Chromium uptake bySaccharomyces cerevisiae and isolation of glucose tolerance factor from yeast biomass

Fermentations with yeastSaccharomyces cerevisiae in semiaerobic and in static conditions with the addition of chromic chloride into the used molasses medium were analysed. It was proved that the addition of optimal amounts of CrCl₃ into the basal medium enhanced the kinetics of alcohol fermentations. The addition of 200 mg/l CrCl₃ into the medium stimulated both the yeast growth and the ethanol production in all experimental conditions. On the other hand, the results showed that Cr³⁺ ions were incorporated into yeast cells during fermentation. Under these conditions the accumulation of Cr³⁺ ions was performed by yeast cells during the exponential growth phase, and with enriched amounts of 30–45 (μg/g_d.m. of cells. Yeast biomass enriched with chromium ions was extracted with 01 mol/l NH₄OH assuming that the extracts had the glucose tolerance factor (GTF). Then the extracts were passed through a gel-filtration column in order to isolate and purify the GTF. The presence of GTF in the purified fractions was determined by measuring the absorbance at 260 nm. It is evident from the obtained results that the added purified fractions enhanced the rates of CO₂ production as well as the glucose utilization during alcoholic fermentation. As expected, the enhancement of both rates depended on the amounts of extracts added to the fermentation substrate. Thus, it is evident that purified extracts contained the GTF compound, and that Cr³⁺ ions were bonded to the protein molecule.     

Differential Cellular Effects of Electroporation and Electrochemotherapy in Monolayers of Human Microvascular Endothelial Cells

In vivo electroporation of tumours shows disruption of blood flow and creates a vascular effect with an initial rapid and transient vasoconstriction phase and a much longer lasting phase with changed microvascular endothelium. These changes are not well understood but are presumed to involve the cytoskeleton. The paper presents for the first time differential in vitro effects describing cytoskeleton changes and monolayer integrity changes by both electroporation and electrochemotherapy of monolayers of human microvascular endothelial cells (HMEC-1). After the application of electric field pulses, the morphology of cells, and both the F-actin and Beta-tubulin cytoskeleton proteins were affected. During both electroporation and electrochemotherapy, the initial phase of cellular damage was noticed at 10 min as swollen cells and honeycomb-like actin bundles. The electroporation-induced cellular effects, observed from electric pulses >150 V, were voltage-dependent and within 24 hrs partly recoverable. The electrochemotherapy-induced cellular effects developed at 2 hrs in spindle-like cells, and more densely packed F-actin and Beta-tubulin were observed, which were dependent on the amount of bleomycin and the voltages applied (>50 V). In addition, for electrochemotherapy with electric pulses >150 V cellular changes were not recoverable within 24 hrs. The effects on monolayer integrity were reflected in the enhanced monolayer permeability, with the electrochemotherapy showing an earlier onset and synergy. We conclude that electrochemotherapy as compared to electroporation leads within 24 hrs to a quicker and more pronounced monolayer integrity damage and endothelial cell death, which together provide further insight into the cellular changes of the vascular disruption of electrochemotherapy.     

MK-801 effect on regional cerebral oxidative stress rate induced by different duration of global ischemia in gerbils

We investigated MK-801 effect on ischemia-induced oxidative stress—the most important factor that exacerbates brain damage by reperfusion. The common carotid arteries of gerbils were occluded for 5, 10, or 15 min. Immediately after the occlusion, MK-801 (3 mg/kg i.p.) or saline were given in normothermic conditions. The MK-801 effects were followed in vivo by monitoring the neurological status of animals and at the intracellular level by standard biochemical assays. We investigated nitric oxide levels, superoxide production, superoxide dismutase activity, index of lipid peroxidation (ILP), and reduced glutathione content in hippocampus, striatum, forebrain cortex, and cerebellum. The measurements took place at different times (1, 2, 4, 7, 14, and 28 days) after reperfusion. Increased duration of cerebral ischemia resulted in a progressive induction of oxidative stress. Our results revealed pattern of dynamic changes in each oxidative stress parameter level which corresponded with ischemia duration in all tested brain structures. Most sensitive oxidative stress parameters were ILP and superoxide production. Our study confirmed spatial distribution of ischemia-induced oxidative stress. Tested brain structures showed different sensitivity to each oxidative stress parameter. As judged by biochemical and neurological data, applied MK-801 showed neuroprotective efficiency by reduction of ischemia-induced oxidative stress in brain.     

Landscape genetics and spatial pattern of phenotypic variation of Eristalis tenax across Europe

A study of population connectivity of the migratory insect species, such as dronefly Eristalis tenax (Diptera, Syrphidae), has an essential importance in understanding the relative influence of the evolutionary forces and environmental features that interact in the spatial distribution of molecular and morphological diversity. However, specific study aiming to understand spatial genetic structure of dronefly populations and its migratory potential is lacking. Hence, we studied a spatial pattern of genetic and phenotypic variation of seven European populations of E. tenax incorporating landscape genetic methods using allozyme data, wing size and shape and abdominal colour pattern. Based on the observed lack of genotypic structuring, we suggested that there has been sufficient long‐distance gene flow to effectively homogenize population structuring at a broader geographical scale. Wing shape similarity among populations and an overlap of abdominal colour variation showed no clear clustering related to geography, which is in congruence with genetic data. However, genetic (F_ST values) and phenotypic (wing size) data and landscape genetics indicated subdivision between the Balkan populations (four Serbian samples) and populations from Central (Germany and Switzerland) and Northern (Finland) Europe. These findings indicated a potential connection between the Central and Northern Europe supporting the Central European origin of the flies caught in Finland. Thus, by performing spatial analysis and combining genetic–morphological approach, we shed light on the movement pattern in complex landscapes and thus provided the necessary guidelines to a broad‐scale analysis of this widespread generalist pollinator.     

