We have previously shown that the 40-residue peptide termed amyloid 13-protein (A,3P ) in solutio... more We have previously shown that the 40-residue peptide termed amyloid 13-protein (A,3P ) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether AP3P[1-40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A,BP leads to the spontaneous formation of cation-selective channels. With Cs' as the main cation in both the external as well as the internal saline, the amplitude of the A,3P[1-40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50-500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid 13-protein in solution, can block the flow of Cs+ through the A13P channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A13P[1-40] channel pore. These properties of the A,BP[1-40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid 1-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid 13-protein and neuronal membranes also occur in vivo, lending further support to the idea that A,BP[1-40] channel formation might be a mechanism of amyloid 1-protein neurotoxicity. . Cortical neurons exposed to A,BP[1-40] exhibit elevated levels of intracellular free calcium ([Ca2+]i), suggesting that toxicity may result from amyloid 13-protein-evoked Ca2+ entry . We have previously shown that when A,3P[1-40] was incorporated into lipid bilayer membranes, the amyloid formed cation-selective (including
Proceedings of The National Academy of Sciences, 1996
The Alzheimer disease 40-residue amyloid (3 protein (APP[1-40]) forms cation-selective channels a... more The Alzheimer disease 40-residue amyloid (3 protein (APP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range ofconductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AI3P[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the A,BP[1-40J channel. Provided the A(3P[1-401 channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose-
Proceedings of The National Academy of Sciences, 1967
The ionic currents that flow during a voltage clamp of the membrane of the squid axon in sea wate... more The ionic currents that flow during a voltage clamp of the membrane of the squid axon in sea water were measured' 2 and analyzed3 into an early transient current of Na+ and a later outward current of K+. In 1952, Hodgkin et al.2 observed a reversal in the early current when the axonal membrane was strongly depolarized. The characteristics of the early current led Hodgkin and Huxley3 to propose that, whether inward or outward, it was carried by the same ion, sodium. The potential at which the early current reversed was called the sodium potential and it was shown to vary with the log of the external sodium concentration.
Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation... more Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.
The main effects of existing socioeconomic arrangements for agricultural land development in the ... more The main effects of existing socioeconomic arrangements for agricultural land development in the Eastern Caribbean are discussed and changes aimed at improving both the efficiency and squity of the land development process are identified. The author argues that the existing 'plantations-small holders' structure is incapable of providing an efficient way of developing the available land resources and of improving the standard of living for the vast rural population. A new set of export oriented agrlcultural enterprises is pinpointed as a requirement in any viable solution aimed at taking land away from its present role as a resource for survival for the many and an economic benefit for the few and to reinstate agricultural lands as one of the key resources for more egalitarian, environmentally sound and efficient sociosconomic devel-
Philosophical Transactions of The Royal Society B: Biological Sciences, 1975
Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated ne... more Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmann&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s law (midpoint potential -33.7 mV;
Pflugers Archiv-european Journal of Physiology, 1978
Asymmetrical displacement currents were studied in myelinated nerve fibres fromRana esculenta wit... more Asymmetrical displacement currents were studied in myelinated nerve fibres fromRana esculenta with a voltage clamp technique. For brief pulses symmetrical with respect to a holding potential of −97 mV, the asymmetry current flowing during pulses (on-response) exhibited a rising phase to a peak followed by an approximately exponential decline. After the pulses the rising phase in the off-response could not be resolved; the time constant varied about 2-fold with either size or duration of the pulse. For longer pulses a second slower component could be detected both in on- and off-responses. The rapidly declining on- and off-responses associated with brief pulses carried about the same chargesQ on andQ off. Increasing the duration of the pulse reducedQ off. For all pulses testedQ off approached about one fifth ofQ max. The reduction ofQ off was roughly characterised by time constants ranging between 1.5 and 0.5 ms for potentials between −25 and +23 mV. Analysis of individual membrane currents confirmed that the capacity current after depolarizing pulses decreased with pulse length. The effects of membrane potential on asymmetry current were studied by varying the level from which pulses were applied during 46.9 ms prepulses in the range from −97 to −29 mV. The fast and slow components of asymmetry current were affected differently. For potentials more positive than −90 mV the fast on-response was reduced and reversed its sign at a potential 25 mV more negative than the potential estimated from the steady-state charge distribution measured from −97 mV.
