KAUSHIK NAG 1*, KARINA RODRIGUEZ-CAPOTE *, AMIYA KUMAR PANDA, LAURA FREDERICK , STEPHEN A. HEARN ... more KAUSHIK NAG 1*, KARINA RODRIGUEZ-CAPOTE *, AMIYA KUMAR PANDA, LAURA FREDERICK , STEPHEN A. HEARN , NILS O. PETERSEN , SAMUEL SCHÜRCH and FRED POSSMAYER 1. 1 Departments of Obstetrics and Gynaecology, 2 Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, 3 Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5A5, Departamento de Bioquímica, Instituto Superior de Ciencias Médicas de la Habana-Instituto de Ciencias Básicas y Preclínicas Victoria de Girón, Habana, Cuba, Department of Chemistry, Behala College, Kolkata 700 060, W.B., India , Department of Pathology, St. Joseph’s Health Centre, London, Ontario, Canada N6A 4V2, 7 Department of Physiology and Biophysics, University of Calgary, Alberta, Canada. * K. Nag and K. Rodriguez-Capote contributed equally to the work described in this paper. † Present address: Department of Biochemistry, Memorial University, St. John’s, Newfoundland, Canada A1B 3X9 Short Title: CRP...
Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosi... more Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosis, differentiation, and proliferation. Several different pathways such as Smad, p38, and PI3/Akt are activated by BMPs. Signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. BMPRs shuttle between membrane domains such as caveolae enriched with caveolin-1 b-isoform and caveolae of the caveolin-1 a/b-isoforms. It is hypothesized that there are other membrane domains to which the receptors localize. We used immunoprecipitation, Western blots, image cross-correlation spectroscopy, and fluorescence resonance energy transfer to investigate the interaction of BMPRs with proteins in clathrin-coated pits (CCPs). Our data indicate that these domains are associated with at least two of the BMPRs: BRIa and BRII. For the first time, to our knowledge, we showed what we believe are specific interactions between BRIa and BRII with a key component of CCPs, adaptor protein 2. Further, disruption of CCPs resulted in increased BRIa aggregation at the cell surface and activation of the BMP pathway even in the absence of BMP2. Therefore, CCPs seem to function as a negative regulatory membrane domain for BMP pathway activation.
Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate ... more Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate processes as diverse as neurogenesis, skeletal formation, and hematopoesis. They signal through a hetero-oligomer complex of BMP receptors. Binding of the ligand to the receptors activates several pathways, including Smad and p38. BMP signaling is controlled in the extracellular space, the plasma membrane, and the intracellular space; however, the mechanism of receptor signaling at the plasma membrane and proteins that regulate this process still need to be identified. The experiments presented here identify the protein kinase casein kinase II (CK2) as a BMP receptor type Ia (BRIa) interacting protein. Fluorescence resonance energy transfer revealed that this interaction occurs at the plasma membrane. BMP2 stimulation of C2C12 cells leads to the release of CK2 from BRIa. Blocking this interaction with specific peptides that inhibit the binding sites for CK2 on BRIa demonstrated a redistribution of BRIa on the plasma membrane. Signaling was initiated once CK2 was released from BRIa, leading to the mineralization of C2C12 cells. These data suggest that CK2 is a negative regulator of BMP signaling and osteoblast differentiation.
Proceedings of the National Academy of Sciences, 1987
Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion throug... more Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic di...
Hydrogen peroxide, formed directly or as a product of Superoxide dismutation, can oxidize ferrocy... more Hydrogen peroxide, formed directly or as a product of Superoxide dismutation, can oxidize ferrocytochrome c at rates comparable to those at which ferricytochrome c is reduced by Superoxide. This reoxidation can significantly affect estimates of rates and amounts of Superoxide production using absorbance changes for cytochrome c at 550 nm as the assay. The oxidation can be inhibited by catalase.
