20 research outputs found
Cloning and characterisation of chlorophyll synthase from Avena sativa
The chlorophyll synthase gene from oat (Avena sativa) was cloned and expressed in Escherichia coli. The deduced amino acid sequence consists of 378 amino acids including a presequence, of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatically active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlorophyll synthase of Synechocystis PCC 6803. The gene is constitutively expressed as the same transcript level is found in dark-grown and in light-grown seedlings. The enzyme requires magnesium ions for activity; manganese ions can reconstitute only part of the activity. Diacetyl and N-phenylmaleimide (NPM) inhibit the enzyme activity. Site-directed mutagenesis reveals that, out of the 4 Arg residues present in the active core protein, Arg-91 and Arg-161 are essential for the activity. Five cysteine residues are present in the core protein, of which only Cys-109 is essential for the enzyme activity. Since the wild-type and all other Cys-mutants with the exception of the mutant C304A are inhibited by N-phenylmaleimide, we conclude that the inhibitor binds to a non-essential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed
IDENTIFICATION OF TOUCHDOWN AND TOE-OFF IN TURF-SPORT SPECIFIC MOVEMENTS USING KINEMATIC DATA
The accurate determination of touchdown and toe-off during the stance phase in human locomotion is important for further motion analysis. The aim of this study was to evaluate the accuracy of using kinematic data to detect these events and therefore ground contact time of movements on artificial turf. Seven athletes performed five different turf-sport specific movements in which a single contact was made on a force plate (1000 Hz), while kinematic data of six markers were recorded (CODA, 400 Hz). A force threshold (20N) was set to determine the events of the touchdown and toe-off for the kinetic data. Comparison was made between the kinetic and kinematic derived event times. The errors between the kinetic and kinematic data ranged from 1.6 to 3.4% for the acceleration, hurdle hop and a turn with change of direction of 135°. It was concluded that kinematic data can accurately determine touchdown and toe-off events for certain movements on artificial turf
A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes
Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems
An ecosystem-scale litter and microplastics monitoring plan under the Arctic Monitoring and Assessment Programme (AMAP)
Lack of knowledge on levels and trends of litter and microplastics in the Arctic, is limiting our understanding of the sources, transport, fate, and effects is hampering global activities aimed at reducing litter and microplastics in the environment. To obtain a holistic view to managing litter and microplastics in the Arctic, we considered the current state of knowledge and methods for litter and microplastics monitoring in eleven environmental compartments representing the marine, freshwater, terrestrial, and atmospheric environments. Based on available harmonized methods, and existing data in the Arctic, we recommend prioritization of implementing litter and microplastics monitoring in the Arctic in four Priority 1 compartments—water, aquatic sediments, shorelines, and seabirds. One or several of these compartments should be monitored to provide benchmark data for litter and microplastics in the Arctic and, in the future, data on spatial and temporal trends. For the other environmental compartments, methods should be refined for future sources and surveillance monitoring, as well as monitoring of effects. Implementation of the monitoring activities should include community-based local components where possible. While organized as national and regional programs, monitoring of litter and microplastics in the Arctic should be coordinated, with a view to future pan-Arctic assessments.publishedVersio
Microplastics in arctic invertebrates- Status on occurrence and recommendations for future monitoring
Few studies have been published on occurrence and distribution on microplastics (MPs) in invertebrates from the Arctic. We still need to develop harmonised methods to enable good comparison between studies taking into account recovery rates, size ranges, shapes and polymer types. Here, we review studies on MPs in invertebrates from the Arctic and present suggestions on sampling protocols and potential indicator species. Since information on MPs in Arctic invertebrates is vastly lacking, we recommend to at least include suspension feeding bivalves like mussels in monitoring programmes to function as indicator species in the Arctic. Mussels have also been suggested as indicator species for MP monitoring in coastal regions further south. Although we recognise the challenge with particle selection and egestion in mussels as well as the relatively low concentrations of MPs in Arctic waters, uptake levels seem to represent recent exposures. More research is needed to understand these selection processes and how they affect the bioaccumulation processes. Future research should include studies on whether different functional groups of invertebrates have different exposures to MPs, e.g., if there are differences between sessile versus motile species or different feeding strategies. More knowledge on monitoring strategies for pelagic and benthic species is needed.Microplastics in arctic invertebrates- Status on occurrence and recommendations for future monitoringpublishedVersio
