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R Rolandi

Publications and source records attributed to R Rolandi.

17 recordsLinked to original sources
PubMed1998

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.

Aging↗
PubMed1995

Structure of rat tail tendon collagen examined by atomic force microscope.

The Atomic Force Microscope (AFM) was used to inspect collagen fibrils deposited on mica sheets at different fibrillogenesis times. Collagen was obtained from rat tail tendon fibers. Various fibril forms were observed, together with the characteristic periodic intra-fibril structure (D-bands). The fibril thickness, width, D-band periodicity and depth were measured and the statistical distribution of these parameters at 1, 2, 5, 10 and 15 days of in vitro fibril formation time was calculated. The fibrils showed an increasing size with time, but the band interval measure remained stable. The band depth, after an initial increase, exhibited a relative steadiness. The results indicate that AFM offers, at low resolution, images qualitatively similar to those obtained with electron microscopy, but with less manipulation of the sample. A quantitative evaluation of collagen structural features in the nanometer scale is made possible by AFM.

Animals↗
PubMed1995

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.

Aging↗
PubMed1992

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.

Adult↗
PubMed1991

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.

Aging↗
PubMed1990

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.

Aging↗
PubMed1985

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.

Adrenal Gland Neoplasms↗
PubMed1983

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.

Archaea↗
PubMed1982

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.

Archaea↗
PubMed1977

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.

Animals↗
PubMed1975

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.

Ammonia↗
PubMedDate not supplied

Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation.

BACKGROUND: The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation. METHODS: Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL¿BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL¿WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. RESULTS: Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modified by non-enzymatic glycation. CONCLUSION: This is the first description obtained with SFM of diabetes-induced ultrastructural changes in collagen fibrils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modifications in collagen fibrils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification.

Animals↗
PubMedDate not supplied

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.

Adsorption↗