In the original theoretical development of fluorescence photobleaching recovery with circular or ... more In the original theoretical development of fluorescence photobleaching recovery with circular or Gaussian laser intensity profiles (Axelrod et al., 1976, Biophys. J.) the bleaching process is assumed to obey first order kinetics in the fluorescent probe. While this is reasonable in most cases where oxygen participates in the photolysis reaction, some processes may obey second order kinetics in the fluorophore concentration due to dimerization. Accordingly, we present here an analysis of the fluorescence recovery when the photobleaching process is taken to be second order in the probe. Analytical solutions for small bleaching levels indicate that the fluorescence recovery curve is very similar to that measured following a bleaching process first order in the probe. Numerical solutions for moderate bleaching levels show that the recovery is qualitatively similar, but quantitatively different. Because the shape of the recovery curve provides no evidence as to the order of photobleaching, we recommend continued use of the previous theoretical analysis. However, it must be borne in mind that the diffusion coefficient is increasingly underestimated as the extent of photobleaching is increased. The true diffusion coefficient is obtained in the limit of small levels of photobleaching. Estimates of the fractional recovery are not affected by this approach.
A series of homologous amphiphilic molecules with surface areas in the range of 0.3 nm2 to 3.0 nm... more A series of homologous amphiphilic molecules with surface areas in the range of 0.3 nm2 to 3.0 nm2 were prepared and used to investigate the diffusion in model dimyristoylphosphatidylcholine membranes as a function of temperature. The diffusion behavior of smaller molecules can be described by the interfacial viscosity limited free area theory promoted by Vaz and his co-workers, and that of the larger molecules can best be modeled by a recent interpretation of the theoretical description proposed by Evans and Sackmann. The experimental data show that the rate of diffusion is controlled by the size of the molecules at the interface of the lipid membrane, and provide evidence for a view of the membrane as a hydrodynamic triple layer with a low-viscosity central layer encased by two more viscous, yet fluid, layers.
Measurement of receptor distributions on cell surfaces is one important aspect of understanding t... more Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.
Pre-fibrillar oligomers of α-synuclein are thought to be pathogenic molecules leading to neurotox... more Pre-fibrillar oligomers of α-synuclein are thought to be pathogenic molecules leading to neurotoxicity associated with Parkinson's disease and other neurodegenerative disorders. However, small oligomers are difficult to isolate for study. To gain better insight into the properties of small α-synuclein oligomers, we investigated engineered oligomers of specific size (dimers, tetramers, and octamers) linked head-to-tail in tandem, comparing the behavior of the oligomers to monomeric α-synuclein. All oligomeric constructs remained largely disordered in solution, as determined from dynamic light scattering and size-exclusion chromatography. Electron microscopy revealed that each construct could aggregate to form fibrils similar to those formed by monomeric α-synuclein. The interactions with large unilamellar vesicles (LUVs) composed of negatively-charged lipids differed depending on size, with smaller oligomers forming more extensive helical structure as determined by CD spectroscop...
Aqueous phase synthesis at 80 o C was carried out to synthesize lead sulfide (PbS) nano-(NC) and ... more Aqueous phase synthesis at 80 o C was carried out to synthesize lead sulfide (PbS) nano-(NC) and micro-crystals (MC) by using cationic twin-tail surfactants (TTS) such as 12-0-12, 10-2
A seed-growth (SG) method has been used to synthesize gold (Au) and Au-silver (Ag) bimetallic pea... more A seed-growth (SG) method has been used to synthesize gold (Au) and Au-silver (Ag) bimetallic pear-necklace-type nanoparticles (NP) as bioconjugate materials by using a series of phospho-glycerol (PG) and phospho-choline (PC) lipids as capping agents. All ...
Two series of macrocyclic polyamine derivatives with various length of linkers were synthesized a... more Two series of macrocyclic polyamine derivatives with various length of linkers were synthesized as dendritic ligands containing two, three, four, five or six terminal nitrilotriacetic acid (NTA) groups through convergent and sequential pathways. Tetrafluorophenyl esters were employed as activating reagents in the coupling step of assembling NTA groups to the cyclic polyamine head. A key step of the sequential synthesis is the use of the coupling reagent, 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) for the formation of amide bonds between the cyclic polyamine and pendant groups. Finally, two representative compounds were used to demonstrate the formation of stoichiometric protein assemblies.
