Morphology, mechanical properties and viability of encapsulated cells.

The morphological and mechanical properties of encapsulated yeast cells (Saccharomyces cerevisiae) have been investigated by atomic force microscopy (AFM). Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte (PE) layers. The properties of cells coated by different numbers of PE layers and from PE solutions of different ionic strength have been investigated. AFM imaging indicates an increase in PE coating stability when decreasing the solution ionic strength. The Young's moduli of the different examined systems have been evaluated through a quantitative analysis of force-distance curves by using the Hertz-Sneddon model. The analysis indicates an increase in hybrid system stiffness when lowering the ionic strength of the PE solution. An evaluation of the viability of encapsulated cells was obtained by confocal laser scanning microscopy (CLSM) measurements. CLSM analysis indicates that cells preserve their subcellular structure and duplication capability after encapsulation. By coupling AFM and CLSM data, a correlation between local stiffness and duplication rate was obtained.

Incorporation into lipid bilayer membranes of a photo--sensitive pigment from the honeybee compound eye.

An increase of electrical conductance up to a factor 10(2)--5-10(2) was obtained by adding, in the dark, the honeybee photopigment to a positively charged lipid bilayer. The increase in conductance was made slower by illuminating the system during the incorporation of the protein into the membrane and it was negligible when the photopigment was bleached before the incorporation. The interaction of the photopigment with the membrane is tentatively interpreted in terms of formation of channels.

Prevention of diabetes-increased aging effect on rat collagen-linked fluorescence by aminoguanidine and rutin.

Products from the advanced Maillard reaction, which increase during aging and diabetes, may contribute to the development of the typical pathology of aging and diabetes. These compounds are detectable only by their characteristic fluorescence, and few data based on long-term studies are available. For this reason, we studied subcutaneous skin collagen fluorescence in 57 nondiabetic (10- to 110-wk-old) and 74 streptozocin-induced diabetic (10- to 22-wk-old) rats. An exponential increase (r = 0.969, P less than 0.001) of collagen-linked fluorescence (excitation at 370 nm, emission at 440 nm) was observed with aging; after a lag, diabetes induced an earlier dramatic elevation of the fluorescence, suggesting a more complicated phenomenon than simple accumulation. To prevent such increases, the effects of 1 g.kg-1.day-1 aminoguanidine, suggested to be an inhibitor of the advanced glycosylation reaction, and 1 g.kg-1.day-1 rutin, an aldose reductase inhibitor, in drinking water were tested. Both treatments had a significant lowering effect on collagen fluorescence in diabetic rats. The mechanisms by which aminoguanidine and rutin prevent the accumulation of fluorescence are unknown, but these observations raise the question of whether they could be identical. If fluorescence is a marker for age-related pathologies and diabetic sequelae, aminoguanidine and rutin could have therapeutic effects in their prevention.

Age-related increase of collagen fluorescence in human subcutaneous tissue.

Nonenzymatic glycation produces compounds with characteristic fluorescence in long-lived proteins. We recently described the influence of age in rat collagen-linked fluorescence. To examine the effect of age in humans, we studied the subcutaneous collagen-linked fluorescence in samples from 26 subjects of both sexes (age range, 42 to 78 years) who were undergoing vascular surgery. Intensity of fluorescence at 385 nm (upon excitation at 335 nm) and 440 nm (upon excitation at 370 nm) increased exponentially with age (r = .827, y = 114 + e0.038x, P less than .001; and r = .905, y = 36 + e0.039x, P less than 0.001, respectively). The two sets of data exhibited a high degree of correlation (r = .980, P less than .001, n = 26). Age-adjusted fluorescence data did not correlate with sex, body weight, or type of vascular pathology. The collagen fluorescence accumulation rate was 3.7% per year, and the characteristic time (CT) was 26 to 27 years. We conclude that the fluorescence measurement is a reliable methodology that can be used as a marker for biological age until new, more-specific tools are available.

Self-assembled monolayers of organosulphur molecules bearing calix[4]arene moieties.

