1,721,184 research outputs found
Flussi di acqua e soluti attraverso n membrane in serie, con trasporto attivo
No full textTrasporto di acqua e soluti attraverso n membrane in serie in presenza di trasporto "attivo" di soluto. L'esperienza mostra che le relazioni fra i flussi di materia attraverso membrane, e le forze generalizzate che li sostengono sono non lineari. Ciò è vero sia per il trasporto attraverso singole membrane biologiche sia per quello attraverso barriere complesse, come gli epiteli, che si possono ritenere costituiti da più membrane semplici in serie. Il modello più semplice, ma poco realistico, costituito da due sole membrane, già consente di spiegare l'accoppiamento fra il trasporto "attivo" (accoppiato a reazioni chimiche) di soluto e il trasporto d'acqua (1-2). La generalizzazione a un sistema costituito da n membrane in serie, ma puramente passivo (3-4) viene ora estesa con l'introduzione del trasporto attivo. Lo studio teorico del modello e la simulazione al calcolatore di tale sistema hanno mostrato che: 1. la non linearità del flusso volumetrico si osserva solo in presenza di soluto e l'equazione si riduce alla legge lineare di Darcy per il solvente puro, 2. la non linearità richiede comunque l'asimmetria del sistema ed è dovuta all'accumulo di soluto nei compartimenti interni, 3. i coefficienti non costanti che correlano i flussi alle loro forze traenti sono funzione, oltre che del flusso stesso, anche delle variabili operative, 4. il comportamento non lineare di membrane semplici può venire spiegato con il fatto che gli strati limite di fluido non mescolato si comportano come membrane non selettive. (1) C. S. Patlack, D. A. Goldstein, J. F. Hoffman: J. Theor. Biol. 5, 426-442 (1963). (2) G. Monticelli, F. C. Celentano: Bull. Math. Biol. 45, 1073-1096 (1983). (3) F. C. Celentano, G. Monticelli: Atti VI Congresso SIBPA, Camogli, 1983, pp 6062. (4) F. C. Celentano, G. Monticelli in V. Capasso, E. Grosso, S. L. Paveri Fontana: Mathematics in Biology and Medicine, Springer Berlino, 1985, pp 293-299
Effect of calcium ions on black lipid membranes of phosphatidylinositol and oxidized cholesterol in presence of protein forming pore
No full textThe divalent calcium ions have been found to affect the surface potential of the membranes of nerve and of the black lipid membranes (BLM). In fact the addition of calcium to the bilayer formed of phosphatidylserine decreases the magnitude of surface potential either by screening and/or binding effects. Furthermore, calcium influences the conductance of the neutral carrier nonactine across BLM (S. Mclaughin, 1977: Current Topics in Membranes and Transport, 9, 71).
We have studied the effect of this ion on the incorporation of the porin in BLM formed by phosphatidylinositol (PI) and oxidized cholesterol (OxChol).
The artificial membranes were formed as previously reported (G. Monticelli et al., 1990, The Tenth School on Biophysics of Membrane Transport, School Proceedings, l: 329, Szczyrk, Poland). PI was extracted from ox brain and was chromatografícally pure; the OxChol was obtained as described by H. T. Tien et al. (Nature, 1966, 212, 718). The porin, purified by heart mithocondria, kindly given by Prof. F. Palmieri. The medium bathing the BLM was KCl 1 M and KCl plus CaCl2 1-10 mM, and porin (at different concentrations) was added at membrane black on the two aqueous bathing solutions. The conductance and the capacitance of the BLM have been measured as previously reported (G. Monticelli et al., 1990, The Tenth School on Biophysics of Membrane Transport, School Proceedings, 1: 329, Szczyrk, Poland).
The presence of calcium ions in the medium makes the BLM more stable.
The addition of porin to the aqueous phases of the both kind of BLM resulted in an increase of the conductance; but the kinetic is "S" shaped for the PI and hyperbolic for the OxChol membranes; furthermore the increase and the responsiveness were different, in that PI membranes needed an higher concentration of porin to determine a large increase of the conductance compared with OxChol membranes.
Calcium ions (at the concentrations used), by exerting a binding and screening effect on the surface of the membrane, affect either the maximum value of the conductance and the speed at which this value is reached when the porin is present
Coefficienti fenomenologici per il trasporto di acqua e soluti in sistemi a n membrane in serie
No full textLe relazioni flusso–forza nelle membrane biologiche risultano sperimentalmente non lineari. Per descrivere tale comportamento è stato proposto (1) di impiegare le equazioni lineari "pratiche" di Kedem e Katchalsky scritte in forma locale e integrate attraverso lo spessore di un sistema di due membrane in serie. Si ottengono equazioni fenomenologiche non lineari per il trasporto di acqua e soluti, che richiedono la ridefinizione come derivate, anziché rapporti semplici, dei coefficienti di permeabilità idraulica, di flusso osmotico e di riflessione. I risultati già ottenuti per due membrane (2) sono stati ora estesi a un sistema di n membrane, nel quale sono assimilabili formalmente a una membrana non selettiva anche gli strati limite. Per la membrana i-esima si scrive la concentrazione nel compartimento di destra i+1 in funzione di quella nel compartimento di sinistra i, del flusso volumetrico Jv e di quello di soluto Js. Mediante sostituzione ricorsiva delle concentrazioni si ottiene la concentrazione nel compartimento estremo di destra n+1 in funzione di quella nel compartimento estremo di sinistra 1. Di qui si ottiene Js in funzione di Jv, di C = C(n+1) - C(1) e di C(1) stessa. Si segue un procedimento analogo per le pressioni nei vari compartimenti e si ottiene la relazione che fornisce Jv. Da quest’ultima si ricava che: 1) il coefficiente di filtrazione Lp non è solo l’inverso della somma degli inversi dei singoli coefficienti Lpi relativi alle singole membrane ma contiene pure un termine additivo non lineare in C(1); 2) anche il coefficiente di flusso osmotico Lpd contiene un termine in C(1); 3) il rapporto -Lpd/Lp è indipendente da C(1) e, come nella teoria classica lineare, fornisce un coefficiente di riflessione per il sistema che risulta pari alla media dei coefficienti di riflessione delle n membrane pesati sull’inverso dei rispettivi coefficienti di permeabilità; 4) il medesimo risultato si ottiene facendo il limite per Jv tendente a zero del rapporto tra le differenze di pressione osmotica e idrostatica, confermando che si tratta effettivamente di un coefficiente di riflessione; 5) la legge di Darcy risulta una legge limite valida solo per il solvente puro, C(1) = 0. (1) C. S. Patlak, D. A. Goldstein, J. F. Hoffman: J. Theor. Biol. 5, 426-442 (1963) (2) G. Monticelli, F. Celentano: Further Properties of the Two-Membrane Model, Bull. Math. Biol, in stampa (1983)
Phenomenological description of selectivity in actively transporting membranes
No full textA phenomenological description of active and passive flows of solute and solvent across a biological membrane can be made explicitly considering the dependence of matter flows upon the rate of metabolic reactions /1/, or introducing a generalized chemical potential including a term accounting for active transport /2/, or making the hypothesis that solute flow can be splitten in two superimposed and thermodynamically couplet active and passive components. With the two latter approaches, by means of a transformation of flows and forces at constant temperature and in absence of electric field, two systems of three interacting flows, sustained by three different forces, can be obtained. The two systems lead to equivalent descriptions of volumetric flow and allow the determination of the reflection coefficient for solute passive transport /3/. The relationship between reflection coefficient and apparent reflection coefficient /4/ is also obtained. 1. A. Katchalsky, P. F. Curran. Nonequilbrium Thermodynamics in Biophysics, Cambridge Mass. (1965); 2. J. M. Diamond. J. Physiol. 161, 503 (1962); 3. F. Celentano, G. Monticelli, G. Torelli. Proc. Ist. Europ. Biophys. Congr. 3, 309 (1971); 4. C. J. Bentzel, M. Davies, W. N. Scott, M. Zatzman, A. K. Solomon. J. Gen. Physiol. 51, 517 (1968
PORIN INCORPORATION KINETICS IN PHOSPHATIDYLINOSITOL AND OXIDIZED CHOLESTEROL MEMBRANES
No full textConductivity of planar lipid bilayer membranes can be modulated by insertion of protein molecules (1).
Mithocondrial porins form water filled pores in the outer membrane and these channels act selectively on the permeation of hydrophilic solutes.
Aims of this research were: 1) the reconstitution of porin channels in planar lipid bilayer membranes, 2) the analysis and the comparison of porin incorporation kinetics in different lipid membranes: phosphatidylinositol (PI) and oxidized cholesterol, 3) the study of protein concentration effect on the incorporation kinetics, 4) the analysis of the channel reconstitution dependence on the applied voltage.
The artificial membranes were formed, with lipid in n-decane (1% w/w), brushing the solution on a small circular hole between two compartments, 4 ml volume, of a teflon experimental chamber. The two compartments were filled with KCl 1M solution and continuously stirred.
Pt electrodes in bathing solutions were connected to a current to voltage converter and to a sinusoidal voltage source (Vs=VS sin2nt; n=1Hz) so that the voltage at the converter (Vl) resulted directly related to the electrical current crossing the membrane (2).
Experiments were performed at room temperature: 24 ± 1 °C.
Membrane electrical conductances and capacitances were higher in oxidized cholesterol than in PI membranes.
Addition of porin to the solutions bathing a completely black membrane induced an increase of membrane current with time indicating protein fusion and channel formation into the lipid matrix.
In PI membranes the time course of current was S-shaped, irrespective of porin addition before or after membrane formation, but in the first case a delay in channel formation and a slower kinetics were observed (3).
In oxidized cholesterol membranes the current time behaviours were S-shaped when porin was added after membrane formation but resulted of exponential type when porin was present before membrane formation. In this second case faster kinetics were observed and the incorporation started with an higher conductance value.
The time lag between porin addition and the first current variation was shorter in oxidized cholesterol than in PI membranes. For a same porin concentration the incorporation kinetics were faster and higher steady levels were reached in oxidized cholesterol than in PI membranes.
In both types of membranes porin incorporation rate decreased lowering porin concentration; five kinetics of porin incorporation (protein added after membrane formation) in oxidized cholesterol membranes are reported in figure.
Besides the porin concentration also the applied voltage affects the incorporation kinetics and the channel formation.
At a given porin concentration faster kinetics are recorded and higher steady levels are reached increasing the voltage to a critical value after which incorporation is less effective, the kinetics are slower and accompanied by lower steady levels.
The voltage dependence of functional pore reconstitution was more evident in PI than in oxidized cholesterol membranes.
In oxidized cholesterol membrane, at the steady state, nonlinear current-voltage characteristics were obtained. The conductance increases with the voltage to reach a critical value after which inactivation occurs.
At a given applied voltage higher membrane conductances were calculated increasing porin concentration.
Significative differences in the current-voltage relations were obtained depending on the absence or the presence of porin at the membrane formation. In this latter case the membranes had higher conductance values but, at the tested voltages, the range of variability was smaller.
The nature of the lipid matrix can be considered responsible of the different protein incorporation; in particular the surface charge of the lipid bilayer membrane and the interface potential, due to the local accumulation of ions at the membrane-solution interface, play a key role in the incorporation of porin in PI planar bilayer lipid membranes.
These observations suggest the possibility to insert protein channels in lipid layers built on a solid substrate finalized to realize organic semiconductors to be used as biosensor or transducers.
Work supported in part by CNR grant 92.02202.CT14.
1) G. Monticelli - "Pore formation in planar lipid bilayer membranes" Proc. X School on Biophysics of Membrane Transport, Szczyrk (Poland) 1990, 1: 315-327.
2) G. Monticelli, E. Gallucci and S. Micelli - "Experimental data on incorporation of porin molecules in lipid bilayers" Proc. X School on Biophysics of Membrane Transport, Szczyrk (Poland) 1990, 1: 328-343.
3) G. Monticelli, E. Gallucci and S. Micelli - "Pore formation in lipid membranes" Proc. Int. Cong. Memb. and Memb. Processes, Chicago 1990, 1: 175-177
A simulation of mass transport across series arrays of membranes with chemical reaction-coupled solute flow
No full textBoth in biology and technology, membranes can be seldom treated as single, thin and linearly behaving barriers. Mass transport across thick membranes, as first suggested by Patlak, Goldstein and Hoffman (1), can be described integrating across the membrane thickness the local, linear, practical equations by Kedem and Katchalsky (2), which have been recently shown to derive directly fom the local energy dissipation function of the membrane (3). When the heat flow associated to chemical reactions or to a temperature gradient can be neglected, the presence of a solute "active" transport can also be accounted for (1,4). In the present paper we extend to n membranes, by means of a recursive procedure, the previous treatment of a series array of few membranes (1, 4) and derive some parameters characterizing the transport properties of the complex barrier. This task has been performed analytically, with the help of the symbolic computation program REDUCE, obtaining a non-linear correlation between the flows and their driving forces, depending on the volume flow and on the solute concentration of the transported solution. The nonlinearity appears to be a consequence of the solute accumulation in the inner compartments of the array. The classical linear law by Darcy is a limiting case of our volume flow equation when only pure solvent is transported. Around the volume and solute flow equations we have written a program in Pascal allowing the simulation of a series array of up to 10 membranes and unstirred layers, assimilated to non-selective membranes. The results of the simulation are in agreement with experimental data obtained using complex biological barriers like epithelia. 1. C.S. Patlak, D.A. Goldstein, J.F. Goldstein: J. Teor. Biol. 5, 426-442 (1963); 2. O. Kedem, A. Katchalsky: Biochim. Biophys. Acta 27, 229-246 (1958); 3. F. Celentano, G. Monticelli: Local Practical Equations for Heat and Mass Transport Driven by Temperature Gradients, Proc. Europe-Japan Congr. Membranes and Membrane Proc., Stresa, June 18-22 1984, in press; 4. G. Monticelli, F. Celentano: Bull. Math. Biol. 45, 1073-1096 (1983)
CALORIMETRIA DIFFERENZIALE A SCANSIONE DI MISCELE FOSFOLIPIDICHE DA MEMBRANE DI GLOBULI ROSSI
No full textNegli ultimi anni particolare attenzione è stata posta alle relazioni tra funzione delle membrane, le loro proprietà chimico-fisiche e la composizione in acidi grassi, prevalentemente della frazione fosfolipidica considerato il fatto che fosfolipidi, trovati nelle membrane biologiche, derivano le loro proprietà idrofobiche dagli acidi grassi costituenti.
Le correlazioni più frequentemente analizzate sono quelle tra componenti di membrana e i loro acidi grassi modificati, in vivo, da alterazioni della composizione lipidica della dieta o, in vitro, mediante opportune modifiche del mezzo di coltura.
Somministrando diete diverse a bovine da latte abbiamo creato una “banca” di campioni di membrane di globuli rossi a diversa composizione lipidica (1, 2).
Scopi di questo studio sono stati: 1) l’analisi della composizione delle miscele di fosfolipidi estratti dalle membrane, 2) la valutazione delle correlazioni tra composizione e proprietà termotropiche delle miscele.
I lipidi totali sono stati estratti dagli eritrociti bovini con miscele di cloroformio/metanolo in diversi rapporti e le miscele lipidiche sono state frazionate mediante cromatografia su colonna.
Le singole specie fosfolipidiche sono state separate in HPTLC e quantificate mediante densitometria a scansione.
La composizione in acidi grassi dei fosfolipidi di membrana è stata valutata mediante gas-liquido cromatografia e dispersioni acquose tamponate sono state esaminate mediante calorimetria differenziale a scansione.
Per ogni campione è stato calcolato il contenuto di acidi grassi saturi (S), insaturi (U) e l’indice di insaturazione (UI).
I principali acidi grassi presenti (mediamente ≥ 10% in peso) sono risultati: C16:0, C18:0, C18:1, C18:2 e C24:0.
Le miscele di fosfolipi mostrano una o due transizioni di fase a temperature comprese tra 17.7 e 40.0 °C. La prima temperatura di transizione è compresa tra 17.7 e 26.0 °C.
UI e U/S non risultano essere correlati al contenuto percentuale delle diverse specie molecolari di fosfolipidi (PC, PE, SM, PS+PI) e di PC+SM, costituenti principali del foglietto esterno del doppio strato lipidico.
Esiste, invece, una correlazione lineare tra la prima temperatura di transizione ed UI (P < 0.05) e U/S (P < 0.01). La prima temperatura di transizione diminuisce all’aumentare di UI o del rapporto U/S.
References
1. G. Monticelli, S. Rapelli, G. Montorfano, P. Magistretti and B. Berra- “Red blood cell membrane composition following diet manipulation in the cow” Riv. Ital. Sostanze Grasse 67: 507-515 (1990)
2. G. Monticelli, M. Masserini,G. Lercker, T. Beringhelli, P. Marciani, E. Calappi and B. Berra – “Red blood cell membrane composition following diet manipulation in the cow. II: phospholipid fatty acid distribution and physico-chemical characteristics of membrane and its constituents” Riv. Ital. Sostanze Grasse 69: 189-199 (1992
EFFECT OF DIVALENT CATIONS ON PORIN INCORPORATION IN PLANAR BLM
No full textBiological membranes contain as a basic structural unit a phospholipid bilayer.
Phospholipid membranes in the form of bilayer films (BLM) are currently used as experimental models for transport phenomena of the biological membranes. In particular, bilayers made of acidic phospholipids, present the advantage to investigate the bioelectrical phenomena across the biological membranes.
This class of phospholipids by having a fixed charge might bind ions that play an important role on many physiological processes. It is known that Ca++ and other alkaline cations change the electrostatic potential of the negatively charged bilayer membranes either absorbing to the phospholipids or accumulating in the aqueous diffuse double layer, and this potential change may induce conductance variation (1).
Cd++ another divalent cation has been shown to exert influence on the permeability of some anaelectrolytes across BLM made of negatively charged phospholipids, but not on the zwitterionic phospholipids (2).
We have studied the effect of divalent cations as Ca++ and Ba++ that exert a cross linking together with a screening effect respectively, on the kinetics of incorporation of the mithocondrial porin in bimolecular lipid membranes made with phosphatydilinositol.
In order to understand how lipids of the bilayer affect protein incorporation at the molecular level we must consider that protein crossing the bilayer has to surmount different regions characterized by different properties as interfacial region, head groups, lipid backbone and hydrocarbon core.
In this study we focused our attention to the role of the interface and head group regions of the BLM in the porin incorporation.
The artificial membranes were made of phosphatydilinositol cromatografically pure in n-decane (1% w/w). The bathing media were KC1 1M and KC1 1M plus CaC12 or BaCl2 10 mM. Porin was added at membrane "black" on the two aqueous bathing solutions. Bilayers conductivity and capacitance measurements were carried out at temperature of 25 ± 2 °C and studied by means of alternate current (f = 1 Hz) recording the voltage after a current to voltage converter in series with the membrane; simultaneously electrical capacitance was measured at 1 KHz before and during porin incorporation (3) .
We noticed that when the membranes are in the presence of Ca++ or Ba++ there was an exceptional stability.
With the different bathing solutions no significant differences were observed in membrane conductance values whereas small capacitance variations were recorded.
Once the membrane has became black porin, at a fixed concentration, was added to the aqueous solutions, and after a lag time due to the diffusion of the protein in the solutions and of the time to overcome the membrane barriers, an increase of the voltage output due to the channels formation was recoded. This phenomenon shows a "S-shaped" kinetics. In contrast to the kinetics of incorporation of the porin in oxidized cholesterol lipid membranes (4), showing an hyperbolic shape. These different kinetics may be explained by the different interface barrier; in fact when the experiment were conducted with porin already present in the bathing solutions before membrane formation, the different kinetics persisted in the phosphatydilinositol and in oxidized cholesterol BLM.
The S shaped kinetics persists also when porin is incorporated in phosphatydilinositol membranes in the presence of Ca++ or Ba++; but the curve in presence of the Ca++ is shifted to the right.
This shift is much more pronounced in the presence of Ba++.
It is known that Ca++ affects the surface potential of BLM containing negatively charged lipids. The protein incorporation into BLM can be regarded as a two steps process: I) the interaction at the surface barrier, and II) the penetration into hydrocarbon region.
In the first step the protein encounter an higher resistance in phosphatydilinositol membrane because it has to overcome the interface region consisting of a diffuse double layer, the membrane surface and the polar head groups.
Ca++, by exerting a cross-linking and screening effect on negatively charged membranes, retards the porin incorporation.
At the moment we are not able to distinguish which of the two effect is prevalent; but results in presence of Ba++, that exerts a screening effect only seem to indicate that the latter effect is prevalent. In order to establish the pure cross-linking effect experiments are in progress.
The influence of the divalent cations on the protein incorporation into BLM, that are sensitive to changes in environmental conditions, make this study appealing in order to provide a better knowledge in the molecular mechanism of the protein incorporation and channel formation.
Work supported by 40% grant from MURST 1991
1) S. G. A. McLaughlin, G. Szabo and G. Eisenman, 1971: J. Gen. physiol., 58, 667.
2) S. Micelli, E. Gallucci and C. Lippe, 1978: Arch. Intern. Physiol. Biochim., 86, 755-759.
3) G. Monticelli, E. Gallucci and S. Micelli, 1990: Proc. X School on Biophysics of Membrane Transport, 1: 328-343, Poland.
4) S. Micelli, G. Monticelli and E. Gallucci, 1990: IBST 1: 221
Fisiologia
No full textLa Fisiologia è la scienza che si occupa di come funzionano, in condizioni normali, gli organismi viventi, le loro parti e le relazioni fra le varie strutture, le relative funzioni e l’integrazione di queste.
Lo studio della Fisiologia umana riguarda tutti i livelli dimensionali e organizzativi: dall’atomico-molecolare al sistemico. La Fisiologia è quindi strettamente correlata a varie discipline e diventa insostituibile per chiarire i processi di sviluppo e di invecchiamento, per comprendere come si instaurano i processi patologici – ovvero le alterazioni dei meccanismi che, modificando le funzioni, portano alla malattia – e per definire l’approccio farmacologico atto a ripristinare la funzionalità. Peraltro, anche i processi di ideazione, sintesi e sviluppo di molecole, agenti come possibili farmaci, non possono fare a meno delle conoscenze, a vario livello strutturale, dei meccanismi di funzionamento dell’organismo umano e delle sue parti.
Questa seconda edizione è stata ampiamente aggiornata e migliorata soprattutto per aiutare gli studenti nel superamento delle difficoltà, sviluppando in modo graduale gli argomenti senza esagerate semplificazioni e conservando il rigore nella trattazione. Analoga attenzione si è posta all’apparato illustrativo, con oltre cinquecento figure, base fondamentale per una comprensione della materia.
Sul sito web, che accompagna il libro, sono disponibili numerosi test di autovalutazione interattivi: un supporto ulteriore per lo studente nell’affrontare lo studio della Fisiologia, così come sarà certamente d’aiuto la possibilità di sfogliare il libro anche in formato digitale
Adaptation in Helix pomatia neurons
No full text1. Spike frequency adaptation has been studied on neurons of Helix pomatia subesophageal ganglia and interpreted by means of a behavioural model describing the phenomenon in neurons either silent or autorhythmic at rest.
2. At low stimulating currents the initial discharge frequency F(0) is linearly related to the current strength G.
3. In the linearity range F(0)/G each neuron was characterized by means of four model parameters: the proportionality constant between F(0) and G, the decay constant of the frequency, the inhibitory current from a single nerve impulse and the decay time constant of the inhibitory current.
4. The four parameters varied in different cells with a range of 0.18-4.98 Hz/nA, 1.02-3.85 sec, 0.05-0.95 nA and 1.74-22.33 sec, respectively.
5. Experimental results have been analyzed considering inhibitory current, electrogenic sodium pump and other proposed adaptation parameters
- …