Pflugers Archiv-european Journal of Physiology, 1975
The time course of the membrane currents in the node of Ranvier in which the sodium and potassium... more The time course of the membrane currents in the node of Ranvier in which the sodium and potassium conductances have been blocked reveals asymmetries during and after the application of depolarizing and hyperpolarizing voltage-clamp pulses of identical size. Since, 1. the integrals of the “on” and “off” current transients were found to be equal and opposite, 2. the charge displaced reached saturation (about 140·10−15 C/node) when the internal potential was taken to a sufficiently positive value during the depolarizing pulses and, 3. the size of the charge transferred was unaffected by temperature although its time constant had a large temperature coefficient (Q 10=2.4), these currents to our opinoon must result from charge movements confined to the membrane and, therefore, can be considered as non-linear displacement currents. The steady-state rearrangement of the charges is consistent with a Boltzmann distribution of charges (effective valencez′=1.65) between two configurations characterized by different energy levels. The midpoint potential of the distribution curve is −33.7 mV and its maximum slope,kT/z′e, is 14.9 mV. Following changes in membrane potential the charges undergo a first order transition between these states. We propose that these displacement currents arise from a redistribution of the charges involved in the sodium gating system.
A controversy of long standing in membrane electrophysio-logy is whether the sodium ion current (... more A controversy of long standing in membrane electrophysio-logy is whether the sodium ion current (I Na) and potassium ion current (I K) pass through the membrane in separate channels, or through a single set of channels which conduct first I Na and then I K. In support of the latter ...
ALTHOUGH there is evidence that axonal membrane potentials are related to sodium and potassium mo... more ALTHOUGH there is evidence that axonal membrane potentials are related to sodium and potassium movements1, there has been little explicit discussion of how these ion movements may be initiated, or of the chemical processes which may permit them to occur2. Because it is not possible to identify the molecules involved by measuring potentials and associated ion movements, there is a
The profits of a sugar plantation in any of our West Indies Colonies are generally much greater t... more The profits of a sugar plantation in any of our West Indies Colonies are generally much greater than those of any other cultivation that is known either in Europe or America.'
We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage... more We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage-clamped squid axons. Internal perfusion of the axons with Pronase rather selectively destroys inactivation of the Na conductance (g.).-At the level of a single channel, Pronase probably acts in an allor-none manner: each channel inactivates normally until its inactivation gate is destroyed, and then it no longer inactivates. Pronase reduces gN., possibly by destroying some of the channels, but after removal of its inactivation gate a Na channel seems no longer vulnerable to Pronase. The turn-off kinetics and the voltage dependence of the Na channel activation gates are not affected by Pronase, and it is probable that the enzyme does not affect these gates in any way. Neither the K channels nor their activation gates are affected in a specific way by Pronase. Tetrodotoxin does not protect the inactivation gates from Pronase, nor does maintained inactivation of the Na channels during exposure to Pronase. Our results suggest that the inactivation gate is a readily accessible protein attached to the inner end of each Na channel. It is shown clearly that activation and inactivation of Na channels are separable processes, and that Na channels are distinct from K channels.
The genus Persea consists of two subgenera, Persea (known as avocados) and Eriodaphne (known as a... more The genus Persea consists of two subgenera, Persea (known as avocados) and Eriodaphne (known as aguacatillos, avocado-like species). The present study aimed to determine whether the genus Persea is a monophyletic group and whether the division into two subgenera is an artificial one. In conjunction with these goals, a hypothesis of the phylogenetic relationships among Persea species is proposed. Our results suggest that Persea is not a monophyletic group. Two clades of Persea can be recognized. The results suggest that Eriodaphne and Persea should be considered to be independent genera. Various leaf and floral characters contributed for separation into groups. The definition of Eriodaphne is based on fruit color, sessile glands at the base of the stamens, vein prominence and leaf shape. Persea is more closely related to genera Nectandra and Ocotea than Eriodaphne. The 11 species included in Persea are recognized as species. The group is defined by fruit flavor, mature leaf color, number of tertiary divergent veins, and pubescent bracts in the inflorescence. Within this group, a clade of six species in which P. guatemalensis is included is recovered by seed shape, venation pattern relief, and number of fruits per cluster. Moreover, the seed shape supports the separation of P. floccosa and P. zentmyerii. A molecular character analysis is necessary to support the Persea clades proposed herein. Nevertheless the phylogenetic relationships revealed by this study provide new bases for the selection and conservation of the species Persea.