Synthetic circuits reveal how mechanisms of gene regulatory networks constrain evolution

Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such biases are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most pertinent evidence is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in Escherichia coli that produce a gene expression stripe—a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution.     

Den 1, Den2 and Den3, ATP-inhibited deoxyribonucleases from Dropsophila embryonic nuclei

Three Drosophila embryonic deoxyribonucleases, designated den1, den2 and den3, are identified in nuclear extracts separated by glycerol density gradient centrifugation. Den1, removes short products from the 5-ends of single-stranded DNA or double-stranded DNA with either blunt or 5-recessed termini. Den2 is inactive with single-stranded DNA and acts as 3-exonuclease with double-stranded DNA possessing either blunt or 3-recessed termini. Den3 preferentially uses partial duplex DNA containing single-stranded gap and it catalyzes hydrolysis, in 3-5 direction, of one of the shorter strands that flank the gap. Nucleolytic activities of den1, den2 and den3 are inhibited with ATP.     

Non-secretory solitary plasmacytoma of the spleen

A case of primary nonsecretory plasmacytoma of the spleen is reported. On laparotomy and splenectomy a 920 g spleen was removed, measuring 16×14×6 cm. The cut surface of the entire spleen showed that the tumour occupied most of the splenic tissue. A bone marrow aspirate and trephine, skeletal survey showed no signs of myeloma. Biopsy of the liver and regional lymph nodes was normal. Immunocytochemistry of the splenic tumour showed positivity for pan-B and plasma cell markers. After splenectomy the patient was treated with chemotherapy according to protocol VBCMP (M2).     

Genetic and phenotypic variation in central and northern European populations of Aedes (Aedimorphus) vexans (Meigen, 1830) (Diptera,Culicidae)

The floodwater mosquito Aedes vexans can be a massive nuisance in the flood plain areas of mainland Europe, and is the vector of Tahyna virus and a potential vector of Dirofilaria immitis. This epidemiologically important species forms three subspecies worldwide, of which Aedes vexans arabiensis has a wide distribution in Europe and Africa. We quantified the genetic and phenotypic variation in Ae. vexans arabiensis in populations from Sweden (northern Europe), Hungary, and Serbia (central Europe). A landscape genetics approach (F_ST, STRUCTURE, BAPS, GENELAND) revealed significant differentiation between northern and southern populations. Similar to genetic data, wing geometric morphometrics revealed two different clusters, one made by Swedish populations, while another included Hungarian and Serbian populations. Moreover, integrated genetic and morphometric data from the spatial analysis suggested groupings of populations into three clusters, one of which was from Swedish and Hungarian populations. Data on spatial analysis regarding an intermediate status of the Hungarian population was supported by observed Isolation‐by‐Distance patterns. Furthermore, a low proportion of interpopulation vs intrapopulation variance revealed by AMOVA and low‐to‐moderate F_ST values on a broader geographical scale indicate a continuous between‐population exchange of individuals, including considerable gene flow on the regional scale, are likely to be responsible for the maintenance of the observed population similarity in Aе. vexans. We discussed data considering population structure in the light of vector control strategies of the mosquito from public health importance.     

The CFTR M470V gene variant as a potential modifier of COPD severity: study of Serbian population

Chronic obstructive pulmonary disease (COPD) is a complex disease influenced by genetic and environmental factors. Cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important component of the lung tissue homeostasis, involved in the regulation of the rate of mucociliary clearance. As it is known that certain CFTR variants have consequences on the function of CFTR protein, the aim of this study was to examine the possible role of F508del, M470V, Tn locus, and R75Q variants in COPD development and modulation. Total number of 86 COPD patients and 102 control subjects were included in the study. Possible association between COPD susceptibility, severity, and onset of the disease and allele or genotype of four analyzed CFTR variants was examined. No associations were detected between COPD development, onset of the disease and tested CFTR alleles and genotypes. However, VV470 genotype was associated with mild/moderate COPD stages in comparison to severe/very severe ones (OR = 0.29, 95%CI = 0.11-0.80, p = 0.016). Our study showed that patients with VV470 genotype had a 3.4-fold decreased risk for the appearance of severe/very severe COPD symptoms, and the obtained results indicate that this genotype may have a protective role. These results also suggest the importance of studying CFTR gene as a modifier of this disease.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. KI, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k3, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. KI values of 1.3 x 10(-4) M(-1), 5.6 x 10(-6) M(-1) and 7.2 x 10(-6)M(-1) were obtained for malathion, malaoxon and isomalathion, respectively. The IC50 values for free/immobilized AChE, (3.7 +/- 0.2) x 10(-4) M/(1.6 +/-0.1) x 10(-4), (2.4 +/- 0.3) x 10(-6)/(3.4 +/- 0.1) x 10(-6)M and (3.2 +/- 0.3) x 10(-6) M/(2.7 +/- 0.2) x 10(-6) M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 x 10(-7) M/2 x 10(-7) M, 2 x 10(-7) M/3 x 10(-7)M and 2 x 10(-7) M/4.5 x 109-7) M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.