We investigate the self-assembly of modified calix[4]arene on gold surfaces. Calix[4]arene was modified through a reaction sequence which led to assembling of the crown-5 moiety and to the insertion of two thioether groups into the starting molecule. The so-obtained calix[4]arene-crown-5 bis(7-thiatridecyloxy) (hereafter called calix[4]arene) was in the stable 1,3-alternate conformation. The calix[4]arene/gold interface was investigated by means of spectroscopic ellipsometry (SE), scanning tunneling microscopy (STM) and cyclic voltammetry (CV). SE data indicate a layer thickness compatible with the formation of a monomolecular layer. This result is confirmed by STM imaging which shows the formation of a high density of small pits, one gold layer deep, a typical feature of self-assembled organosulphur monolayers on gold. CV measurements performed in presence of the [Ru(NH(3))(6)(2+/3+)] redox couple indicate a passivation of the metal electrode, resulting in a reduction of the redox current, after the layer deposition. CV has also been used to investigate the selectivity properties of calix[4]arene-covered gold electrodes by measuring the redox current decrease in the presence of different salt solutions. It is found that calix[4]arene-covered electrodes are able to complex K(+) and Ba(2+), while no complexation is observed in the case of Li(+), Na(+), Cs(+), Mg(2+) and Ca(2+).

Microtopography of the eye surface of the crab Carcinus maenas: an atomic force microscope study suggesting a possible antifouling potential.

Marine biofouling causes problems for technologies based on the sea, including ships, power plants and marine sensors. Several antifouling techniques have been applied to marine sensors, but most of these methodologies are environmentally unfriendly or ineffective. Bioinspiration, seeking guidance from natural solutions, is a promising approach to antifouling. Here, the eye of the green crab Carcinus maenas was regarded as a marine sensor model and its surface characterized by means of atomic force microscopy. Engineered surface micro- and nanotopography is a new mechanism found to limit biofouling, promising an effective solution with much reduced environmental impact. Besides giving a new insight into the morphology of C. maenas eye and its characterization, our study indicates that the eye surface probably has antifouling/fouling-release potential. Furthermore, the topographical features of the surface may influence the wettability properties of the structure and its interaction with organic molecules. Results indicate that the eye surface micro- and nanotopography may lead to bioinspired solutions to antifouling protection.

A thermodynamic and structural study of myelin basic protein in lipid membrane models.

Myelin basic protein (MBP) is a major protein of the myelin membrane in the central nervous system. It is believed to play a relevant role in the structure and function of the myelin sheath and is a candidate autoantigen in demyelinating processes such as multiple sclerosis. MBP has many features typical of soluble proteins but is capable of strongly interacting with lipids, probably via a conformation change. Its structure in the lipid membrane as well as the details of its interaction with the lipid membrane are still to be resolved. In this article we study the interaction of MBP with Langmuir films of anionic and neutral phospholipids, used as experimental models of the lipid membrane. By analyzing the equilibrium surface pressure/area isotherms of these films, we measured the protein partition coefficient between the aqueous solution and the lipid membrane, the mixing ratio between protein and lipid, and the area of the protein molecules inserted in the lipid film. The penetration depth of MBP in the lipid monolayer was evaluated by x-ray reflectivity measurements. The mixing ratio and the MBP molecular area decrease as the surface pressure increases, and at high surface pressure the protein is preferentially located at the lipid/water interface for both anionic and neutral lipids. The morphology of MBP adsorbed on lipid films was studied by atomic force microscopy. MBP forms bean-like structures and induces a lateral compaction of the lipid surface. Scattered MBP particles have also been observed. These particles, which are 2.35-nm high, 4.7-nm wide, and 13.3-nm long, could be formed by protein-lipid complexes. On the basis of their size, they could also be either single MBP molecules or pairs of c-shaped interpenetrating molecules.

Correlation of collagen-linked fluorescence and tendon fiber breaking time.

We review in this paper some reported data on age-dependent modification of proteins pointing out the relationship between the increase of nonenzymatic glycation in abdominal skin collagen of Wistar rats, evaluated by fluorescence intensity, and tendon breaking time, used as a parameter of collagen stiffness. Fluorescence intensity data linearly correlate with the breaking times of collagen fibers from Wistar rats reported from different sources, according to the hypothesis of a common etiological mechanism. It is possible to suppose that posttranslational modifications of proteins play a role in the tissue aging and their level in collagen may be used as a parameter for quantitation.

