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Biomedical subjects

E Bertoli

Publications and source records attributed to E Bertoli.

At least 19 recordsLinked to original sources
PubMed1992

Structural and functional relationships in DnaK and DnaK756 heat-shock proteins from Escherichia coli.

The secondary structures of DnaK and the mutant DnaK756 heat-shock proteins from Escherichia coli have been investigated by Fourier transform infrared spectroscopy. The analysis of infrared data showed that DnaK and DnaK756 proteins have different secondary structures that are not affected by the presence of ATP or beta, gamma-methyleneadenosine 5'-triphosphate. The infrared data indicate also that the tertiary structures of DnaK and DnaK756 proteins are different and that DnaK protein undergoes conformational changes in its tertiary structure not only during binding of ATP but also during ATP hydrolysis. Using fluorescence spectroscopy of a single tryptophan located in the N-terminal domain of DnaK protein and fluorescence of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which interacts with hydrophobic domains of DnaK protein, we were able to distinguish between two conformational states of DnaK protein. After binding of triphosphonucleotides, the C-terminal domain of DnaK protein changes in tertiary structure in such a way that fewer hydrophobic segments are exposed on the surface of the protein. After ATP hydrolysis, the number of hydrophobic segments on the surface of the protein is further reduced, and moreover the tertiary structure of the N-terminal domain of the protein changes. These data are discussed in terms of structural and functional relationships of both DnaK and DnaK756 proteins.

Adenosine Triphosphate↗
PubMed1992

Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study.

Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta-turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme.

5'-Nucleotidase↗
PubMed1992

Diabetes mellitus induces red blood cell plasma membrane alterations possibly affecting the aging process.

Various alterations of red blood cell (RBC) plasma membrane appear both in diabetes mellitus and during the physiological aging process. Diabetes mellitus decreases RBC life-span; therefore, it may change the plasma membrane by acting through its effect on the aging process. In order to clarify the issue, RBCs from normal subjects and insulin-dependent diabetic patients were fractionated in five subpopulations of different mean age (fraction 1: early young RBC, fraction 5: mature RBC). Thereafter, plasma membranes were prepared and enzymatic activities, membrane fluidity and lipid peroxidation were evaluated. NA+, K(+)-ATPase activity decreased during aging and it was higher in all RBC subpopulations from normal subjects in comparison to diabetic patients. Next, lipid peroxidation and fluidity increased during aging in both the study groups; in this case, however, in all subpopulations, except for that from fraction 1, RBCs from diabetic patients showed higher membrane fluidity and lipid peroxidation in comparison to normal subjects. Data herein reported suggest that diabetes mellitus affects the plasma membrane independently of (lipid peroxidation and fluidity) or dependently on (Na+, K(+)-ATPase) its effect on aging. In the case of lipid peroxidation and fluidity diabetes mellitus seems to affect the membrane by decreasing RBC life span, whereas in the case of Na+K(+)-ATPase it seems to alter this enzymatic activity which in turn might affect RBC aging. Acetylcholinesterase activity decreased during aging in RBCs from normal subjects, but it increased in RBCs from diabetic patients; RBC subpopulation from fraction 1, on the other hand, showed similar values in normal subjects and diabetic patients. In this case the effect of diabetes mellitus appears only during aging.

Acetylcholinesterase↗
PubMed1992

Lifetime determination of low sample concentrations: a new cuvette for highly accurate and sensitive fluorescence measurements.

A new cuvette was developed for spectrofluorometric measurements which increases the efficiency of recorded fluorescence emission by at least two-fold, decreasing, at the same time, the noise component of the record. It is suited for both steady-state and dynamic fluorescence measurements. Comparative data for both the classical and the novel cuvettes are presented here.

Data Interpretation, Statistical↗
PubMed1992

Biochemical characterization of density-separated trout erythrocytes.

1. A discontinuous gradient in the region of 45-65% "Percoll" has been utilized for the separation of trout erythrocytes. Three different fractions were obtained. 2. We have evaluated antioxidant enzyme activities and membrane fluidity. The results indicated that catalase and glutathione peroxidase activities increased with the density of the fraction while the membrane fluidity was unchanged. 3. The observed results show a marked difference between nucleated (fish) and unnucleated (human) separated erythrocytes.

