Thermal fluctuations of haemoglobin from different species: adaptation to temperature via conformational dynamics.

Thermodynamic stability, configurational motions and internal forces of haemoglobin (Hb) of three endotherms (platypus, Ornithorhynchus anatinus; domestic chicken, Gallus gallus domesticus and human, Homo sapiens) and an ectotherm (salt water crocodile, Crocodylus porosus) were investigated using circular dichroism, incoherent elastic neutron scattering and coarse-grained Brownian dynamics simulations. The experimental results from Hb solutions revealed a direct correlation between protein resilience, melting temperature and average body temperature of the different species on the 0.1 ns time scale. Molecular forces appeared to be adapted to permit conformational fluctuations with a root mean square displacement close to 1.2 Å at the corresponding average body temperature of the endotherms. Strong forces within crocodile Hb maintain the amplitudes of motion within a narrow limit over the entire temperature range in which the animal lives. In fully hydrated powder samples of human and chicken, Hb mean square displacements and effective force constants on the 1 ns time scale showed no differences over the whole temperature range from 10 to 300 K, in contrast to the solution case. A complementary result of the study, therefore, is that one hydration layer is not sufficient to activate all conformational fluctuations of Hb in the pico- to nanosecond time scale which might be relevant for biological function. Coarse-grained Brownian dynamics simulations permitted to explore residue-specific effects. They indicated that temperature sensing of human and chicken Hb occurs mainly at residues lining internal cavities in the ß-subunits.

Contractile tension and beating rates of self-exciting monolayers and 3D-tissue constructs of neonatal rat cardiomyocytes.

The CellDrum technology (The term 'CellDrum technology' includes a couple of slightly different technological setups for measuring lateral mechanical tension in various types of cell monolayers or 3D-tissue constructs) was designed to quantify the contraction rate and mechanical tension of self-exciting cardiac myocytes. Cells were grown either within flexible, circular collagen gels or as monolayer on top of respective 1-mum thin silicone membranes. Membrane and cells were bulged outwards by air pressure. This biaxial strain distribution is rather similar the beating, blood-filled heart. The setup allowed presetting the mechanical residual stress level externally by adjusting the centre deflection, thus, mimicking hypertension in vitro. Tension was measured as oscillating differential pressure change between chamber and environment. A 0.5-mm thick collagen-cardiac myocyte tissue construct induced after 2 days of culturing (initial cell density 2 x 10(4) cells/ml), a mechanical tension of 1.62 +/- 0.17 microN/mm(2). Mechanical load is an important growth regulator in the developing heart, and the orientation and alignment of cardiomyocytes is stress sensitive. Therefore, it was necessary to develop the CellDrum technology with its biaxial stress-strain distribution and defined mechanical boundary conditions. Cells were exposed to strain in two directions, radially and circumferentially, which is similar to biaxial loading in real heart tissues. Thus, from a biomechanical point of view, the system is preferable to previous setups based on uniaxial stretching.

From powder to solution: hydration dependence of human hemoglobin dynamics correlated to body temperature.

A transition in hemoglobin (Hb), involving partial unfolding and aggregation, has been shown previously by various biophysical methods. The correlation between the transition temperature and body temperature for Hb from different species, suggested that it might be significant for biological function. To focus on such biologically relevant human Hb dynamics, we studied the protein internal picosecond motions as a response to hydration, by elastic and quasielastic neutron scattering. Rates of fast diffusive motions were found to be significantly enhanced with increasing hydration from fully hydrated powder to concentrated Hb solution. In concentrated protein solution, the data showed that amino acid side chains can explore larger volumes above body temperature than expected from normal temperature dependence. The body temperature transition in protein dynamics was absent in fully hydrated powder, indicating that picosecond protein dynamics responsible for the transition is activated only at a sufficient level of hydration. A collateral result from the study is that fully hydrated protein powder samples do not accurately describe all aspects of protein picosecond dynamics that might be necessary for biological function.

Hemoglobin senses body temperature.

