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  • 1.
    Karlsson, Annika M.
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bjuhr, Katarina
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Testorf, Martin
    Linköping University, Department of Biomedical Engineering, Physiological Measurements. Linköping University, The Institute of Technology.
    Öberg, Åke
    Linköping University, Department of Biomedical Engineering, Physiological Measurements. Linköping University, The Institute of Technology.
    Lerner, Ethan
    Bunsen Rush Laboratories, Dallas, TX, USA.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Svensson, Samuel P.S.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Biosensing of opioids using frog melanophores2002In: Biosensors and Bioelectronics, ISSN 0956-5663, Vol. 17, no 4, p. 331-335Article in journal (Refereed)
    Abstract [en]

    Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.

  • 2.
    Testorf, Martin
    Linköping University, Department of Biomedical Engineering. Linköping University, The Institute of Technology.
    Melanophores: cell biophysics and sensor applications2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis deals with studies of melanophores and melanosomes by means of a physical approach. Melanophores are pigment cells that give the black colour to many vertebrates, e.g. fishes, frogs, and reptiles. Covering large areas of the skin, these cells are approximately 0.1 mm in diameter, and they contain black pigment granules called melanosomes.

    The geometry and the electric charge of isolated melanosomes were two physical properties that were studied. The electric charge was measured by electrophoresis and was found to be (-1.7 ± 0.2)·10-16 Coulomb in average. The geometry of melanosomes was measured using Scanning Force Microscopy, SFM, and resulted in an ellipsoidal shape with an average major diameter of 840 ± 20 nm.

    Under nervous and hormonal control, melanophores rearrange the intracellular melanosomes from a scattered distribution, called dispersed, to a state where all melanosomes are accumulated in the cell centre, called the aggregated state. In this way, melanophores change from black towards transparent. This gives an animal the ability to change not only between being pale or dark, but also between different colours by using melanophores to cover and uncover the colours of different types of pigment cells from underlying layers.

    The volume of melanosomes was measured with SFM. This study resulted in a difference of 18% when individual melanosomes from aggregated and dispersed melanophores were measured separately.

    Magnetic field exposure of melanophores has been reported to affect the aggregation. However, contradicting results are presented in the literature. To clarify the possible effect of magnetic fields on melanophores, experiments by aggregating fish melanophores under exposure to strong (8 and 14 Tesla), homogenous, static magnetic fields were carried out. Both the magnetic field-induced Lorentz force on the charged melanosomes and the reorientation of the cytoskeleton were considered as possible explanations of any effects. Whenfield experiments were compared to control experiments with zero field, no difference in aggregation levels were found. However, a more irregular speed of aggregation was seen in the 8 Tesla field than in the control experiments.

    A theoretical model was developed to explain switch-like responses in biological systems. A switch-like response to a graded stimulation was sometimes seen in the case of melanophores but was shown not to have a very large so-called Hill coefficient. The model is simple in its approach. It may be applied to general phenomena and is based on the assumption of a simultaneous desorption of an activator (agonist; substrate molecule; ... ) and an inactivator (antagonist, inhibitor; ... ) caused by a collision or interaction between two effector molecules (e.g. receptors or enzymes).

    Melanosomes are also found in the human body and have a remarkably capacity to bind other molecules. A well-established forensic application of this is to detect (illegal) drugs that have bound to melanosomes in hair shafts. So far this application is only qualitative. This thesis includes a characterisation of the binding of flunitrazepam to melanin. Flunitrazepam is the active substance of Rohypnol, which is a sedative that is illegal in several countries and sometimes called the "date-rape-drug".

    Melanophores are excellent model systems for studies of cellular phenomena. Moreover, melanophores are also interesting in sensing aspects. The change from black to transparent is a highly visible response to substances in their surroundings and has previously been the measurand in melanophore-based biosensors. The physical approach of these studies of melanophores also had the objective of evaluating new biosensor solutions.