Study of aging rat tail collagen using atomic force microscope.

Collagen structure of young and old rats was examined by using atomic force microscope (AFM) images. Rat tail tendons of eight and twenty-four month-old Wistar rats were digested enzymatically (pepsin), and allowed to refibrillate for 24 hours at 37 degrees C. The samples were examined using a Nanoscope III (Digital Instruments, Santa Barbara, CA, U.S.A.) with a J scanning head and a 200 microns silicon nitride cantilever. The study was performed in air and without filters. The AFM inspection of refibrillated collagen produced images showing long fibrils with relatively homogeneous heights and widths, characterized by clear banding with a periodic interval (D band) of 67 nm. With respect to collagen extracted from young rats, collagen extracted from old rats revealed fibrils exhibiting the same band interval, but with lower widths and heights. Furthermore, the depth of gap between two overlaps showed a higher mean value in the aged rats. These data are consistent with biochemical reports of collagen modifications during aging; we suggest that post-synthetic reactions might be responsible for this as they interfere with the refibrillation process and also modify the three-dimensional structure of fibrils.

Monolayer black membranes from bipolar lipids of archaebacteria and their temperature-induced structural changes.

The membrane of Caldariella acidophila, an extreme thermophilic archaebacterium, is characterized by unusual bipolar complex lipids. They consist of two nonequivalent polar heads, linked by a C40 alkylic component. The molecular organization of these lipids in the plasma membrane is still a matter of study. In this paper, we present current-voltage measurements on artificial bipolar lipid membranes, indicating that molecules are indeed organized as a covalently bound bilayer, in which each molecule is completely stretched and spans its entire thickness. Furthermore, conformational transitions of these artificial membranes (which could be formed only above 70 degrees C from a lipid/squalene dispersion) are analyzed in the 80 to 15 degrees C temperature range. Abrupt variations in capacitance and valinomycin-induced conductance seem to indicate the occurrence of at least two structural changes. Measurements are also extended to different solvent systems. Results are consistent with the picture of a monolayer bipolar lipid membrane in which few solvent molecules align themselves parallel to the lipophilic chains. The amount of solvent as well as the temperature at which conformational transitions occur, depend on the solvent system in which the lipid is dispersed.

Effects of unstirred layers on the steady-state zero-current conductance of bilayer membranes mediated by neutral carriers of ions.

Some effects of diffusion polarization and chemical reactions on the steady-state zero-current conductance of lipid bilayers mediated by neutral carriers of ions have been studied theoretically and experimentally. Assuming that ion permeation across the interfaces occurs via a heterogeneous reaction between ions in the solution and carriers in the membrane, the relationship between the conductance and the aqueous concentration of carriers is shown to be linear only in a limited range of sufficiently low concentrations. At higher carrier concentrations, which for the most strongly bound cations are within the range of the experimentally accessible values, the conductance is expected to become limited by diffusion of the carried ion in the unstirred layers and therefore reach an upper limiting value independent of the membrane properties. This expectation has been successfully verified for glyceryl-monooleate membranes in the presence of the ions K+, Rb+ and NH+4 and carriers such as valinomycin and trinactin. The experimental results support, at least for the present system, the generally accepted view that complexation between ions and the macrocyclic antibiotics occurs at the membrane surface; it is shown, in fact, that for a different mechanism, such as that by which the complexes would form in the aqueous solutions and cross the interfaces as lipid-soluble ions, the same type of saturation would be expected to be observable only for unrealistically high values of the rate constants of the ion-carrier association. A previously proposed criterion to distinguish between these two mechanisms, based on the dependence of the conductance on the ion concentration, is discussed from the viewpoint of this more comprehensive model.

Artificial black membranes from bipolar lipids of thermophilic Archaebacteria.

