Obstacles and limitations of transfer factor biological activity assay design.

Transfer factor (TF) is a heterogeneous mix of low-molecular weight molecules obtained from dialyzed leukocyte extract that is capable of transferring cell-mediated immunity. As an immunostimulatory drug TF is used to improve treatment of infectious diseases, allergies, cancer and immune deficiencies. The main benefit of TF preparations as immunotherapeutic agents is the induction of a rapid immune response and the potential of TF as an adjuvant in combination with other drugs might lead to development of novel approaches to combat various diseases in the future. The process of TF preparation is rather simple. However, with respect to fact that TF is composed by several multifunction molecules, it is likely that during the activity measurement based only on one single parameter, other TF biological activities might be overlooked. In addition, separated TF components might display synergetic activity effect. According to recent European Pharmacopoeia there is no general protocol for immuno-stimulatory drugs (including TF) activity measurement available. Nevertheless, for the process of TF preparation, control of input material and for final pharmaceutical product batches it is inevitable to guaranty proper quality control including appropriate in vivo or in vitro test(s) for TF biological activity assay. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic problem, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. The currently used method for testing biological activity of TF is the in vitro MTT cells proliferation assay that is recognized by control authorities in Slovak Republic. Keywords: immune system; transfer factor; dialysable leukocyte extract; diseases; MTT cells proliferation assay.

Changes in surface water chemistry caused by natural forest dieback in an unmanaged mountain catchment.

Ionic and nutrient compositions of throughfall, tributaries and lake outlet were analysed in the Plesné catchment-lake system (an unmanaged mountain forest in Central Europe) from 1997 to 2016. The aim was to evaluate changes in surface water chemistry after natural forest dieback. In the 2004-2008, 93% of the Norway spruce trees were killed by bark beetle outbreak, and all dead biomass remained in the catchment. Forest dieback changed the chemistry of all water fluxes, and the magnitude, timing, and duration of these changes differed for individual water constituents. The most pronounced decreases in throughfall concentrations occurred for K+, dissolved organic carbon (DOC), Ca2+ and Mg2+, i.e. elements mostly originating from canopy leaching, while concentrations of NH4+ and soluble reactive phosphorus (SRP) remained almost unaffected. In tributaries, the most rapid changes were increases in NO3-, K+, H+ and ionic aluminium (Ali) concentrations, while terrestrial export of DOC and P forms started more slowly. Immediately after the forest dieback, increase in NO3- concentrations was delayed by elevated DOC availability in soils. NO3- became the dominant anion, with maximum concentrations up to 346µeqL-1 within 5-7years after the bark beetle outbreak, and then started to decrease. Terrestrial exports of Ali, K+, H+, Mg2+, and Ca2+ accompanied NO3- leaching, but their trends differed due to their different sources. Elevated losses of SRP, DOC, and dissolved organic nitrogen continued until the end of the study. In the lake, microbial processes significantly decreased concentrations of NO3-, organic acid anions, H+ and Ali, and confounded the chemical trends observed in tributaries. Our results suggest that terrestrial losses of elements and the deterioration of waters after forest dieback are less pronounced in unmanaged than managed (clear-cut) catchments.

Enhanced metabolism as a common feature of cancer plasticity.

Cancer cells often rely on glycolytic metabolism in order to fulfill high demands of ATP and macromolecules for the sustained growth and proliferation. However, glycolysis is not necessarily the main source of energy for all cancer cells. Some of them rather depend on glutamine or lactate that favor the utilization of oxidative metabolic pathway. Different employment rate of metabolism creates variable products that participate in the formation of environmental milieu, which in turn triggers broad spectrum of cellular signaling pathways leading to migration, invasion, or proliferation. In this review we discuss different metabolic pathways promoted in tumor cells and describe the possibilities of their targeting as therapeutic strategies.

Modelling inorganic nitrogen in runoff: Seasonal dynamics at four European catchments as simulated by the MAGIC model.