    List of papers
    1. The electric charge of pigment granules in pigment cells
    Open this publication in new window or tab >>The electric charge of pigment granules in pigment cells
    2001 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 16, no 1-2, p. 31-36Article in journal (Refereed) Published
    Abstract [en]

    Black pigment cells called melanophores change colour in response to environmental changes and have lately been studied as promising biosensors. To further elucidate the intracellular processes involved in the colour changes of these cells, and to find optimal biosensing principles, the electric charge of intracellular pigment granules, melanosomes, has been determined in vitro by electrophoresis. Melanosomes from the two extreme states in the cell colour change (aggregated and dispersed melanosomes) were measured. The charge was found to be −1.5·10−16 and −1.7·10−16 C, aggregated and dispersed melanosomes, respectively, without significant difference between the two conditions. This charge is of the same order of magnitude as the one of 1000 electrons. The origin of the melanosome charge, and the use of these findings in new biosensor principles, is discussed.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-32560 (URN)10.1016/S0956-5663(00)00130-5 (DOI)18473 (Local ID)18473 (Archive number)18473 (OAI)
    Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13
    2. Melanophore aggregation in strong static magnetic fields
    Open this publication in new window or tab >>Melanophore aggregation in strong static magnetic fields
    2002 (English)In: Bioelectromagnetics, ISSN 0197-8462, E-ISSN 1521-186X, Vol. 23, no 6, p. 444-449Article in journal (Refereed) Published
    Abstract [en]

    Contradicting results can be found in the literature on effects from magnetic exposure to pigment cells. We have studied the influence of strong, static, homogenous magnetic fields of 8 and 14 T on melanophore aggregation during exposure to the field. Melanophores, black pigment cells, in fish are large flat cells having intracellular black pigment granules. Due to large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes. This is especially true for modeling nerve cells, since melanophores share stem cells with axons. Twenty experiments on black tetra fish fins were carried out in the two magnetic flux densities. The same number of control experiments were carried out in the magnet with the magnetic field turned off. Several factors, such as the degree of maximal aggregation, speed of aggregation, and irregularity of the speed, were examined. The statistical analysis showed no significant field effects on the aggregation, with one exception: the irregularity in aggregation speed in the 8 T field, compared to control. The believed reorientation of the cytoskeleton (microtubules) in the field or the induced Lorentz force on moving pigment granules, did not affect the aggregation.

    Keywords
    Pigment Cell, DC field, Lorentz force, Melanosome, Nerve stem cell, Pigment cell
    National Category
    Engineering and Technology
    Identifiers
    urn:nbn:se:liu:diva-46794 (URN)10.1002/bem.10040 (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
    3. Volume changes of individual melanosomes measured by scanning force microscopy
    Open this publication in new window or tab >>Volume changes of individual melanosomes measured by scanning force microscopy
    2001 (English)In: Pigment Cell Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 14, no 6, p. 445-449Article in journal (Refereed) Published
    Abstract [en]

    Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G-protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three-dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P<0.05, n=200). The average ellipsoidal volume was 0.14±0.01 (aggregated) and 0.17±0.01 μm3 (dispersed), a difference of 18%. The average major diameter was 810±20 and 880±20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non-destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-26717 (URN)10.1034/j.1600-0749.2001.140604.x (DOI)11311 (Local ID)11311 (Archive number)11311 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13
    4. Characterization of [3H]flunitrazepam binding to melanin
    Open this publication in new window or tab >>Characterization of [3H]flunitrazepam binding to melanin
    Show others...
    2001 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 298, no 2, p. 259-264Article in journal (Refereed) Published
    Abstract [en]