The membrane of thermophilic archaebacteria is characterized by the presence of unusual isoprenoid bipolar lipids. The molecular organization of these lipids is still a matter of study. Important information could come from forming artificial black membranes. Black films can be formed from n-alkane or squalene dispersions of bipolar lipids extracted from the membrane of Caldariella acidophila. Membrane formation occurred only above a critical temperature (approximately 70 degrees C) corresponding to the physiological one. At lower temperatures, special solvent systems (n-alkanes or squalene, butanol and n-alkanes or squalene, butanol chloroform) were required. To characterize the physical parameters of these membranes, conductance and capacitance measurements were performed. Conductance was in the range of 10(-8) - 10(-7) omega -1 cm -2 , where specific capacitance at T = 72 degrees C was Cs = 0.685 +/- 0.004 microF/cm2 and Cs = 0.658 +/- 0.08 microF/cm2, corresponding to a dielectric thickness of 27 and 29 A for squalene and dodecane dispersions, respectively. Capacitance was shown to vary as the square of membrane potential, as usual in lipid bilayers. Values of the proportionality constant alpha have been compared to those of solvent-containing and solvent-free bilayers. The behavior of capacitance as a function of temperature is also shown by lowering temperature; the occurrence of complex structural changes was indicated. All the experimental data suggest that the presence of solvent is very low. Two possible molecular configurations of the films are discussed.

Measurements of surface roughness: use of a CCD camera to correlate doubly scattered speckle patterns.

We describe an instrument, built around a commercial CCD camera and some fast image-processing boards, that evaluates roughness height by measuring the average size of doubly scattered speckle patterns. The device is a variant of a recent proposal that was based on the use of a spatial modulator to perform the Fourier transform of a speckle image. In the present setup, the Fourier transform is replaced by the direct evaluation of a second-order correlation function. Strictly speaking, the device proposed in this paper is not a real-time device but its response time (approximately 10 s) is sufficiently short to be of practical value for many applications. Updated CCD cameras that will significantly improve the performance of our prototype are already on the market.

Leptinotoxin-h action in synaptosomes, neurosecretory cells, and artificial membranes: stimulation of ion fluxes.

Leptinotoxin-h (LPTx), a neurotoxin (otherwise designated beta-leptinotarsin-h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry 23, 734-741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+-containing Ringer medium, at concentrations in the 10(-11) - 10(-10) M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage-dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx; and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+-free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (approximately 10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono- and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of alpha-latrotoxin (alpha-LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit alpha-LTx binding.

Role of advanced glycation end products in aging collagen. A scanning force microscope study.

The collagen structure of young and old rats was examined using a scanning force microscope (SFM). Rat tail tendons of 8- and 24-month-old Wistar rats were frayed by two blades and examined using a Nanoscope III SFM. In the same tendons, the pentosidine concentrations, a marker of the Maillard reaction, were determined by HPLC. The SFM inspection of native fibrils produces images of collagen bundles, with parallel fibrils. The diameters of old rat collagen fibrils were large in comparison to the young ones. Moreover, fibrils obtained from old rats exhibited the same band interval, while the depth of the gap between two overlap zones showed a higher mean value with respect to young collagen. The pentosidine concentration was also higher in the old than in the young tendons. In conclusion, in the presence of an increased concentration of advanced glycation end products, significant structural alterations have been observed in old fibrils.

Adsorption of gamma-aminobutyric acid to phosphatidylserine membranes.

The interaction of the negatively-charged phosphatidylserine (PS) and gamma-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles. GABA does not permeate through PS membranes and, in concentrations of 10(-5)-10(-4) M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of 14C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.

An atomic force microscopy investigation of protein crystal surface topography.

Tapping mode atomic force microscopy was employed to study the surface structure of different protein crystals in a liquid environment. The (101) face of hen egg-white lysozyme crystals and the (111) face of horse spleen ferritin crystals were studied. On the (101) face of lysozyme crystals we observed islands delimitated by micro-steps and elongated in the [010] direction. The elongation direction coincides with the preferential growth direction predicted by a growth model reported in the literature. The islands observed on the ferritin (111) face are also delimitated by micro-steps but have circular symmetry. Sectioning of the images allowed us to measure the step heights. The surface free energy was estimated from the growth step morphology. Molecular resolution was achieved for ferritin crystals, showing a hexagonal surface packing, as expected for the molecular lattice of a (111) face in a fcc crystal.