Experiences from the implementation of a biosafety system in Slovenia

The development and implementation of an effective national biosafety system is important for several key reasons: to ensure safe access to products of modern biotechnology, to build public confidence, to encourage the growth of domestic modern biotechnology, and to comply with international standards and agreements. There is no single best approach in the development and implementation of a national biosafety system and each country is faced with unique challenges. Slovenia is a small country and a new EU Member State. However, it has developed and implemented an efficient national biosafety system. The key elements of this system are administrative procedure, risk assessment, enforcement, and public participation and information.     

Voltametric monitoring of enzyme-mediated indigo reduction in the presence of various fibre materials

A comparative study of enzyme-mediated indigo reduction is presented as an environmentally-friendly alternative to alkaline sodium dithionite reduction. The effect of the mediator 1,8-dihydroxy-9,10-anthraquinone in enzymatic reduction was studied by means of voltammetry, both in the presence and absence of different textile materials (polyamide 6, polyamide 6,6 and cotton), and compared to chemically reduced indigo. It was observed that bio-catalytic formation of leuco indigo and its exhaustion on substrates is inversely proportional to the pH within the range of 7–11. Additionally, substrate coloration was strongly influenced by the mediator, resulting in in situ formation of leuco indigo. This effect was most pronounced for polyamide substrates. The reuse of an enzyme-mediated reduction bath for dyeing was assessed showing that the levelness of the obtained shade was either excellent or good at pH 9 and 11, respectively. The wash, perspiration, and light color fastness properties of all textile materials dyed with enzymatically-reduced indigo were comparable or even better than those obtained with chemically reduced indigo. The use of enzyme-mediated reduction of indigo combined with potential reuse of the reduction bath represents a cost effective and environmentally-friendly dyeing process that can be applied for the dyeing of natural cellulosic and synthetic polyamide fibres.     

Interaction Between Equally Charged Membrane Surfaces Mediated by Positively and Negatively Charged Macro-Ions

In biological systems, charged membrane surfaces are surrounded by charged molecules such as electrolyte ions and proteins. Our recent experiments in the systems of giant phospholipid vesicles indicated that some of the blood plasma proteins (macro-ions) may promote adhesion between equally charged membrane surfaces. In this work, theory was put forward to describe an IgG antibody-mediated attractive interaction between negatively charged membrane surfaces which was observed in experiments on giant phospholipid vesicles with cardiolipin-containing membranes. The attractive interactions between negatively charged membrane surfaces in the presence of negatively and positively charged spherical macro-ions are explained using functional density theory and Monte Carlo simulations. Both, the rigorous solution of the variational problem within the functional density theory and the Monte Carlo simulations show that spatial and orientational ordering of macro-ions may give rise to an attractive interaction between negatively charged membrane surfaces. It is also shown that the distinctive spatial distribution of the charge within the macro-ions (proteins) is essential in this process.     

Environmental changes affect ecosystem services of the intermittent Lake Cerknica

Lake Cerknica is an intermittent wetland with seasonal water level fluctuations. This paper discusses the possible changes of ecosystem services due to altered ecosystem functions caused by regional climate change. For this purpose, the lake's water regime was analysed and biomass production of common reed (Phragmites australis), the prevailing species, was related to ambient temperatures and water level. The effect of the latter was also examined for reed transpiration rate and plant diversity. The results revealed a gradual loss of seasonality of floods and droughts. High water level in winter months and temperatures at the beginning as well as the end of vegetation period were found to have a significant influence on reed biomass. The plant diversity research in six subsequent years in three different aquatic habitats revealed trends of a decrease due to increased water depth during the vegetation period. The complexity of the system and consequently ecosystem services might also be affected due to changes of other processes, which revealed to be related to water level; namely transpiration rate, plant mycorrhizal colonisation and soil mineralisation.     

