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Biomedical subjects

Ingemar Lundström

Publications and source records attributed to Ingemar Lundström.

14 recordsLinked to original sources
PubMed2005

Generation of biochemical response patterns of different substances using a whole cell assay with multiple signaling pathways.

Distinctive generation of biochemical response patterns of eight different substances, using an assay based on pigment containing cells, was demonstrated. Xenopus laevis melanophores, transfected with human beta(2)-adrenergic receptor, were seeded in a 96 well microplate and used to generate individual biochemical images through a two transient measuring protocol that contributes to highlight the response signatures of the agents. Adequate signal processing creates distinctive patterns in a time-concentration response space suitable for substance classification. The concept of biochemical images is introduced here. The assays were evaluated both with a standard microplate reader and with a computer screen photo-assisted technique (CSPT) yielding similar results. Since CSPT platforms only demand standard computer sets and web cameras as measuring setup, applications for these kind of assays outside main-laboratories were discussed.

Adrenergic beta-2 Receptor Antagonists↗
PubMed2005

Frog melanophores cultured on fluorescent microbeads: biomimic-based biosensing.

Melanophores are pigmented cells in lower vertebrates capable of quick color changes and thereby suitable as whole cell biosensors. In the frog dermis skin layer, the large and dark pigmented melanophore surrounds a core of other pigmented cells. Upon hormonal stimulation the black-brown pigment organelles will redistribute within the melanophore, and thereby cover or uncover the core, making complex color changes possible in the dermis. Previously, melanophores have only been cultured on flat surfaces. Here we mimic the three dimensional biological geometry in the frog dermis by culturing melanophores on fluorescent plastic microbeads. To demonstrate biosensing we use the hormones melatonin and alpha-melanocyte stimulating hormone (alpha-MSH) as lightening or darkening stimuli, respectively. Cellular responses were successfully demonstrated on single cell level by fluorescence microscopy, and in cell suspension by a fluorescence microplate reader and a previously demonstrated computer screen photo-assisted technique. The demonstrated principle is the first step towards "single well/multiple read-out" biosensor arrays based on suspensions of different selective-responding melanophores, each cultured on microbeads with distinctive spectral characteristics. By applying small amount of a clinical sample, or a candidate substance in early drug screening, to a single well containing combinations of melanophores on beads, multiple parameter read-outs will be possible.

Animals↗
PubMed2005

Hepatocyte growth factor (HGF) in fecal samples: rapid detection by surface plasmon resonance.

BACKGROUND: The development of biosensors, based on surface plasmon resonance (SPR) technology, enables monitoring of a variety of biospecific interactions without the need for chemical-, biological- or radiological-labelled reagents. METHOD: We utilised SPR to detect hepatocyte growth factor (HGF) in reconstituted faecal samples and studied samples from patients with infectious gastroenteritis (n = 20) and normal controls (n = 10). Mouse anti-human HGF monoclonal antibodies and recombinant human HGF receptor (c-Met)/Fc chimera were immobilised in flow cells of a CM5 biosensor chip. RESULTS: We found that infectious gastroenteritis produced a higher signal response compared to controls, due to binding of HGF to monoclonal anti-HGF antibody as well as binding of HGF to c-Met receptor (p < 0.01). The SPR signal response correlated with results from ELISA (r = 72%, p > 0.001). The signal response decreased significantly (p < 0.05) when samples were diluted with dextran, because of reduction in both specific as well as unspecific binding of HGF to dextran. The decrease in the specific response might imply that the dextran- binding site for HGF overlaps with the antibody binding epitope, or that dextran binding induces a conformational change of the HGF molecule. Bands corresponding to HGF were found by gel electrophoresis of purified faeces in an affinity chromatography column immobilised by HGF ligands. CONCLUSION: Determination of HGF by SPR might be beneficial in diagnosis of acute situations that present with symptoms of gastroenteritis and may, possibly, guide appropriate medical treatments. This is to our knowledge the first report on the use of SPR for detection of HGF in faeces samples.

Adult↗
PubMed2005

Molecular gradients: an efficient approach for optimizing the surface properties of biomaterials and biochips.