Spatial control of cell growth on surfaces can be achieved by the selective deposition of molecul... more Spatial control of cell growth on surfaces can be achieved by the selective deposition of molecules that influence cell adhesion. The fabrication of such substrates often relies upon photolithography and requires complex surface chemistry to anchor adhesive and inhibitory molecules. The production of simple, cost-effective substrates for cell patterning would benefit numerous areas of bioanalytical research including tissue engineering and biosensor development. Poly(dimethylsiloxane) (PDMS) is routinely used as a biomedical implant material and as a substrate for microfluidic device fabrication; however, the low surface energy and hydrophobic nature of PDMS inhibits its bioactivity. We present a method for the surface modification of PDMS to promote localized cell adhesion and proliferation. Thin metal films are deposited onto PDMS through a physical mask in the presence of a gaseous plasma. This treatment generates topographical and chemical modifications of the polymer surface. Removal of the deposited metal exposes roughened PDMS regions enriched with hydrophilic oxygen-containing species. The morphology and chemical composition of the patterned substrates were assessed by optical and atomic force microscopies as well as X-ray photoelectron spectroscopy. We observed a direct correlation between the surface modification of PDMS and the micropatterned adhesion of fibroblast cells. This simple protocol generates inexpensive, single-component substrates capable of directing cell attachment and growth.
A seed-mediated approach was applied to synthesize gold (Au) nanoparticles (NP) by using twin tai... more A seed-mediated approach was applied to synthesize gold (Au) nanoparticles (NP) by using twin tail alkylammonium cationic surfactants such as 12-6-12 and 12-0-12 as capping agents in aqueous phase at ambient conditions. The growth of Au NP was monitored by changing the amount of seed. Spherical NP (10-50nm) and nanorods (aspect ratio) 2-3) were obtained in the presence of 12-6-12 as capping agent; their shape and size systematically deformed because of anisotropic growth with a decrease in the amount of seed. In contrast, when 12-0-12 was used as a capping agent, no anisotropic growth was observed. An effective liquid/solid interfacial adsorption of 12-0-12 prevented anisotropic growth which led to precise morphologies. This was not observed in the case of 12-6-12 because of the presence of a spacer which restricted an effective interfacial adsorption because of the steric factors. XPS and FTIR studies clearly indicated the presence of a surfactant film on the surface of Au NP, while XRD analysis demonstrated a difference in the preferential adsorption of 12-6-12 and 12-0-12 at different crystal planes of fcc geometry which resulted in a difference in their capping behaviors.
Reverse phase high pressure liquid chromatography of Amphotericin B samples provides one means of... more Reverse phase high pressure liquid chromatography of Amphotericin B samples provides one means of cleanly separating Amphotericin B from a strongly fluorescent contaminant. Purified Amphotericin B does not exhibit any fluorescence.
The functional diversity of red blood cells needs to be taken into account in future studies, whi... more The functional diversity of red blood cells needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified lysophosphatidic acid signalling cascade provides as a multicomponent system the potential for delays and gains and such provokes a tremendous variability. Heterogeneity in red blood cell responses is important for the basic understanding of red blood cell signalling and their contribution to numerous diseases, especially with respect to calcium influx and the associated pro-thrombotic activity.
Proceedings of the National Academy of Sciences, 1982
We describe an approach to exploring cell surface-cytoskeleton interactions through direct measur... more We describe an approach to exploring cell surface-cytoskeleton interactions through direct measurements of the mechanical resistance of living cells to locally applied forces. These measurements are sensitive to variations in structure across the cell and at various depths below its surface. We find that local cellular deformability depends on the temperature and on the integrity of the cytoskeleton. Cytochalasin B increases the deformability of all regions of the cell except the nucleus.