Nitrogen (N) deposition is globally considered as a major threat to ecosystem functioning with important consequences for biodiversity, carbon sequestration and N retention. Lowered N retention as manifested by elevated concentrations of inorganic N in surface waters indicates ecosystem N saturation. Nitrate (NO3) concentrations in runoff from semi-natural catchments typically show an annual cycle, with low concentrations during the summer and high concentrations during the winter. Process-oriented catchment-scale biogeochemical models provide tools for simulation and testing changes in surface water and soil chemistry in response to changes in sulphur (S) and N deposition and climate. Here we examine the ability of MAGIC to simulate the observed monthly as well as the long-term trends over 10-35 years of inorganic N concentrations in streamwaters from four monitored headwater catchments in Europe: Certovo Lake in the Czech Republic, Afon Gwy at Plynlimon, UK, Storgama, Norway and G2 NITREX at Gårdsjön, Sweden. The balance between N inputs (mineralization+deposition) and microbial immobilization and plant uptake defined the seasonal pattern of NO3 leaching. N mineralization and N uptake were assumed to be governed by temperature, described by Q10 functions. Seasonality in NO3 concentration and fluxes were satisfactorily reproduced at three sites (R2 of predicted vs. modelled concentrations varied between 0.32 and 0.47 and for fluxes between 0.36 and 0.88). The model was less successful in reproducing the observed NO3 concentrations and fluxes at the experimental N addition site G2 NITREX (R2=0.01 and R2=0.19, respectively). In contrast to the three monitored sites, Gårdsjön is in a state of change from a N-limited to N-rich ecosystem due to 20 years of experimental N addition. At Gårdsjön the measured NO3 seasonal pattern did not follow typical annual cycle for reasons which are not well understood, and thus not simulated by the model. CAPSULE: The MAGIC model is able to simulate NO3 leaching on a monthly as well as an annual basis, and thus to reproduce the seasonal and short-term variations in N dynamics.

Ovine herpesvirus 1 (OVHV-1) thymidine kinase locus sequence analysis: evidence that OVHV-1 belongs to the Macavirus genus of the Gammaherpesvirinae subfamily.

The HindIII-HincII fragment of the 5.5 kbp H11 HindIII clone of ovine herpesvirus 1 (OvHV-1) was cloned and its primary structure was determined by preparation of nested deletion subclones and their sequencing. Sequence analysis of the overlapping clones revealed that 3239 bp OvHV-1 fragment contains complete thymidine kinase (TK) gene, a partial open reading frame of ORF20 and that encoding glycoprotein H (gH). The conserved OvHV-1 TK displayed the highest similarity to homologous TK proteins encoded by members of the Macavirus genus of the Gammaherpesvirinae subfamily. These data including our previous analysis of the partial sequence of VP23 homologue might serve as further evidence that OvHV-1 should be categorized within the genus Macavirus of the Herpesviridae family.

Modelling soil nitrogen: the MAGIC model with nitrogen retention linked to carbon turnover using decomposer dynamics.

We present a new formulation of the acidification model MAGIC that uses decomposer dynamics to link nitrogen (N) cycling to carbon (C) turnover in soils. The new model is evaluated by application to 15-30 years of water chemistry data at three coniferous-forested sites in the Czech Republic where deposition of sulphur (S) and N have decreased by >80% and 40%, respectively. Sulphate concentrations in waters have declined commensurately with S deposition, but nitrate concentrations have shown much larger decreases relative to N deposition. This behaviour is inconsistent with most conceptual models of N saturation, and with earlier versions of MAGIC which assume N retention to be a first-order function of N deposition and/or controlled by the soil C/N ratio. In comparison with earlier versions, the new formulation more correctly simulates observed short-term changes in nitrate leaching, as well as long-term retention of N in soils. The model suggests that, despite recent deposition reductions and recovery, progressive N saturation will lead to increased future nitrate leaching, ecosystem eutrophication and re-acidification.

UV photoinitiated changes of humic fluorophores, influence of metal ions.

Fluorophore types and their photochemical stability have been tested in two samples of humic acids (HA) and four types of fulvic acids (FA) extracted from upper soil horizons (O and A horizons) in Norway spruce forest mountain ecosystems. Only one type of fluorophore occurred in all samples, with an excitation maximum at 310 nm for both HA and FA samples and emission maxima between 420-435 and 440-450 for HA and FA, respectively. HA weak native fluorescence increased significantly during irradiation in the first 12 h. Fluorophores in FA were uniformly degraded from the beginning of irradiation. Addition of metal (aluminium or ferric) ions did not affect the positions of fluorescence maxima in any of the studied samples; mild effects on fluorescence intensities were observed.

Antioxidant enzymes in barley green biomass.