UNLABELLED: When aspirating human red blood cells (RBCs) into 1.3 mum pipettes (DeltaP = -2.3 kPa), a transition from blocking the pipette below a critical temperature T(c) = 36.3 +/- 0.3 degrees C to passing it above the T(c) occurred (micropipette passage transition). With a 1.1 mum pipette no passage was seen which enabled RBC volume measurements also above T(c). With increasing temperature RBCs lost volume significantly faster below than above a T(c) = 36.4 +/- 0.7 (volume transition). Colloid osmotic pressure (COP) measurements of RBCs in autologous plasma (25 degrees C < or = T < or = 39.5 degrees C) showed a T (c) at 37.1 +/- 0.2 degrees C above which the COP rapidly decreased (COP transition). In NMR T(1)-relaxation time measurements, the T(1) of RBCs in autologous plasma changed from a linear (r = 0.99) increment below T(c) = 37 +/- 1 degrees C at a rate of 0.023 s/K into zero slope above T(c) (RBC T(1) transition). IN CONCLUSION: An amorphous hemoglobin-water gel formed in the spherical trail, the residual partial sphere of the aspirated RBC. At T(c), a sudden fluidization of the gel occurs. All changes mentioned above happen at a distinct T(c) close to body temperature. The T(c) is moved +0.8 degrees C to higher temperatures when a D(2)O buffer is used. We suggest a mechanism similar to a "glass transition" or a "colloidal phase transition". At T(c), the stabilizing Hb bound water molecules reach a threshold number enabling a partial Hb unfolding. Thus, Hb senses body temperature which must be inscribed in the primary structure of hemoglobin and possibly other proteins.

Hemoglobin dynamics in red blood cells: correlation to body temperature.

A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9 degrees C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature.

Molecular processes in biological thermosensation.

Since thermal gradients are almost everywhere, thermosensation could represent one of the oldest sensory transduction processes that evolved in organisms. There are many examples of temperature changes affecting the physiology of living cells. Almost all classes of biological macromolecules in a cell (nucleic acids, lipids, proteins) can present a target of the temperature-related stimuli. This review discusses some features of different classes of temperature-sensing molecules as well as molecular and biological processes that involve thermosensation. Biochemical, structural, and thermodynamic approaches are applied in the paper to organize the existing knowledge on molecular mechanisms of thermosensation. Special attention is paid to the fact that thermosensitive function cannot be assigned to any particular functional group or spatial structure but is rather of universal nature. For instance, the complex of thermodynamic, structural, and functional features of hemoglobin family proteins suggests their possible accessory role as "molecular thermometers".

Decrease in extracellular collagen crosslinking after NMR magnetic field application in skin fibroblasts.

Although biological effects of electromagnetic fields were investigated intensively, there is still no agreement on the significance of their effects. The underlying mechanisms and therapeutic importance are still mostly unknown too. In this study, primary cultures of human dermal fibroblasts were exposed to magnetic field at nuclear magnetic resonance (NMR) conditions for in total 5 days and 4 h/day. Among the investigated parameters were: cell proliferation rate, cell morphology, total protein concentration as well as content of skin-specific collagen types I, III, IV. NMR exposure induced distinct changes both in cellular and extracellular components. The extracellular matrix (ECM) of NMR-exposed cells had less cross-linked collagen. In particular, the increase of collagen of the soluble fraction was at 17.2 +/- 2.9% for type I, 27.0 +/- 1.86% for type III, 17.3 +/- 1.46% for type IV (N = 6). In the absence of resonance frequency, the effects of magnetic field on ECM were less profound.

Body temperature-related structural transitions of monotremal and human hemoglobin.

In this study, temperature-related structural changes were investigated in human, duck-billed platypus (Ornithorhynchus anatinus, body temperature T(b) = 31-33 degrees C), and echidna (Tachyglossus aculeatus, body temperature T(b) = 32-33 degrees C) hemoglobin using circular dichroism spectroscopy and dynamic light scattering. The average hydrodynamic radius (R(h)) and fractional (normalized) change in the ellipticity (F(obs)) at 222 +/- 2 nm of hemoglobin were measured. The temperature was varied stepwise from 25 degrees C to 45 degrees C. The existence of a structural transition of human hemoglobin at the critical temperature T(c) between 36-37 degrees C was previously shown by micropipette aspiration experiments, viscosimetry, and circular dichroism spectroscopy. Based on light-scattering measurements, this study proves the onset of molecular aggregation at T(c). In two different monotremal hemoglobins (echidna and platypus), the critical transition temperatures were found between 32-33 degrees C, which are close to the species' body temperature T(b). The data suggest that the correlation of the structural transition's critical temperature T(c) and the species' body temperature T(b) is not mere coincidence but, instead, is a more widespread structural phenomenon possibly including many other proteins.