Animals↗
PubMed1991

A new fluorescence method to detect singlet oxygen inside phospholipid model membranes.

A fluorescence method for detecting singlet oxygen (1O2) in model membranes is proposed. 1O2 was generated by hydrogen peroxide/sodium hypochlorite system. 1,3-Diphenylisobenzofuran (DPBF), a specific 1O2 trap, dissolved in organic solvents gives a strong fluorescence spectrum when excited at 410 nm. A similar spectrum, with a maximum at 455 nm, is obtained when DPBF is incorporated in unilamellar dipalmitoylphosphatidylcholine liposomes. The intensity of fluorescence spectrum decreases when DPBF-labeled liposomes are exposed to singlet oxygen. This decrease is sensitive to 1O2 traps and quenchers like tryptophan and sodium azide, to lipid membrane fluidity and to the concentration of sodium hypochlorite and hydrogen peroxide.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1991

Structural and functional aspects of the respiratory chain of synaptic and nonsynaptic mitochondria derived from selected brain regions.

Studies on brain mitochondria are complicated by the regional, cellular, and subcellular heterogeneity of the central nervous system. This study was performed using synaptic and nonsynaptic mitochondria obtained from cortex, hippocampus, and striatum of male Sprague-Dawley rats (3 months old). Ubiquinone content, detected by HPLC analysis, was about 1.5 nmol/mg protein with an approximate CoQ9/CoQ10 molecular ratio of 2:1. The activities of several respiratory chain complexes were also studied (succinate-cyt. c reductase, NADH-cyt. c reductase, succinate-DCIP, ubiquinol2-cyt. c reductase, and cytochrome oxidase), and generally found to be higher in mitochondria from cortex than from other regions. Study of the activities of some of these enzymes vs. 1/T (Arrhenius plots) showed a straight line with an activation energy between 7 and 10 kcal/mol in all the three areas considered. Only CoQ2H2-cyt. c reductase activity revealed a biphasic temperature dependence. Also anisotropy (as fluorescence polarization) of the hydrophobic probe DPH showed a deviation from linearity; the break points for both enzymatic activity and anisotropy were found at about 23-24 degrees C.

Animals↗
PubMed1991

Effect of the fungicides tributyltin acetate and tributyltin chloride on multilamellar liposomes: fluorescence studies.

The influence of tri-n-butyltin acetate (TBTA) and tri-n-butyltin chloride (TBTC) on the physico-chemical state of charged and neutral phospholipids was investigated using multilamellar liposomes. The thermal dependence of steady state fluorescence polarization of DPH and its charged derivative TMA-DPH was recorded. The two fungicides lowered DPPC phase transition temperature and broadened the temperature range of the transition in different ways. The effects were concentration-dependent. The results show that TBTC interacts more effectively with DPPC model membranes rather than TBTA. Moreover, TBTC broadens and shifts the main phase transition (Tm) more effectively in DPPC rather than in DMPC liposomes. Below Tm, TBTC decreases fluorescence polarization (P) in all phospholipids used. Above Tm P is almost constant in phospholipids with saturated acyl chains, except for DMPG. In fact, an increase of P is detectable in this lipid as in PLs with unsaturated acyl chains. It is suggested that the effects of TBT on liposomal membranes are dependent on the anion moiety and phospholipids characteristics.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1979

Uncoupling effect of a low homolog of unbiquinone (UQ-3) in rabbit heart mitochondria.

Short-chain ubiquinone (UQ-3) inhibits ADP-stimulated respiration (state 3) in intact rabbit heart mitochondria. This effect appears to be similar in all the three sites of oxidative phosphorylation by using different respiratory substrates. Ubiquinone-3 also immobilizes lipids into mitochondrial membranes indicating that the uncoupling effect might be a consequence of an altered physical state of membrane lipids.

Animals↗
PubMed1979

Effect of ubiquinone-homologs on the sensitivity of mitochondrial ATPase to energy transfer inhibitors.

Short-chain ubiquinone (UQ-3) abolishes oligomycin sensitivity of ATPase in submitochondrial particles and the effect is reversed by long-chain ubiquinone (UQ-7). Ubiquinone-3 also abolishes DCCD sensitivity of ATPase in submitochondrial particles but the effect is not reversed by long-chain ubiquinones. These data suggest that ubiquinone interferes with energy transfer process by interaction with mitochondrial ATPase.