Green biomass of young barley plants exhibited statistically significant higher activity of superoxide dismutase (SOD) and catalase (CAT) at sampling I (in the phase of plant development DC 29) compared to the later sampling II (DC 31). Significant effects of varieties, years and interactions of the studied factors on the activity of the studied antioxidants were determined. During the experiment period (2005-2007), the variety Sebastian provided statistically significant higher average SOD activity (486 U.g-1) versus the variety Malz (416 U.g-1 dry matter) and line KM1910 (418 U.g-1 dry matter). No statistically significant difference was recorded between the latter two varieties. Average catalase activity of the varieties did not show any significant difference. Significantly higher CAT activity in the sampling I was recorded on average of years and locations in the variety Sebastian and hull-less line KM1910 (935 and 907 U.g-1) compared to the variety Malz (675 U.g-1). We can state that green biomass of young spring barley plants taken during the growth phase DC 29 was a significant source of enzymes catalase and superoxide dismutase in the course of the experiment (2005-2007).

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2.

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

Alternative splicing variant of the hypoxia marker carbonic anhydrase IX expressed independently of hypoxia and tumour phenotype.

CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8-9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia- and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control.

Sequence analysis of the regions flanking terminal repeats of the genome of umava isolate of murine gammaherpesvirus.

The umava isolate of murine gammaherpesvirus (MHV-umava) slightly differs from Murine gammaherpesvirus 68 (MHV-68) and two other isolates of murine gammaherpesvirus (MHV), MHV-76 and MHV-72 in some biological properties. To identify the region(s) in the MHV-umava genome responsible for this phenomenon, we compared the sequences flanking terminal repeats (TRs) of the MHV-umava genome with those of MHV-68, MHV-76 and MHV-72. Restriction and sequence analyses revealed in MHV-umava as compared to MHV-68 approximately 9.3 kbp deletion at the left end of the genome and approximately 1.5 kbp deletion at the right end of the genome. While the approximately 9.3 kbp deletion was similar to that in MHV-76, the approximately 1.5 kbp deletion was unique for MHV-umava.

Modulation of expression of the sigma receptors in the heart of rat and mouse in normal and pathological conditions.

The aim of the present work was to study the effect of various stressors (hypoxia, cold, immobilization) on the gene expression of sigma receptors in the left ventricles of rat heart. We have clearly shown that gene expression of sigma receptors is upregulated by strong stress stimuli, such as immobilization and/or hypoxia. Nevertheless, cold as a milder stressor has no effect on sigma receptor's mRNA levels. Signalling cascade of sigma receptors is dependent on IP(3) receptors, since silencing of both, type 1 and 2 IP(3) receptors resulted in decreased mRNA levels of sigma receptors. Physiological relevance of sigma receptors in the heart is not clear yet. Nevertheless, based on the already published data we can assume that sigma receptors might participate in contractile responses in cardiomyocytes.

Modulation of expression of Na+/Ca2+ exchanger in heart of rat and mouse under stress.

AIM: The Na(+)/Ca(2+) exchanger (NCX) is a major Ca(2+) extrusion system in the plasma membrane of cardiomyocytes and an important component participating on the excitation-contraction coupling process in muscle cells. NCX1 isoform is the most abundant in the heart and is known to be changed after development of ischaemia or myocardial infarction. Objective of this study was to investigate the effect of stress factors (immobilization, cold and short-term hypoxia) on the expression of NCX1, in vivo, in the heart of rat and mouse. METHODS: We compared gene expression and protein levels of control and stressed animals. The activity of NCX was measured by the whole cell configuration using the patch clamp. We also measured physiological parameters of the heart in physiological conditions and under ischaemia-reperfusion to compare response of control and stressed hearts. RESULTS: We have found that only strong stress stimulus (hypoxia, immobilization) applied repeatedly for several days elevated the NCX1 mRNA level. Cold, which is a weaker stressor that activates mainly sympathoneural, and only marginally adrenomedullary system did not affect the gene expression of NCX1. Thus, from these results it appears that hormones produced by the adrenal medulla (mainly adrenaline) might be involved in this process. To study possible mechanism of the NCX1 regulation by stress, we focused on the possible role of the hypothalamo-pituitary-adrenocortical pathway in the activation of catecholamine synthesis in the adrenal medulla. We have already published that cortisol affects activity, but not the gene expression of NCX1. In this work, we used corticotropin-releasing hormone (CRH) knockout mice, where secretion of corticosterone and subsequently adrenaline is significantly suppressed. As no increase in NCX1 mRNA was observed in CRH knockout mice due to immobilization stress, we proposed that adrenaline (probably regulated via corticosterone) is involved in the regulation of NCX1 gene expression during stress. CONCLUSIONS: The gene expression and protein levels of the NCX1 are increased by the strong stress stimuli, e.g. hypoxia, or immobilization stress. The activity of NCX1 is decreased. Based on these results, we assume that the gene expression of NCX is increased as a consequence of suppressed activity of this transport system.