We have previously shown that the 40-residue peptide termed amyloid 13-protein (A,3P ) in solutio... more We have previously shown that the 40-residue peptide termed amyloid 13-protein (A,3P ) in solution forms cation-selective channels across artificial phospholipid bilayer membranes. To determine whether AP3P[1-40] also forms channels across natural membranes, we used electrically silent excised membrane patches from a cell line derived from hypothalamic gonadotrophin-releasing hormone GnRH neurons. We found that exposing either the internal or the external side of excised membrane patches to A,BP leads to the spontaneous formation of cation-selective channels. With Cs' as the main cation in both the external as well as the internal saline, the amplitude of the A,3P[1-40] channel currents was found to follow the Cs+ gradient and to exhibit spontaneous conductance changes over a wide range (50-500 pS). We also found that free zinc (Zn2+), reported to bind to amyloid 13-protein in solution, can block the flow of Cs+ through the A13P channel. Because the Zn2+ chelator o-phenanthroline can reverse this blockade, we conclude that the underlying mechanism involves a direct interaction between the transition element Zn2+ and sites in the A13P[1-40] channel pore. These properties of the A,BP[1-40] channel are rather similar to those observed in the artificial bilayer system. We also show here, by immunocytochemical confocal microscopy, that amyloid 1-protein molecules form deposits closely associated with the plasma membrane of a substantial fraction of the GnRH neurons. Taken together, these results suggest that the interactions between amyloid 13-protein and neuronal membranes also occur in vivo, lending further support to the idea that A,BP[1-40] channel formation might be a mechanism of amyloid 1-protein neurotoxicity. . Cortical neurons exposed to A,BP[1-40] exhibit elevated levels of intracellular free calcium ([Ca2+]i), suggesting that toxicity may result from amyloid 13-protein-evoked Ca2+ entry . We have previously shown that when A,3P[1-40] was incorporated into lipid bilayer membranes, the amyloid formed cation-selective (including
Proceedings of The National Academy of Sciences, 1996
The Alzheimer disease 40-residue amyloid (3 protein (APP[1-40]) forms cation-selective channels a... more The Alzheimer disease 40-residue amyloid (3 protein (APP[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range ofconductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to AI3P[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the A,BP[1-40J channel. Provided the A(3P[1-401 channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dose-
Proceedings of The National Academy of Sciences, 1967
The ionic currents that flow during a voltage clamp of the membrane of the squid axon in sea wate... more The ionic currents that flow during a voltage clamp of the membrane of the squid axon in sea water were measured' 2 and analyzed3 into an early transient current of Na+ and a later outward current of K+. In 1952, Hodgkin et al.2 observed a reversal in the early current when the axonal membrane was strongly depolarized. The characteristics of the early current led Hodgkin and Huxley3 to propose that, whether inward or outward, it was carried by the same ion, sodium. The potential at which the early current reversed was called the sodium potential and it was shown to vary with the log of the external sodium concentration.
Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation... more Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.