NMR in vitro effects on proliferation, apoptosis, and viability of human chondrocytes and osteoblasts.

This study presents findings on the proliferation rate, cellular apoptosis, and viability of human chondrocyte and osteoblast cultures before and after treatment with NMR pulse sequences. A commercially available nuclear magnetic resonance machine (MBST(R)-Nuclear Magnetic Resonance Therapy) was used for treatment. The study was carried out for 19 days, including 9 days of NMR exposure in a controlled, double-blind, randomized manner, using commercially available human cell lines. The study revealed that NMR treatment did not induce apoptosis or inhibit cell viability, but revealed a tendency of an elevated cell proliferation rate as observed by cell count.

Bactericidal effects of plasma-generated cluster ions.

Air purification by plasma-generated cluster ions (PCIs) relies on a novel technology producing hydrated positive and negative ions. Phenomenological tests have shown strong evidence of lethal effects of the PCIs on various micro-organisms. However, the mechanisms of PCI action are still widely unknown. The aim was thus to test the bactericidal efficacy of PCI technology on common indoor micro-organisms and to explore possible PCI mechanisms of action. According to time/dose-dependent experiments with Staphylococcus, Enterococcus, Micrococcus and Bacillus, the inhibiting effects became apparent within the first few minutes of PCI exposure and led to an irreversible 99.9% destruction within the following 2-8 h of exposure. The destructive effect of the PCIs corresponded to membrane damage of the bacteria. Use of the techniques of both SDS PAGE and 2D PAGE revealed changes in the bacterial surface protein composition induced by the PCIs. In contrast, neither DNA nor cytoplasm protein damage was detected electrophoretically. The antimicrobial action of the PCIs seems to occur because of chemical modification of the surface proteins of bacteria. In situ hydroxyl radical formation on the surface of bacteria was proposed as the leading mechanism of the protein damage caused by the PCIs. At the same time, DNA damage seems not to be involved in the antibacterial action of the PCIs. The data obtained would broaden the knowledge concerning the antibacterial effects of air-born plasma-generated cluster ions and help to produce more efficient air-cleaning devices.

Evaluation of lateral mechanical tension in thin-film tissue constructs.

Fibroblast-populated collagen matrices provide a simplified tissue model for wound healing and development processes. A technology (CELLDRUM Technology) evaluating lateral mechanical tension in fibroblast-populated collagen matrices (tissue constructs) with a thickness of 1 mm was introduced. Defined mechanical boundary conditions together with the known number and orientation of the cells revealed precise data on the average tension exerted by a single cell. Circular cell-populated collagen gels were manufactured inside the CELLDRUM on top of a flexible membrane. The collagen matrix was then excited by a sound pulse. The resulting resonance oscillation was monitored by a laser-based deflection sensor and frequency and damping were analyzed giving information on mechanical properties of the tissue construct. Several evaluation experiments were performed. Calf serum enhanced contractile forces of fibroblasts dose dependently. After the gels were treated with cytochalasin D for 24 h, the cell forces were reduced by 42% of control. The remaining tension was attributed to the extracellular matrix remodeling occurring during cell growth and to other cytoskeletal structures like microtubules and intermediate filaments. We also found that only after a few hours of culture fibroblast-seeded collagen gels began developing significant mechanical tension. A mechanical tension profile of proliferating fibroblasts in collagen gels over culture time was obtained.

Biological and mechanical quality of red blood cells cultured from human umbilical cord blood stem cells.

Human umbilical cord blood (CB) has moved from the status of biological waste to that of a valuable source of haematopoietic stem (HS) cells. There are potentially three major clinical applications for HS cells and ex vivo-expanded HS cells: reconstitution of haematopoiesis in patients undergoing chemotherapy; gene therapy (e.g. in thalassaemia, sickle cell anaemia); and large-scale production of mature blood cells. Erythropoiesis is accomplished by highly complex interactions of haematopoietic progenitor cells, stromal cells and cytokines in the bone marrow. Among them, erythropoietin is the principal regulator. Ex vivo cell culture experiments to obtain mature red blood cells were the focus of this study. Attempts to elucidate appropriate medium components and amounts of haematopoietic growth factors were successful: enucleated and haemoglobin-filled erythroid cells were obtained from primitive HS cells. Dimethylsulphoxide (DMSO) was found to be of particular importance as an efficient differentiation inducer. The differentiation process was followed microscopically and by fluorescence-activated cell sorting (FACS). Using the micropipette aspiration technique, the elastic properties of erythroid cells were evaluated as erythropoiesis progressed. Discocyte-like cells, comprising reticulocytes and finally differentiated red blood cells, showed an about ten-fold higher membrane shear modulus compared with control cells.

Determination of the elastic shear modulus of cultured human red blood cells.

In this study we investigated the mechanical properties of in vitro cultured red blood cells (RBCs) in a liquid system. We used human umbilical cord blood as a highly efficient source of hematopoietic stem cells (HS). Our first goal was to establish an optimal medium composition in order to yield finally differentiated RBCs, i.e. enucleated and hemoglobin-filled cells. Different stages of cell differentiation were distinguished based on morphological observations and flow cytometry measurements. By means of the micropipette aspiration technique we estimated the deformability characteristics of the cultured cells. Up to the stage of oxiphilic normoblasts they readily deformed. Reticulocytes and mature RBCs showed an enhanced stiffness as compared to RBCs obtained from donors.

A novel method to quantify mechanical tension in cell monolayers.

A new technology to analyze mechanical properties of adherent cell monolayers grown on elastic silicon membranes is introduced. Measurements were performed using 3T3 (NIH) fibroblasts under the influence of Cytochalasin D and Thrombin. The stress-strain relation of the cell monolayer-silicon-composite was monitored. The drum-like construct of the culture chamber opens new roads for studying the mechanics of cell monolayers and of ultra flat tissue constructs. Steady state as well dynamic mechanical studies can be performed. Defined mechanical boundary conditions together with the known number and orientation of the cells allow precise information on the average tension exerted by a single cell within the monolayer.

Evidence for a second valve system in lymphatics: endothelial microvalves.

The mechanism for interstitial fluid uptake into the lymphatics remains speculative and unresolved. A system of intralymphatic valves exists that prevents reflow along the length of the lymphatic channels. However, these valves are not sufficient to provide unidirectional flow at the level of the initial lymphatics. We investigate here the hypothesis that initial lymphatics have a second, separate valve system that permits fluid to enter from the interstitium into the initial lymph channels but prevents escape back out into the tissue. The transport of fluorescent microspheres (0.31 microm) across endothelium of initial lymphatics in rat cremaster muscle was investigated with micropipette manipulation techniques. The results indicate that microspheres can readily pass from the interstitium across the endothelium into the lumen of the initial lymphatics. Once inside the lymphatic lumen, the microspheres cannot be forced out of the lumen even after elevation of the lymphatic pressure by outflow obstruction. Reaspiration of the microspheres inside the lymphatic lumen with a micropipette is blocked by the lymphatic endothelium. This blockade exists whether the aspiration is carried out at the microsphere entry site or anywhere along the initial lymphatics. Nevertheless, puncture of the initial lymphatic endothelium with the micropipette leads to rapid aspiration of intralymphatic microspheres. Investigation of lymphatic endothelial sections fixed during lymph pumping shows open interendothelial junctions not found in resting initial lymphatics. These results suggest that initial lymphatics have a (primary) valve system at the level of the endothelium. In conjunction with the classical (secondary) intralymphatic valves, the primary valves provide the mechanism that facilitates the unidirectional flow during periodic compression and expansion of initial lymphatics.

Temperature transition of human hemoglobin at body temperature: effects of calcium.

We studied the effects of calcium ion concentration on the temperature dependence of rheological behavior of human red blood cells (RBCs) and concentrated hemoglobin solutions. Our previous study (G. M. Artmann, C. Kelemen, D. Porst, G. Büldt, and S. Chien, 1998, Biophys. J., 75:3179-3183) showed a critical temperature (Tc) of 36.4 +/- 0.3 degrees C at which the RBCs underwent a transition from non-passage to passage through 1.3 microm micropipettes in response to an aspiration pressure of -2.3 kPa. An increase in intracellular Ca2+ concentration by using the ionophore A23187 reduced the passability of intact RBCs through small micropipettes above T(c); the micropipette diameter needed for >90% passage increased to 1.7 microm. Viscometry of concentrated hemoglobin solutions (45 and 50 g/dl) showed a sudden viscosity transition at 36 +/- 1 degrees C (Tc(eta)) at all calcium concentrations investigated. Below Tc(eta), the viscosity value of the concentrated hemoglobin solution at 1.8 mM Ca(2+) was higher than that at other concentrations (0.2 microM, 9 mM, and 18 mM). Above Tc(eta), the viscosity was almost Ca2+ independent. At 1.8 mM Ca2+ and 36 +/- 1 degrees C, the activation energy calculated from the viscometry data showed a strong dependence on the hemoglobin concentration. We propose that the transition of rheological behavior is attributable to a high-to-low viscosity transition mediated by a partial release of the hemoglobin-bound water.

Hypochlorous acid and human blood low density lipoproteins modified by hypochlorous acid increase erythrocyte adhesion to endothelial cells.

The ability of hypochlorous acid (HOCl) (anion form - hypochlorite, OCl-) and HOCl/OCl- -modified human blood low density lipoproteins (HOCl-LDLs) to stimulate erythrocyte adhesion to endothelial cell monolayers was studied. LDLs were modified by incubating at different HOCl/OC- concentrations. This led to a damage of proteins and lipids. We found (1) a more than 20-fold decrease of LDL fluorescence intensity (extinction at 285 nm, emission at 340 nm), (2) accumulation of secondary (TBA-reactive substances) and final (Schiff bases) products of lipid peroxidation, and (3) increase in the electrophoretic mobility of LDLs. Preincubation of endothelial cells (ECs) with HOCI/OCl- (up to 50 microM) enhanced erythrocyte adhesion to the EC monolayer. Preincubation of ECs with HOCl-LDLs (up to 250 microM of HOCI//OCl- during LDL modification) (1) caused an increase in the cholesterol/phospholipid molar ratio in EC and (2) enhanced adhesion of erythrocytes to endothelium. Application of HOCl/OCl- at concentrations above 50 microM or treatment of LDLs with 500 microM HOCl resulted in the cytotoxic effect on ECs and led to a decrease in the molar cholesterol/phospholipid ratio in ECs and adhesion of erythrocytes to endothelium. The results suggest that HOCl/OCl- at physiological concentrations stimulates the adhesion of blood cells to the endothelium and cholesterol accumulation in the vessel wall ECs either directly or due to LDL modification. Both effects could be important in the development of many vascular diseases.

Chlorpromazine modulates the morphological macro- and microstructure of endothelial cells.

Chlorpromazine (CP), an amphipathic, antipsychotic agent, causes concave membrane bending in red blood cells with formation of stomatocytic shapes by modulation of the phospholipid bilayer. This study was designed to investigate the effects of CP on the shape of bovine aortic endothelial cells (BAEC) and their membranes in confluent monolayers with phase-contrast and transmission electron microscopy. Exposure of BAECs to nanomolar levels of CP leads to membrane curvature changes. With increasing CP concentrations, the membrane assumed a shape with enhanced numbers of intracellular caveolae and projection of pseudopodia at all junctions. At higher CP concentrations (up to 150 microM), the endothelial cells assumed almost spherical shapes. The evidence suggests that CP may affect lipid bilayer bending of BAECs in analogy with previous observations on erythrocytes, supporting the formation of caveolae and pseudopodia in BAECs due to the induction of concave membrane bending, as well as an effect on endothelial cell membrane adhesion at higher CP concentrations with loss of cellular attachment at junctions.

Temperature transitions of protein properties in human red blood cells.

Human red blood cells (RBC) undergo a sudden change from blocking to passing through 1.3 +/- 0.2-micrometer micropipettes at a transition temperature (Tc) of 36.4 degrees C. For resealed RBC ghosts this transition occurs at 28.3 degrees C (Tg). These findings are attributed to an elastomeric transition of hemoglobin from being gel-like to a fluid and to an elastomeric transition of membrane proteins such as spectrin. Spectrin shows a uniform distribution along the aspirated RBC tongue above Tg in contrast to the linear gradient below Tg.