KAUSHIK NAG 1*, KARINA RODRIGUEZ-CAPOTE *, AMIYA KUMAR PANDA, LAURA FREDERICK , STEPHEN A. HEARN ... more KAUSHIK NAG 1*, KARINA RODRIGUEZ-CAPOTE *, AMIYA KUMAR PANDA, LAURA FREDERICK , STEPHEN A. HEARN , NILS O. PETERSEN , SAMUEL SCHÜRCH and FRED POSSMAYER 1. 1 Departments of Obstetrics and Gynaecology, 2 Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, 3 Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5A5, Departamento de Bioquímica, Instituto Superior de Ciencias Médicas de la Habana-Instituto de Ciencias Básicas y Preclínicas Victoria de Girón, Habana, Cuba, Department of Chemistry, Behala College, Kolkata 700 060, W.B., India , Department of Pathology, St. Joseph’s Health Centre, London, Ontario, Canada N6A 4V2, 7 Department of Physiology and Biophysics, University of Calgary, Alberta, Canada. * K. Nag and K. Rodriguez-Capote contributed equally to the work described in this paper. † Present address: Department of Biochemistry, Memorial University, St. John’s, Newfoundland, Canada A1B 3X9 Short Title: CRP...
Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosi... more Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosis, differentiation, and proliferation. Several different pathways such as Smad, p38, and PI3/Akt are activated by BMPs. Signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. BMPRs shuttle between membrane domains such as caveolae enriched with caveolin-1 b-isoform and caveolae of the caveolin-1 a/b-isoforms. It is hypothesized that there are other membrane domains to which the receptors localize. We used immunoprecipitation, Western blots, image cross-correlation spectroscopy, and fluorescence resonance energy transfer to investigate the interaction of BMPRs with proteins in clathrin-coated pits (CCPs). Our data indicate that these domains are associated with at least two of the BMPRs: BRIa and BRII. For the first time, to our knowledge, we showed what we believe are specific interactions between BRIa and BRII with a key component of CCPs, adaptor protein 2. Further, disruption of CCPs resulted in increased BRIa aggregation at the cell surface and activation of the BMP pathway even in the absence of BMP2. Therefore, CCPs seem to function as a negative regulatory membrane domain for BMP pathway activation.
Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate ... more Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate processes as diverse as neurogenesis, skeletal formation, and hematopoesis. They signal through a hetero-oligomer complex of BMP receptors. Binding of the ligand to the receptors activates several pathways, including Smad and p38. BMP signaling is controlled in the extracellular space, the plasma membrane, and the intracellular space; however, the mechanism of receptor signaling at the plasma membrane and proteins that regulate this process still need to be identified. The experiments presented here identify the protein kinase casein kinase II (CK2) as a BMP receptor type Ia (BRIa) interacting protein. Fluorescence resonance energy transfer revealed that this interaction occurs at the plasma membrane. BMP2 stimulation of C2C12 cells leads to the release of CK2 from BRIa. Blocking this interaction with specific peptides that inhibit the binding sites for CK2 on BRIa demonstrated a redistribution of BRIa on the plasma membrane. Signaling was initiated once CK2 was released from BRIa, leading to the mineralization of C2C12 cells. These data suggest that CK2 is a negative regulator of BMP signaling and osteoblast differentiation.
Proceedings of the National Academy of Sciences, 1987
Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion throug... more Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic di...
Hydrogen peroxide, formed directly or as a product of Superoxide dismutation, can oxidize ferrocy... more Hydrogen peroxide, formed directly or as a product of Superoxide dismutation, can oxidize ferrocytochrome c at rates comparable to those at which ferricytochrome c is reduced by Superoxide. This reoxidation can significantly affect estimates of rates and amounts of Superoxide production using absorbance changes for cytochrome c at 550 nm as the assay. The oxidation can be inhibited by catalase.