    In both clinical and forensic toxicology, the analysis of hair for drugs is an important tool to determine drug use in the past or to verify abstinence from illegal drugs during extended periods. Melanin is proposed as one of the factors that influences drug incorporation to hair and we have characterized the binding of the drug flunitrazepam to melanin in vitro. The drug was 3H labeled and melanin granules from cuttlefish, Sepia officinalis, were used according to the suggested standard for melanin studies. We observed a rapid Langmuir-like binding followed by a slower diffusion-limited binding that may be interpreted as an initial surface binding followed by deeper bulk binding. From three concentrations of melanin, with a 60-min incubation time, a mean saturation value of 180 ± 20 pmol/mg was calculated. The binding of a group of benzodiazepines and tranquilizers was compared to the binding of [3H]flunitrazepam by means of displacement experiments. These drugs showed binding characteristics similar to [3H]flunitrazepam except phenobarbital, which had a lower affinity to melanin. The method presented in this study allowed measurements with low melanin and drug concentrations and it has the strength of directly measuring the amount of drug bound to melanin, in contrast to previous indirect methods.

    Keywords
    Affinity, Benzodiazepines, Binding, Hair, Melanin
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-47156 (URN)10.1006/abio.2001.5364 (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
    5. A model for switch-like phenomena in biological systems
    Open this publication in new window or tab >>A model for switch-like phenomena in biological systems
    Show others...
    2001 (English)In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 94, no 1-2, p. 1-9Article in journal (Refereed) Published
    Abstract [en]

    We present a model for the activity of protein clusters based on a simultaneous desorption of an activator (agonist, substrate molecule, etc.) and an inactivator (antagonist, inhibitor, etc.) caused by the collision or interaction between two effector molecules (e.g. receptors, enzymes). This model gives rise to switch-like dose–response curves, which are difficult to explain by ordinary co-operativity. It fits with recent experimental results obtained on single cells. Some other interesting aspects of the model are also pointed out. The model is similar to the model used to explain steep ‘dose–response curves’ in heterogeneous catalysis, caused by the reaction between two different molecules or atoms on the surface of the catalyst.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-26718 (URN)10.1016/S0301-4622(01)00231-9 (DOI)11312 (Local ID)11312 (Archive number)11312 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13
  • 3.
    Testorf, Martin
    et al.
    Linköping University, Department of Biomedical Engineering. Linköping University, The Institute of Technology.
    Karlsson, Annika M.
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Svensson, Samuel
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Öberg, Åke
    Linköping University, Department of Biomedical Engineering. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    A model for switch-like phenomena in biological systems2001In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 94, no 1-2, p. 1-9Article in journal (Refereed)
    Abstract [en]

    We present a model for the activity of protein clusters based on a simultaneous desorption of an activator (agonist, substrate molecule, etc.) and an inactivator (antagonist, inhibitor, etc.) caused by the collision or interaction between two effector molecules (e.g. receptors, enzymes). This model gives rise to switch-like dose–response curves, which are difficult to explain by ordinary co-operativity. It fits with recent experimental results obtained on single cells. Some other interesting aspects of the model are also pointed out. The model is similar to the model used to explain steep ‘dose–response curves’ in heterogeneous catalysis, caused by the reaction between two different molecules or atoms on the surface of the catalyst.

  • 4.
    Testorf, Martin
    et al.
    Linköping University, Department of Biomedical Engineering. Linköping University, The Institute of Technology.
    Kronstrand, Robert
    Linköping University, Department of Medical and Health Sciences, Forensic Science and Toxicology . Linköping University, Faculty of Health Sciences.
    Svensson, Samuel
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Ahlner, Johan
    Linköping University, Department of Medical and Health Sciences, Forensic Science and Toxicology . Linköping University, Faculty of Health Sciences.
    Characterization of [3H]flunitrazepam binding to melanin2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 298, no 2, p. 259-264Article in journal (Refereed)
    Abstract [en]

    In both clinical and forensic toxicology, the analysis of hair for drugs is an important tool to determine drug use in the past or to verify abstinence from illegal drugs during extended periods. Melanin is proposed as one of the factors that influences drug incorporation to hair and we have characterized the binding of the drug flunitrazepam to melanin in vitro. The drug was 3H labeled and melanin granules from cuttlefish, Sepia officinalis, were used according to the suggested standard for melanin studies. We observed a rapid Langmuir-like binding followed by a slower diffusion-limited binding that may be interpreted as an initial surface binding followed by deeper bulk binding. From three concentrations of melanin, with a 60-min incubation time, a mean saturation value of 180 ± 20 pmol/mg was calculated. The binding of a group of benzodiazepines and tranquilizers was compared to the binding of [3H]flunitrazepam by means of displacement experiments. These drugs showed binding characteristics similar to [3H]flunitrazepam except phenobarbital, which had a lower affinity to melanin. The method presented in this study allowed measurements with low melanin and drug concentrations and it has the strength of directly measuring the amount of drug bound to melanin, in contrast to previous indirect methods.

  • 5.
    Testorf, Martin
    et al.
    Linköping University, Department of Biomedical Engineering. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Öberg, Åke
    Linköping University, Department of Biomedical Engineering. Linköping University, The Institute of Technology.
    The electric charge of pigment granules in pigment cells2001In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 16, no 1-2, p. 31-36Article in journal (Refereed)
    Abstract [en]

    Black pigment cells called melanophores change colour in response to environmental changes and have lately been studied as promising biosensors. To further elucidate the intracellular processes involved in the colour changes of these cells, and to find optimal biosensing principles, the electric charge of intracellular pigment granules, melanosomes, has been determined in vitro by electrophoresis. Melanosomes from the two extreme states in the cell colour change (aggregated and dispersed melanosomes) were measured. The charge was found to be −1.5·10−16 and −1.7·10−16 C, aggregated and dispersed melanosomes, respectively, without significant difference between the two conditions. This charge is of the same order of magnitude as the one of 1000 electrons. The origin of the melanosome charge, and the use of these findings in new biosensor principles, is discussed.

  • 6.
    Testorf, Martin
    et al.
    Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Health Sciences.
    Roback, Kerstin
    Linköping University, Department of Biomedical Engineering. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Svensson, Samuel
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Volume changes of individual melanosomes measured by scanning force microscopy2001In: Pigment Cell Research, ISSN 1755-1471, E-ISSN 1755-148X, Vol. 14, no 6, p. 445-449Article in journal (Refereed)
    Abstract [en]

    Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G-protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three-dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P<0.05, n=200). The average ellipsoidal volume was 0.14±0.01 (aggregated) and 0.17±0.01 μm3 (dispersed), a difference of 18%. The average major diameter was 810±20 and 880±20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non-destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.

  • 7.
    Testorf, Martin
    et al.
    Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Health Sciences.
    Öberg, P. Åke
    Linköping University, Department of Biomedical Engineering. Linköping University, Faculty of Health Sciences.
    Iwasaka, Masakazu
    Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
    Ueno, Shoogo
    Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
    Melanophore aggregation in strong static magnetic fields2002In: Bioelectromagnetics, ISSN 0197-8462, E-ISSN 1521-186X, Vol. 23, no 6, p. 444-449Article in journal (Refereed)
    Abstract [en]

    Contradicting results can be found in the literature on effects from magnetic exposure to pigment cells. We have studied the influence of strong, static, homogenous magnetic fields of 8 and 14 T on melanophore aggregation during exposure to the field. Melanophores, black pigment cells, in fish are large flat cells having intracellular black pigment granules. Due to large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes. This is especially true for modeling nerve cells, since melanophores share stem cells with axons. Twenty experiments on black tetra fish fins were carried out in the two magnetic flux densities. The same number of control experiments were carried out in the magnet with the magnetic field turned off. Several factors, such as the degree of maximal aggregation, speed of aggregation, and irregularity of the speed, were examined. The statistical analysis showed no significant field effects on the aggregation, with one exception: the irregularity in aggregation speed in the 8 T field, compared to control. The believed reorientation of the cytoskeleton (microtubules) in the field or the induced Lorentz force on moving pigment granules, did not affect the aggregation.

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