates metabolized solely by HCAEC were mannan, pectin, gelatin and prevalently tricarballylic acid. HUVEC did not show any uniquely metabolized substrates whereas NHLF exhibited strong utilization of sugars and carboxylic acids along with amino acids and peptides.

Conclusion

Taken together, we show for the first time that this simple energetics assay platform enables metabolic characterization of primary cells and that each of the three human cell types examined gives a unique and distinguishable profile.

     

Fractionation of Phenolic Compounds Extracted from Propolis and Their Activity in the Yeast Saccharomyces cerevisiae

We have here investigated the activities of Slovenian propolis extracts in the yeast Saccharomyces cerevisiae, and identified the phenolic compounds that appear to contribute to these activities. We correlated changes in intracellular oxidation and cellular metabolic energy in these yeasts with the individual fractions of the propolis extracts obtained following solid-phase extraction. The most effective fraction was further investigated according to its phenolic compounds.     

Exoskeleton anchoring to tendon cells and muscles in molting isopod crustaceans

Specialized mechanical connection between exoskeleton and underlying muscles in arthropods is a complex network of interconnected matrix constituents, junctions and associated cytoskeletal elements, which provides prominent mechanical attachment of the epidermis to the cuticle and transmits muscle tensions to the exoskeleton. This linkage involves anchoring of the complex extracellular matrix composing the cuticle to the apical membrane of tendon cells and linking of tendon cells to muscles basally. The ultrastructural arhitecture of these attachment complexes during molting is an important issue in relation to integument integrity maintenance in the course of cuticle replacement and in relation to movement ability. The aim of this work was to determine the ultrastructural organization of exoskeleton - muscles attachment complexes in the molting terrestrial isopod crustaceans, in the stage when integumental epithelium is covered by both, the newly forming cuticle and the old detached cuticle. We show that the old exoskeleton is extensively mechanically connected to the underlying epithelium in the regions of muscle attachment sites by massive arrays of fibers in adult premolt Ligia italica and in prehatching embryos and premolt marsupial mancas of Porcellio scaber. Fibers expand from the tendon cells, traverse the new cuticle and ecdysal space and protrude into the distal layers of the detached cuticle. They likely serve as final anchoring sites before exuviation and may be involved in animal movements in this stage. Tendon cells in the prehatching embryo and in marsupial mancas display a substantial apicobasally oriented transcellular arrays of microtubules, evidently engaged in myotendinous junctions and in apical anchoring of the cuticular matrix. The structural framework of musculoskeletal linkage is basically established in described intramarsupial developmental stages, suggesting its involvement in animal motility within the marsupium.     

Dynamics of responses in compatible potato-Potato virus Y interaction are modulated by salicylic acid

To investigate the dynamics of the potato-Potato virus Y (PVY) compatible interaction in relation to salicylic acid-controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVY(NTN), the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid-analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level.     

Increased glutathione content in yeastSaccharomyces cerevisiae exposed to NaCl

Glutathione is one of the major antioxidant molecules of cells and is thought to play a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. Since an overlap between osmotic and oxidative stress response is already know, the aim of the work was to determine the role of glutathione in yeast stress response to NaCl. YeastSaccharomyces cerevisiae ZIM 2155 was exposed to NaCl in concentration of 1–8% (w/v). Measuring cell cultivability showed a significant decrease appeared in cell exposed to 5–8% NaCl for 1 h. Cultivable cells were about 50% of control. Increased production of reactive oxygen species in cells exposed to 6, 7 and 8% NaCl for 1 h (1.3-fold, 1.9-fold and 2.8-fold increase, respectively) led to elevated glutathione content in reduce d form (119.1%, 122.6%, 141.5%, respectively). Two hours from NaCl addition intracellular oxidant level was slightly elevated compared to 1-h exposure, while glutathione content in reduced form was almost the same. We demonstrated that glutathione plays an important role in yeast stress response to NaCl.     

Genetic diversity of autochthonous pig breeds analyzed by microsatellite markers and mitochondrial DNA D-loop sequence polymorphism

The evaluation of the genetic structure of autochthonous pig breeds is very important for conservation of local pig breeds and preservation of diversity. In this study, 18 microsatellite loci were used to detect genetic relationship between autochthonous pig breeds [Black Slavonian (BS), Turopolje pig (TP), and Croatian wild boar] and to determine phylogenetic relationship among Croatian autochthonous pig breeds and certain Asian and European pigs using the mitochondrial DNA (mtDNA) D-loop sequence polymorphism. Relatively high degree of genetic variation was found between the observed populations. The analysis of mtDNA showed that haplotypes of the studied pig populations are different from the other European and Chinese haplotypes. BS pigs showed some similarities with Mangalitsa and Duroc breeds. The genetic distances of TP can be explained by high degree of inbreeding during the past century. Despite the European origin of Croatian pig breeds with some impact of Chinese breeds in the past, the results of present study show that genetic diversity is still pronounced within investigated breeds. Furthermore, the genetic diversity is even more pronounced between Croatian breeds and other European and Chinese pig breeds. Thus, conservation of Croatian pig breeds will contribute to overall genetic diversity preservation of pig breeds.     

A population genomics insight into the Mediterranean origins of wine yeast domestication

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole‐genome data of oak‐associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.