Hypoxia modulates gene expression of IP3 receptors in rodent cerebellum.

Hypoxic brain cell injury is a complex process that results from a series of intracellular events. In this work, we tested whether severe hypoxia for 6 h can affect gene expression and protein levels of intracellular calcium channels, ryanodine receptors, and inositol 1,4,5-trisphosphate receptors in mouse cerebellum. In addition, we tested the effect of hypoxia on cerebellar granular cells of rats. We have found that gene expression of types 1 and 2 IP(3) receptors is significantly increased after the exposure of mice to hypoxic stimulus for 6 h and also in rat cerebellar granular cells. Increased gene expression of IP(3) receptors was reflected in increased protein levels of these channels as well. In this process, reactive oxygen species are most probably involved, as antioxidant quercetin abolished hypoxia-induced increase in both types 1 and 2 IP3 receptor. Ryanodine receptors of types 1 and 2 and sarco(endo)plasmic reticulum Ca(2+)-ATPase were not affected by hypoxia on the level of messenger RNA. To test physiological consequences, we measured levels of intracellular calcium. We observed significantly elevated calcium level in hypoxic compared to normoxic cells. Deeper understanding of mechanisms, through which hypoxia regulates intracellular calcium, could point towards the development of new therapeutic approaches to reduce or suppress the pathological effects of cellular hypoxia, such as those seen in stroke or ischemia.

Modelling the effect of climate change on recovery of acidified freshwaters: relative sensitivity of individual processes in the MAGIC model.

The MAGIC model was used to evaluate the relative sensitivity of several possible climate-induced effects on the recovery of soil and surface water from acidification. A common protocol was used at 14 intensively studied sites in Europe and eastern North America. The results show that several of the factors are of only minor importance (increase in pCO(2) in soil air and runoff, for example), several are important at only a few sites (seasalts at near-coastal sites, for example) and several are important at nearly all sites (increased concentrations of organic acids in soil solution and runoff, for example). In addition changes in forest growth and decomposition of soil organic matter are important at forested sites and sites at risk of nitrogen saturation. The trials suggest that in future modelling of recovery from acidification should take into account possible concurrent climate changes and focus specially on the climate-induced changes in organic acids and nitrogen retention.

Gene expression of the phenylethanolamine N-methyltransferase is differently modulated in cardiac atria and ventricles.

Phenylethanolamine N-methyltransferase (PNMT) is a final enzyme in catecholamine synthesizing cascade that converts noradrenaline to adrenaline. Although most profuse in adrenal medulla, PNMT is expressed also in the heart, particularly in cardiac atria and ventricles. In atria, the PNMT mRNA is much more abundant compared to ventricles. In present study we aimed to find out whether there is a difference in modulation of the PNMT gene expression in cardiac atria and ventricles. We used three methodological approaches: cold as a model of mild stress, hypoxia as a model of cardiac ischemic injury, and transgenic rats (TGR) with incorporated mouse renin gene (mREN-2)27, to determine involvement of renin-angiotensin pathway in the PNMT gene expression. We have found that PNMT gene expression was modulated differently in cardiac atria and ventricles. In atria, PNMT mRNA levels were increased by hypoxia, while cold stress decreased PNMT mRNA levels. In ventricles, no significant changes were observed by cold or hypoxia. On the other hand, angiotensin II elevated PNMT gene expression in ventricles, but not in atria. These results suggest that PNMT gene expression is modulated differently in cardiac atria and ventricles and might result in different physiological consequences.

Impact of ionic aluminium on extracellular phosphatases in acidified lakes.