A variety of molecular gradients of alkanethiols with the structure HS-(CH(2))(m)-X (m = 15; X = COOH, CH(2)NH(2), or CH(3)) and oligo(ethylene glycol)-terminated alkanethiols with the structures HS-(CH(2))(15)-CO-NH-Eg(n) (n = 2, 4, or 6), HS-(CH(2))(15)-CO-NH-Eg(2)-(CH(2))(2)-NH-CO-(CH(2))(4)-biotin, and HS-(CH(2))(15)-CO-NH-Eg(6)-CH(2)-COOH were prepared on polycrystalline gold films. These gradients were designed to serve as model surfaces for fundamental studies of protein adsorption and immobilization phenomena. Ellipsometry, infrared spectroscopy, and X-ray photoelectron spectroscopy, operating in scanning mode, were used to monitor the layer composition, gradient profiles, tail group conformation, and overall structural quality of the gradient assemblies. The gradient profiles were found to be 4-10 mm wide, and they increased in width with increasing difference in molecular complexity between the thiols used to form the gradient. The oligo(ethylene glycol) thiols are particularly interesting because they can be used to prepare so-called conformational gradients, that is, gradients that display a variation in oligo(ethylene glycol) chain conformation from all trans on the extreme Eg(2,4) sides, via an amorphous-like phase in the mixing regimes, to helical at the extreme Eg(6) sides. We demonstrate herein a series of experiments where the above gradients are used to evaluate nonspecific binding of the plasma protein fibrinogen, and in agreement with previous studies, the highest amounts of nonspecifically bound fibrinogen were observed on all-trans monolayers, that is, on the extreme Eg(2,4) sides. Moreover, gradients between Eg(2) and a biotinylated analogue have been prepared to optimize the conditions for the immobilization of streptavidin. Ellipsometry and infrared spectroscopy reveal high levels of immobilization over a fairly broad range of compositions in the gradient regime, with a maximum between 50 and 60% of the biotinylated analogue in the monolayer. A pI gradient composed of (NH(3)(+)/COO(-))-terminated thiols was also prepared and evaluated with respect to its ability to separate differently charged proteins, pepsin, and lysozyme, on a solid surface.

Adsorption↗
PubMed2005

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization.

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.

Amino Acid Sequence↗
PubMed2004

A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis.

A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.

Acetazolamide↗
PubMed2003

Microplate based biosensing with a computer screen aided technique.

Melanophores, dark pigment cells from the frog Xenopus laevis, have the ability to change light absorbance upon stimulation by different biological agents. Hormone exposure (e.g. melatonin or alpha-melanocyte stimulating hormone) has been used here as a reversible stimulus to test a new compact microplate reading platform. As an application, the detection of the asthma drug formoterol in blood plasma samples is demonstrated. The present system utilizes a computer screen as a (programmable) large area light source, and a standard web camera as recording media enabling even kinetic microplate reading with a versatile and broadly available platform, which suffices to evaluate numerous bioassays. Especially in the context of point of care testing or self testing applications these possibilities become advantageous compared with highly dedicated comparatively expensive commercial systems.

Animals↗
PubMed2003

Computer screen as a programmable light source for visible absorption characterization of (bio)chemical assays.

Visible absorption features suitable for color recognition and micro-plate reading of a standard bioassay are performed by the combination of a computer screen used as a programmable light source and a web camera as detector. The method provides in this way a highly available platform for 'home tests' or 'self-tests', where the requirement is to monitor well defined assays and the use of economical instrumentation is advantageous.

Automation↗
PubMed2002

Monitoring the interfacial capacitance at self-assembled phosphate monolayers on gold electrodes upon interaction with calcium and magnesium.

Electrochemical impedance spectroscopy has been used to evaluate the change in interfacial capacitance upon calcium and magnesium coordination to a phosphate-modified electrode. The phosphate electrode was prepared via immobilization of phosphorylated, thiol-containing, serine analogues onto gold. Upon subjection to calcium and magnesium, a substantial drop in capacitance was observed. Magnesium displayed the largest influence on the capacitance: a 27% capacitance drop was observed upon introduction of a 1 mM solution of magnesium ions. The lowered capacitance is a result of a change in the potential and charge distribution at the film/electrolyte interface as the cations coordinate to the phosphate groups. Moreover, the relationship between electrode potential and capacitance has been investigated and reveals a significant difference between monovalent and divalent cations. As complementary information, infrared reflection absorption spectra of the phosphorylated monolayer having different counterions are presented. The results reported in this paper indicate that the phosphorylated amino acid analogue monolayers could be used in investigations of the biochemically important coordination of calcium and magnesium to phosphates and phosphorylated amino acids.