Measurements of ATP in mammalian cells

Levels of phosphorylated adenosine nucleotides, including the universal energy carrier adenosine 5(')-triphosphate (ATP) and its metabolites adenosine 5(')-diphosphate (ADP) and adenosine 5(')-monophosphate (AMP), define the energy state in living cells and are dependent mainly on mitochondrial function. In this article, we describe a method based on the luciferase-luciferin system used to measure mitochondrial ATP synthesis continuously in permeabilized mammalian cells and mitochondria isolated from animal tissues. We also describe a technique that uses the expression of recombinant targeted luciferase to report ATP content in different cell compartments. Finally, we describe an HPLC-based method for accurate measurement of ATP, ADP, and AMP in cultured cells and animal tissues.     

Differences in the oxygenation of the forearm muscle during isometric contraction in trained and untrained subjects

The aim of this study was to test a hypothesis that a longer duration of isometric contraction is related to an increased oxygenation status of the muscle. A group of trained rock climbers and untrained subjects performed 15 kp sustained isometric contraction until fatigue set in. The oxygenation status was assessed using near infrared spectroscopy (ISS, USA). The results support the hypothesis. The concentration of relative oxygenated hemoglobin was higher in the rock climbers than in the untrained subjects. The relative total hemoglobin did not differentiate the two groups enough to show that blood volume also strongly influences contraction time.     

Cross-presentation of Listeria-derived CD8 T cell epitopes requires unstable bacterial translation products

Presentation of bacteria-derived CD8 T cell epitopes by dendritic cells (DC) requires either their direct infection or that DC acquire and cross-present Ags from other infected cells. We found that cross-presentation of Listeria monocytogenes-derived CD8 T cell epitopes was much stronger than direct Ag presentation by infected murine DC. Cross-presentation of Listeria-derived CD8 T cell epitopes showed unique physiological requirements. It was dependent upon the delivery of unstable bacterial translation products by infected, but still viable, Ag donor cells. Cross-presentation was enhanced both when unstable translation products in infected Ag donor cells were protected from proteasomal degradation and when the production of misfolded bacterial proteins was increased. The requirement of unstable translation products for cross-presentation may represent a novel pathway that functions to focus the CD8 T cell response toward epitopes derived from newly synthesized proteins.     

A comparative genomic analysis of the calcium signaling machinery in Neurospora crassa, Magnaporthe grisea, and Saccharomyces cerevisiae

A large number of Ca2+ -signaling proteins have been previously identified and characterized in Saccharomyces cerevisiae but relatively few have been discovered in filamentous fungi. In this study, a detailed, comparative genomic analysis of Ca2+ -signaling proteins in Neurospora crassa, Magnaporthe grisea, and S. cerevisiae has been made. Our BLAST analysis identified 48, 42, and 40 Ca2+ -signaling proteins in N. crassa, M. grisea, and S. cerevisiae, respectively. In N. crassa, M. grisea, and S. cerevisiae, 79, 100, and 13% of these proteins, respectively, were previously unknown. For N. crassa, M. grisea, and S. cerevisiae, respectively, we have identified: three Ca2+ -permeable channels in each species; 9, 12, and 5 Ca2+/cation-ATPases; eight, six, and four Ca2+ -exchangers; four, four, and two phospholipase C's; one calmodulin in each species; and 23, 21, and 29 Ca2+/calmodulin-regulated proteins. Homologs of a number of key proteins involved in the release of Ca2+ from intracellular stores, and in the sensing of extracellular Ca2+, in animal and plant cells, were not identified. The greater complexity of the Ca2+ -signaling machinery in N. crassa and M. grisea over that in S. cerevisiae probably reflects their more complex cellular organization and behavior, and the greater range of external signals which filamentous fungi have to respond to in their natural habitats. To complement the data presented in this paper, a comprehensive web-based database resource (http://www.fungalcell.org/fdf/) of all Ca2+ -signaling proteins identified in N. crassa, M. grisea, and S. cerevisiae has been provided.     

Numerical optimization of gene electrotransfer into muscle tissue

Background  

Electroporation-based gene therapy and DNA vaccination are promising medical applications that depend on transfer of pDNA into target tissues with use of electric pulses. Gene electrotransfer efficiency depends on electrode configuration and electric pulse parameters, which determine the electric field distribution. Numerical modeling represents a fast and convenient method for optimization of gene electrotransfer parameters. We used numerical modeling, parameterization and numerical optimization to determine the optimum parameters for gene electrotransfer in muscle tissue.