We studied direct inhibiting effects of aluminium (Al) on extracellular phosphatases produced by the plankton of acidified lakes in the Bohemian Forest. In laboratory experiments we tested the effect of different Al concentrations (0-1000 microg l(-1)) on kinetic parameters of acid phosphatases (pH optimum approximately 5.0) at pH between 4.5 and 5.2. We observed a significant reduction of an apparent substrate affinity at Al concentrations between 300 and 1000 microg l(-1) at pH 4.5 and 4.8 (but not at 5.2). In contrast, maximum acid phosphatase activity (AcPA) remained unchanged. Such behaviour of saturation kinetics is compatible with the assumption that ionic Al acts as a competitive inhibitor of acid phosphatases. To decide whether the observed Al effects could be explained alternatively by complexation of Al with substrate, we tested statistically the best fits of data with both possible models (competitive versus complexation). Experimental results supported the competitive hypothesis rather than the complexation model suggested originally by some authors. Furthermore, we tested the Al effect within a wide range of pH from 4.0 to 6.0. For pH values < 5.2, the results of an Al-pH matrix experiment gave a more detailed picture: the higher the Al concentration, the wider the pH range in which Al could negatively affect AcPA. The ecological ramifications of this effect were evaluated in the context of field AcPA data on three strongly acidified lakes.

Natural inactivation of phosphorus by aluminum in atmospherically acidified water bodies.

Atmospheric acidification of catchment-lake ecosystems may provide natural conditions for the in-lake control of P cycling. This process is based on the elevated transport of aluminum from acidified soils and its subsequent precipitation in the water body and is described for strongly acidified forest lakes, acidified and circumneutral reservoirs, and a moderately acidified alpine lake. In water bodies with episodically or permanently acidified inflows a pH gradient develops between lake water and tributaries due to: (i) neutralization of acidic inflows after mixing with waters with undepleted carbonate buffering system, and/or (ii) the in-lake alkalinity generation dominated by biochemical removal of NO3- and SO4(2-). With the pH increasing towards neutrality, ionic Al species hydrolyze and form colloidal Al hydroxides (Al(part)) with large specific surfaces and strong ability to bind orthophosphate from the liquid phase. Moreover, Alpart settles and increases the P sorption capacity of the sediment. The presence of Al(part) on the bottom reduces orthophosphate release from sediments after its liberation from ferric oxyhydroxides during anoxia because Al(part) is not sensitive to redox changes. Consequently, the natural in-lake P inactivation may be expected in any water body with elevated Al input and a pH gradient between its inlet and outlet.

Abnormal activation of glial cells in the brains of prion protein-deficient mice ectopically expressing prion protein-like protein, PrPLP/Dpl.

BACKGROUND: Some lines of mice homozygous for a disrupted prion protein gene (Prnp), including Ngsk Prnp(0/0) mice, exhibit Purkinje cell degeneration as a consequence of the ectopic overexpression of the downstream gene for prion protein-like protein (PrPLP/Dpl) in the brain, but others, such as Zrch I Prnp(0/0) mice, show neither the neurodegeneration nor the expression of PrPLP/Dpl. In the present study, we found that Ngsk Prnp(0/0), but not Zrch I Prnp(0/0) mice, developed gliosis involving both astrocytes and microglia in the brain. MATERIALS AND METHODS: The brains from wild-type (Prnp(+/+)), Ngsk Prnp(0/0), Zrch I Prnp(0/0), and reconstituted Ngsk Prnp(0/0) mice carrying a mouse PrP transgene, designated Tg(P) Ngsk Prnp(0/0) mice, were subjected into Northern blotting and in situ hybridization using probes of glial fibrillary acidic protein (GFAP) and lysozyme M (LM) specific for astrocytes and microglia, respectively. Immunohistochemistry was also performed on the brain sections using anti-GFAP and anti-F4/80 antibodies. RESULTS: Northern blotting demonstrated upregulated expression of the genes for GFAP and LM in the brains of Ngsk Prnp(0/0), but not in Zrch I Prnp(0/0) mice. A transgene for normal mouse PrP(C) successfully rescued Ngsk Prnp(0/0) mice from the glial activation. In situ hybridization and immunohistochemistry revealed activated astrocytes and microglia mainly in the white matter of both the forebrains and cerebella. In contrast, there was no evidence of neuronal injury except for the Purkinje cell degeneration. Moreover, the glial cell activation was notable well before the onset of the Purkinje cell degeneration. CONCLUSIONS: These findings strongly suggest that ectopic PrPLP/Dpl in the absence of PrP(C) is actively involved in the glial-cell activation in the brain.

PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C).

A hydrophobic, fibrillogenic peptide fragment of human prion protein (PrP106-126) had in vitro toxicity to neurons expressing cellular prion protein (PrP(C)). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrP(C). Incubation of MCEC with PrP106-126 (25-200 microM) caused a dose-dependent toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50-100 microg/ml), a drug effective in scrapie prophylaxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP106-126. In conclusion, cerebral endothelium expressing PrP(C) may be directly damaged during spongiform encephalopathies.