Adenosine Triphosphatases↗
PubMed1979

Studies on incorporation of CoQ-homologs in mitochondrial membranes.

In this work we have made a systematic study on the incorporation of different homologs of Coenzyme Q in mitochondrial membranes. We have used a diluted mitochondrial suspension constituting a biphasic system membrane/H2O in which exogenous Q will be distributed depending on its water solubility and on its affinity to the mitochondrial membrane. It was found that Ubiquinones are incorporated into mitochondria in different extents ranging from zero (Q1) to 10 fold (Q10) the concentration of endogenous Q per mg of mitochondrial protein. In Q-depleted mitochondria the extents of incorporation are greater for all the quinones. This study points out that there is a reflection between the isoprenoid units of each Q-homolog and its incorporation into mitochondrial membranes.

Animals↗
PubMed1979

Progressive muscular dystrophy type Duchenne. I. Spin label studies on the physico-chemical state of erythrocyte membranes.

Electron spin resonance studies of erythrocyte membranes from patients with Duchenne muscular dystrophy exhibit changes in the physical state of lipids and proteins in membranes when compared to membranes from normal subjects. The results suggest that the alterations in membrane lipid-protein organization are present in this disease.

Electron Spin Resonance Spectroscopy↗
PubMed1979

Progressive muscular dystrophy type Duchenne. II. Viscosity and resistence to erythrolytic compounds of erythrocyte membranes.

The transition temperature of erythrocyte ghosts of normal subjects is about 18-20 degrees C. We have studied the viscosity of erythrocyte ghosts of dystrophic children, showing that the transition shifts to lower temperatures (17-18 degrees C). After treatment with erythrocytic compounds like L-Lyso phosphatidyl-Choline dystrophic erythrocytes hemolize at lower Lysophosphatidyl-Choline concentration and at a greater extents than these of normal and carriers subjects.

Erythrocyte Membrane↗
PubMed1979

A role of ubiquinone in energy conservation in mitochondria.

Short chain ubiquinones (Q-3) uncouple oxidative phosphorylation in rat heart mitochondria, as shown by polarimetric experiments, and abolish P:O ratios in succinate driven oxidative phosphorylaton. The uncoupling is reversed by long chain ubiquinones (Q-7). Furthermore, short chain ubiquinones abolish oligomycin sensitivity of ATPase; the inhibition is restored by Q-7. The extraction of endogenous ubiquinone from mitochondria reversibly lowers oligomycin sensitivity of ATPase.

Animals↗
PubMed1979

Molecular mechanism of general anesthesia: II. Spin label studies on synaptic membranes.

In this communication we report the effects of general anesthetics on the mobility and order of spin labeled stearic acid derivatives in synaptic membranes and in bilayers formed from the lipids extracted therefrom. The anesthetics studied abolish the immobilization induced by synaptic membrane proteins on the membrane lipids : this effect, observed particularly in the bilayer core, is interpreted as a labilization of lipid-protein interactions induced by anesthetics.

Anesthesia, General↗
PubMed1979

Lipid protein interactions in mitochondria. VIII. Effect of general anesthetics on the mobility of spin labels in lipid vesicles and mitochondrial membranes.

We have studied the effect of general anesthetics on the mobility of two stearic acid spin labels (5-doxyl stearic acid and 16-doxyl stearic acid) in bovine heart mitochondria and in phospholipid vesicles made from either mitochondrial lipids or commercial soybean phospholipids. The general anesthetics used include nonpolar compounds (alcohols, halothane, pentane, diethyl ether, chloroform) and the amphiphatic compound, ketamine. All anesthetics tested increase the mobility of the spin labels in phospholipid vesicles to a limited extent up to a concentration where the ESR spectra become those of free spin labels. On the other hand, anesthetics have a pronounced effect on mitochondrial membranes at concentrations as low as those known to produce general anesthesia; the effect is lower near the bilayer surface (5-doxyl stearic acid) and very strong in the bilayer core (16-doxyl stearic acid). The effects of anesthetics are mimicked by the detergent, Triton X-100. We suggest that the discrepancy between the action of anesthetics in mobilizing the spin labels in lipid vesicles and in membranes results from labilization of lipid protein interactions.

Anesthetics↗