Biosensing Techniques↗
PubMed2002

Interactions between the juxtamembrane domain of the EGFR and calmodulin measured by surface plasmon resonance.

One early response to epidermal growth factor receptor (EGFR) activation is an increase in intracellular calcium. We have used surface plasmon resonance (SPR) to study real-time interactions between the intracellular juxtamembrane (JM) region of EGFR and calmodulin. The EGFR-JM (Met(644)-Phe(688)) was expressed as a GST fusion protein and immobilised on a sensor chip surface. Calmodulin specifically interacts with EGFR-JM in a calcium-dependent manner with a high on and high off rate. Chemical modification of EGFR-JM by using arginine-selective phenylglyoxal or deletion of the basic segment Arg(645)-Arg(657) inhibits the interaction. Phosphorylation of EGFR-JM by protein kinase C (PKC) or glutamate substitution of Thr(654) inhibits the interaction, suggesting that PKC phosphorylation electrostatically interferes with calmodulin binding to basic arginine residues. Calmodulin binding was also inhibited by suramin. Our results suggest that EGFR-JM is essential for epidermal growth factor (EGF)-mediated calcium-calmodulin signalling and for signal integration between other signalling pathways.

Amino Acid Sequence↗
PubMed2002

Surface plasmon resonance and free oscillation rheometry in combination: a useful approach for studies on haemostasis and interactions between whole blood and artificial surfaces.

In haemostatic and biomaterial research biological processes at surfaces and in the bulk phase of the surface-contacting medium are important. The present work demonstrates the usefulness of the combination of surface plasmon resonance (SPR), sensitive to changes in refractive index at surfaces, and free oscillation rheometry (FOR), sensitive to rheological properties of the bulk, for simultaneous real-time measurements on coagulation and fibrinolysis of blood plasma and coagulation of whole blood. SFLLRN stimulated coagulation of native whole blood presented a higher SPR signal with different appearance than plasma coagulation, while the FOR signals corresponding to plasma and whole blood coagulation were similar. This indicated that the SPR technique was more sensitive to cell-surface interactions than to fibrin formation in whole blood during coagulation, while the FOR technique were equally sensitive to coagulation in whole blood and plasma. Spontaneous coagulation of native whole blood in contact with methyl- and hydroxyl-terminated self-assembled monolayers (SAM) on gold and gold surfaces regenerated after coagulation were also studied. The regenerated gold surfaces displayed the shortest coagulation times, although the contact-activation of blood coagulation for these surfaces was low. The methylated and hydroxylated surfaces were comparable in terms of coagulation activation, while the hydroxylated surfaces presented FOR signals that indicated detaching of the coagulum from the surface. The combination of SPR and FOR is well suited for studies of cell- and protein-surface interactions and simultaneous bulk processes. Possible applications are investigations of blood cell defects in patients and monitoring of native whole blood interactions with artificial surfaces.

Biocompatible Materials↗
PubMed2002

Comparative studies with surface plasmon resonance and free oscillation rheometry on the inhibition of platelets with cytochalasin E and monoclonal antibodies towards GPIIb/IIIa.

In the haemostatic system a multitude of processes are intertwined in fine-tuned interactions that arrest bleeding, keep the circulatory system open, and the blood flowing. The occurrence of both surface and bulk interactions adds an additional dimension of complexity. These insights have led to the belief that global overall procedures can inform on the likely behaviour of the system in health and disease. Two sensing procedures: surface plasmon resonance (SPR), which senses surface interactions, and free oscillation rheometry (FOR), which senses interactions within the bulk, have been combined and evaluated. The contribution of blood cells, mainly platelets, to the SPR and FOR signals was explored by simultaneous SPR and FOR measurement during native whole blood coagulation, accelerated via the platelets through addition of SFLLRN peptide and inhibition of platelet aggregation with abciximab (ReoPro) and of shape change with cytochalasin E. The SPR technique was found to be sensitive to inhibition of blood cell functions such as adhesion to and spreading on surfaces, as well as platelet aggregation. SPR seemed not to be directly sensitive to fibrin polymerisation in coagulating whole blood. The FOR technique detected the coagulation as a bulk phenomenon, i.e. the gelation of the blood due to fibrin formation was detected. The combination of SPR and FOR may therefore be suitable for studies on blood cell functions during coagulation.

Abciximab↗