In the original theoretical development of fluorescence photobleaching recovery with circular or ... more In the original theoretical development of fluorescence photobleaching recovery with circular or Gaussian laser intensity profiles (Axelrod et al., 1976, Biophys. J.) the bleaching process is assumed to obey first order kinetics in the fluorescent probe. While this is reasonable in most cases where oxygen participates in the photolysis reaction, some processes may obey second order kinetics in the fluorophore concentration due to dimerization. Accordingly, we present here an analysis of the fluorescence recovery when the photobleaching process is taken to be second order in the probe. Analytical solutions for small bleaching levels indicate that the fluorescence recovery curve is very similar to that measured following a bleaching process first order in the probe. Numerical solutions for moderate bleaching levels show that the recovery is qualitatively similar, but quantitatively different. Because the shape of the recovery curve provides no evidence as to the order of photobleaching, we recommend continued use of the previous theoretical analysis. However, it must be borne in mind that the diffusion coefficient is increasingly underestimated as the extent of photobleaching is increased. The true diffusion coefficient is obtained in the limit of small levels of photobleaching. Estimates of the fractional recovery are not affected by this approach.
A series of homologous amphiphilic molecules with surface areas in the range of 0.3 nm2 to 3.0 nm... more A series of homologous amphiphilic molecules with surface areas in the range of 0.3 nm2 to 3.0 nm2 were prepared and used to investigate the diffusion in model dimyristoylphosphatidylcholine membranes as a function of temperature. The diffusion behavior of smaller molecules can be described by the interfacial viscosity limited free area theory promoted by Vaz and his co-workers, and that of the larger molecules can best be modeled by a recent interpretation of the theoretical description proposed by Evans and Sackmann. The experimental data show that the rate of diffusion is controlled by the size of the molecules at the interface of the lipid membrane, and provide evidence for a view of the membrane as a hydrodynamic triple layer with a low-viscosity central layer encased by two more viscous, yet fluid, layers.
Measurement of receptor distributions on cell surfaces is one important aspect of understanding t... more Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.
Pre-fibrillar oligomers of α-synuclein are thought to be pathogenic molecules leading to neurotox... more Pre-fibrillar oligomers of α-synuclein are thought to be pathogenic molecules leading to neurotoxicity associated with Parkinson's disease and other neurodegenerative disorders. However, small oligomers are difficult to isolate for study. To gain better insight into the properties of small α-synuclein oligomers, we investigated engineered oligomers of specific size (dimers, tetramers, and octamers) linked head-to-tail in tandem, comparing the behavior of the oligomers to monomeric α-synuclein. All oligomeric constructs remained largely disordered in solution, as determined from dynamic light scattering and size-exclusion chromatography. Electron microscopy revealed that each construct could aggregate to form fibrils similar to those formed by monomeric α-synuclein. The interactions with large unilamellar vesicles (LUVs) composed of negatively-charged lipids differed depending on size, with smaller oligomers forming more extensive helical structure as determined by CD spectroscop...
Aqueous phase synthesis at 80 o C was carried out to synthesize lead sulfide (PbS) nano-(NC) and ... more Aqueous phase synthesis at 80 o C was carried out to synthesize lead sulfide (PbS) nano-(NC) and micro-crystals (MC) by using cationic twin-tail surfactants (TTS) such as 12-0-12, 10-2
A seed-growth (SG) method has been used to synthesize gold (Au) and Au-silver (Ag) bimetallic pea... more A seed-growth (SG) method has been used to synthesize gold (Au) and Au-silver (Ag) bimetallic pear-necklace-type nanoparticles (NP) as bioconjugate materials by using a series of phospho-glycerol (PG) and phospho-choline (PC) lipids as capping agents. All ...
Two series of macrocyclic polyamine derivatives with various length of linkers were synthesized a... more Two series of macrocyclic polyamine derivatives with various length of linkers were synthesized as dendritic ligands containing two, three, four, five or six terminal nitrilotriacetic acid (NTA) groups through convergent and sequential pathways. Tetrafluorophenyl esters were employed as activating reagents in the coupling step of assembling NTA groups to the cyclic polyamine head. A key step of the sequential synthesis is the use of the coupling reagent, 2-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) for the formation of amide bonds between the cyclic polyamine and pendant groups. Finally, two representative compounds were used to demonstrate the formation of stoichiometric protein assemblies.