Cloning and characterisation of chlorophyll synthase from Avena sativa
The chlorophyll synthase gene from oat (Avena sativa) was cloned and expressed in Escherichia coli. The deduced amino acid sequence consists of 378 amino acids including a presequence, of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatically active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlorophyll synthase of Synechocystis PCC 6803. The gene is constitutively expressed as the same transcript level is found in dark-grown and in light-grown seedlings. The enzyme requires magnesium ions for activity; manganese ions can reconstitute only part of the activity. Diacetyl and N-phenylmaleimide (NPM) inhibit the enzyme activity. Site-directed mutagenesis reveals that, out of the 4 Arg residues present in the active core protein, Arg-91 and Arg-161 are essential for the activity. Five cysteine residues are present in the core protein, of which only Cys-109 is essential for the enzyme activity. Since the wild-type and all other Cys-mutants with the exception of the mutant C304A are inhibited by N-phenylmaleimide, we conclude that the inhibitor binds to a non-essential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed
An ecosystem-scale litter and microplastics monitoring plan under the Arctic Monitoring and Assessment Programme (AMAP)
Lack of knowledge on levels and trends of litter and microplastics in the Arctic, is limiting our understanding of the sources, transport, fate, and effects is hampering global activities aimed at reducing litter and microplastics in the environment. To obtain a holistic view to managing litter and microplastics in the Arctic, we considered the current state of knowledge and methods for litter and microplastics monitoring in eleven environmental compartments representing the marine, freshwater, terrestrial, and atmospheric environments. Based on available harmonized methods, and existing data in the Arctic, we recommend prioritization of implementing litter and microplastics monitoring in the Arctic in four Priority 1 compartments—water, aquatic sediments, shorelines, and seabirds. One or several of these compartments should be monitored to provide benchmark data for litter and microplastics in the Arctic and, in the future, data on spatial and temporal trends. For the other environmental compartments, methods should be refined for future sources and surveillance monitoring, as well as monitoring of effects. Implementation of the monitoring activities should include community-based local components where possible. While organized as national and regional programs, monitoring of litter and microplastics in the Arctic should be coordinated, with a view to future pan-Arctic assessments
An ecosystem-scale litter and microplastics monitoring plan under the Arctic Monitoring and Assessment Programme (AMAP)
Lack of knowledge on levels and trends of litter and microplastics in the Arctic, is limiting our understanding of the sources, transport, fate, and effects is hampering global activities aimed at reducing litter and microplastics in the environment. To obtain a holistic view to managing litter and microplastics in the Arctic, we considered the current state of knowledge and methods for litter and microplastics monitoring in eleven environmental compartments representing the marine, freshwater, terrestrial, and atmospheric environments. Based on available harmonized methods, and existing data in the Arctic, we recommend prioritization of implementing litter and microplastics monitoring in the Arctic in four Priority 1 compartments—water, aquatic sediments, shorelines, and seabirds. One or several of these compartments should be monitored to provide benchmark data for litter and microplastics in the Arctic and, in the future, data on spatial and temporal trends. For the other environmental compartments, methods should be refined for future sources and surveillance monitoring, as well as monitoring of effects. Implementation of the monitoring activities should include community-based local components where possible. While organized as national and regional programs, monitoring of litter and microplastics in the Arctic should be coordinated, with a view to future pan-Arctic assessments
Novel microscopy-based screening method reveals regulators of contact-dependent intercellular transfer
Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT