Gerhard Michael Artmann

True date palms (Phoenix dactylifera L.) are impressive trees and have served as an indispensable source of food for mankind in tropical and subtropical countries for centuries. The aim of this study is to differentiate date palm tree varieties by analysing leaflet cross sections with technical/optical methods and artificial neural networks (ANN).

Regulating protein function in the cell by small molecules, provide a rapid, reversible and tunable tool of metabolic control. However, due to its complexity the issue is poorly studied so far. The effects of small solutes on protein behavior can be studied by examining changes of protein secondary structure, in its hydrodynamic radius as well as its thermal aggregation. The study aim was to investigate effects of adenosine-5'-triphosphate (ATP), spermine NONOate (NO donor) as well as sodium/potassium ions on thermal aggregation of albumin and hemoglobin. To follow aggregation of the proteins, their diffusion coefficients were measured by quasi-elastic light scattering (QELS) at constant pH (7.4) in the presence of solutes over a temperature range from 25°C to 80°C. 1) Spermine NONOate persistently decreased the hemoglobin aggregation temperature Tairrespectively of the Na+/K+ environment, 2) ATP alone had no effect on the protein's thermal stability but it facilitated protein's destabilization in the presence of spermine NONOate and 3) mutual effects of ATP and NO were strongly influenced by particular buffer ionic compositions. The ATP effect on protein aggregation was ambiguous: ATP alone had no effect on the protein's thermal stability but it facilitated protein's destabilization in the presence of nitric oxide. The magnitude and direction of the observed effects strongly depended on concentrations of K+ and Na+ in the solution.

Minor changes in protein structure induced by small organic and inorganic molecules can result in significant metabolic effects. The effects can be even more profound if the molecular players are chemically active and present in the cell in considerable amounts. The aim of our study was to investigate effects of a nitric oxide donor (spermine NONOate), ATP and sodium/potassium environment on the dynamics of thermal unfolding of human hemoglobin (Hb). The effect of these molecules was examined by means of circular dichroism spectrometry (CD) in the temperature range between 25°C and 70°C. The alpha-helical content of buffered hemoglobin samples (0.1 mg/ml) was estimated via ellipticity change measurements at a heating rate of 1°C/min.

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-μm 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 × 10⁴ cells/ml), a mechanical tension of 1.62 ± 0.17 μN/mm². 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.

When aspirating human red blood cells (RBCs) into 1.3 μm pipettes (ΔP = −2.3 kPa), a transition from blocking the pipette below a critical temperature T _c = 36.3 ± 0.3°C to passing it above the T _c occurred (micropipette passage transition). With a 1.1 μm 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°C ≤ T ≤ 39.5°C) showed a T _c at 37.1 ± 0.2°C above which the COP rapidly decreased (COP transition). In NMR T₁-relaxation time measurements, the T₁ of RBCs in autologous plasma changed from a linear (r = 0.99) increment below T _c = 37 ± 1°C at a rate of 0.023 s/K into zero slope above T _c (RBC T₁ 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°C to higher temperatures when a D₂O 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.

Human red blood cells (RBCs) exhibit sudden changes in their biophysical properties at body temperature (T _B). RBCs were seen to undergo a spontaneous transition from blockage to passage at T _C = 36.4 ± 0.3°C, when the temperature dependency of RBC-passages through 1.3 μm narrow micropipettes was observed. Moreover, concentrated hemoglobin solutions (45 g/dl) showed a viscosity breakdown between 36 and 37°C. With human hemoglobin, a structural transition was observed at T _B as circular dichroism (CD) experiments revealed. This leads to the assumption that a species’ body temperature occupies a unique position on the temperature scale and may even be imprinted in the structure of certain proteins. In this study, it was investigated whether hemoglobins of species with a T _B different from those of human show temperature transitions and whether those were also linked to the species’ T _B. The main conclusion was drawn from dynamic light scattering (DLS) and CD experiments. It was observed that such structural temperature transitions did occur in hemoglobins from all studied species and were correlated linearly (slope 0.81, r = 0.95) with the species’ body temperature. We presumed that α-helices of hemoglobin were able to unfold more readily around T _B. α-helical unfolding would initiate molecular aggregation causing RBC passage and viscosity breakdown as mentioned above. Thus, structural molecular changes of hemoglobin could determine biophysical effects visible on a macroscopic scale. It is hypothesized that the species’ body temperature was imprinted into the structure of hemoglobins.

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.

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.

Intact red blood cells (RBCs) undergo a sudden change from blocking to passing through 1.3 /spl mu/m-micropipettes at a transition temperature (T/sub c/) of 36.4/spl deg/C and -2.3 kPa aspiration pressure. This was attributed to a change of the physical state of hemoglobin. Since Ca/sup 2+/-ions induce conformational changes of proteins, we studied how elevated intracellular Ca/sup 2+/ would effect the RBC passage through micropipettes and T/sub c/. Transitions occurred in A23187-treated RBCs at 1 mM extraneous Ca/sup 2+/ as well. Surprisingly, T/sub c/ remained at 36.4/spl deg/C. However, higher aspiration forces (pipette diameter: 1.7 /spl mu/m) were needed for passages. To look at hemoglobin only, low shear viscosity measurements of hemoglobin solutions at 50 g/dl were carried out as a function of temperature and different Ca concentrations (1.8, 9 and 18 mM). When raising the temperature, a sudden viscosity drop occurred between 35/spl deg/C-38/spl deg/C. However, the absolute difference of viscosities at 35/spl deg/C compared to 38/spl deg/C was lower at 9 mM and had almost disappeared at 18 mM. Furthermore, higher Ca/sup 2+/-levels led to lower viscosities at all temperatures investigated. This may be due to the binding of calcium to hemoglobin molecules, which in turn may have weakened intermolecular bonds leading to a fluidization of hemoglobin solutions at high calcium levels.

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 μM), 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.