Spatial control of cell growth on surfaces can be achieved by the selective deposition of molecul... more Spatial control of cell growth on surfaces can be achieved by the selective deposition of molecules that influence cell adhesion. The fabrication of such substrates often relies upon photolithography and requires complex surface chemistry to anchor adhesive and inhibitory molecules. The production of simple, cost-effective substrates for cell patterning would benefit numerous areas of bioanalytical research including tissue engineering and biosensor development. Poly(dimethylsiloxane) (PDMS) is routinely used as a biomedical implant material and as a substrate for microfluidic device fabrication; however, the low surface energy and hydrophobic nature of PDMS inhibits its bioactivity. We present a method for the surface modification of PDMS to promote localized cell adhesion and proliferation. Thin metal films are deposited onto PDMS through a physical mask in the presence of a gaseous plasma. This treatment generates topographical and chemical modifications of the polymer surface. Removal of the deposited metal exposes roughened PDMS regions enriched with hydrophilic oxygen-containing species. The morphology and chemical composition of the patterned substrates were assessed by optical and atomic force microscopies as well as X-ray photoelectron spectroscopy. We observed a direct correlation between the surface modification of PDMS and the micropatterned adhesion of fibroblast cells. This simple protocol generates inexpensive, single-component substrates capable of directing cell attachment and growth.
A seed-mediated approach was applied to synthesize gold (Au) nanoparticles (NP) by using twin tai... more A seed-mediated approach was applied to synthesize gold (Au) nanoparticles (NP) by using twin tail alkylammonium cationic surfactants such as 12-6-12 and 12-0-12 as capping agents in aqueous phase at ambient conditions. The growth of Au NP was monitored by changing the amount of seed. Spherical NP (10-50nm) and nanorods (aspect ratio) 2-3) were obtained in the presence of 12-6-12 as capping agent; their shape and size systematically deformed because of anisotropic growth with a decrease in the amount of seed. In contrast, when 12-0-12 was used as a capping agent, no anisotropic growth was observed. An effective liquid/solid interfacial adsorption of 12-0-12 prevented anisotropic growth which led to precise morphologies. This was not observed in the case of 12-6-12 because of the presence of a spacer which restricted an effective interfacial adsorption because of the steric factors. XPS and FTIR studies clearly indicated the presence of a surfactant film on the surface of Au NP, while XRD analysis demonstrated a difference in the preferential adsorption of 12-6-12 and 12-0-12 at different crystal planes of fcc geometry which resulted in a difference in their capping behaviors.
Reverse phase high pressure liquid chromatography of Amphotericin B samples provides one means of... more Reverse phase high pressure liquid chromatography of Amphotericin B samples provides one means of cleanly separating Amphotericin B from a strongly fluorescent contaminant. Purified Amphotericin B does not exhibit any fluorescence.
The functional diversity of red blood cells needs to be taken into account in future studies, whi... more The functional diversity of red blood cells needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified lysophosphatidic acid signalling cascade provides as a multicomponent system the potential for delays and gains and such provokes a tremendous variability. Heterogeneity in red blood cell responses is important for the basic understanding of red blood cell signalling and their contribution to numerous diseases, especially with respect to calcium influx and the associated pro-thrombotic activity.
Proceedings of the National Academy of Sciences, 1982
We describe an approach to exploring cell surface-cytoskeleton interactions through direct measur... more We describe an approach to exploring cell surface-cytoskeleton interactions through direct measurements of the mechanical resistance of living cells to locally applied forces. These measurements are sensitive to variations in structure across the cell and at various depths below its surface. We find that local cellular deformability depends on the temperature and on the integrity of the cytoskeleton. Cytochalasin B increases the deformability of all regions of the cell except the nucleus.
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Papers by Nils Petersen