The main effects of existing socioeconomic arrangements for agricultural land development in the ... more The main effects of existing socioeconomic arrangements for agricultural land development in the Eastern Caribbean are discussed and changes aimed at improving both the efficiency and squity of the land development process are identified. The author argues that the existing 'plantations-small holders' structure is incapable of providing an efficient way of developing the available land resources and of improving the standard of living for the vast rural population. A new set of export oriented agrlcultural enterprises is pinpointed as a requirement in any viable solution aimed at taking land away from its present role as a resource for survival for the many and an economic benefit for the few and to reinstate agricultural lands as one of the key resources for more egalitarian, environmentally sound and efficient sociosconomic devel-
Philosophical Transactions of The Royal Society B: Biological Sciences, 1975
Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated ne... more Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmann&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s law (midpoint potential -33.7 mV;
Pflugers Archiv-european Journal of Physiology, 1978
Asymmetrical displacement currents were studied in myelinated nerve fibres fromRana esculenta wit... more Asymmetrical displacement currents were studied in myelinated nerve fibres fromRana esculenta with a voltage clamp technique. For brief pulses symmetrical with respect to a holding potential of −97 mV, the asymmetry current flowing during pulses (on-response) exhibited a rising phase to a peak followed by an approximately exponential decline. After the pulses the rising phase in the off-response could not be resolved; the time constant varied about 2-fold with either size or duration of the pulse. For longer pulses a second slower component could be detected both in on- and off-responses. The rapidly declining on- and off-responses associated with brief pulses carried about the same chargesQ on andQ off. Increasing the duration of the pulse reducedQ off. For all pulses testedQ off approached about one fifth ofQ max. The reduction ofQ off was roughly characterised by time constants ranging between 1.5 and 0.5 ms for potentials between −25 and +23 mV. Analysis of individual membrane currents confirmed that the capacity current after depolarizing pulses decreased with pulse length. The effects of membrane potential on asymmetry current were studied by varying the level from which pulses were applied during 46.9 ms prepulses in the range from −97 to −29 mV. The fast and slow components of asymmetry current were affected differently. For potentials more positive than −90 mV the fast on-response was reduced and reversed its sign at a potential 25 mV more negative than the potential estimated from the steady-state charge distribution measured from −97 mV.
Pflugers Archiv-european Journal of Physiology, 1975
The time course of the membrane currents in the node of Ranvier in which the sodium and potassium... more The time course of the membrane currents in the node of Ranvier in which the sodium and potassium conductances have been blocked reveals asymmetries during and after the application of depolarizing and hyperpolarizing voltage-clamp pulses of identical size. Since, 1. the integrals of the “on” and “off” current transients were found to be equal and opposite, 2. the charge displaced reached saturation (about 140·10−15 C/node) when the internal potential was taken to a sufficiently positive value during the depolarizing pulses and, 3. the size of the charge transferred was unaffected by temperature although its time constant had a large temperature coefficient (Q 10=2.4), these currents to our opinoon must result from charge movements confined to the membrane and, therefore, can be considered as non-linear displacement currents. The steady-state rearrangement of the charges is consistent with a Boltzmann distribution of charges (effective valencez′=1.65) between two configurations characterized by different energy levels. The midpoint potential of the distribution curve is −33.7 mV and its maximum slope,kT/z′e, is 14.9 mV. Following changes in membrane potential the charges undergo a first order transition between these states. We propose that these displacement currents arise from a redistribution of the charges involved in the sodium gating system.
A controversy of long standing in membrane electrophysio-logy is whether the sodium ion current (... more A controversy of long standing in membrane electrophysio-logy is whether the sodium ion current (I Na) and potassium ion current (I K) pass through the membrane in separate channels, or through a single set of channels which conduct first I Na and then I K. In support of the latter ...
ALTHOUGH there is evidence that axonal membrane potentials are related to sodium and potassium mo... more ALTHOUGH there is evidence that axonal membrane potentials are related to sodium and potassium movements1, there has been little explicit discussion of how these ion movements may be initiated, or of the chemical processes which may permit them to occur2. Because it is not possible to identify the molecules involved by measuring potentials and associated ion movements, there is a
The profits of a sugar plantation in any of our West Indies Colonies are generally much greater t... more The profits of a sugar plantation in any of our West Indies Colonies are generally much greater than those of any other cultivation that is known either in Europe or America.'
We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage... more We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage-clamped squid axons. Internal perfusion of the axons with Pronase rather selectively destroys inactivation of the Na conductance (g.).-At the level of a single channel, Pronase probably acts in an allor-none manner: each channel inactivates normally until its inactivation gate is destroyed, and then it no longer inactivates. Pronase reduces gN., possibly by destroying some of the channels, but after removal of its inactivation gate a Na channel seems no longer vulnerable to Pronase. The turn-off kinetics and the voltage dependence of the Na channel activation gates are not affected by Pronase, and it is probable that the enzyme does not affect these gates in any way. Neither the K channels nor their activation gates are affected in a specific way by Pronase. Tetrodotoxin does not protect the inactivation gates from Pronase, nor does maintained inactivation of the Na channels during exposure to Pronase. Our results suggest that the inactivation gate is a readily accessible protein attached to the inner end of each Na channel. It is shown clearly that activation and inactivation of Na channels are separable processes, and that Na channels are distinct from K channels.
The genus Persea consists of two subgenera, Persea (known as avocados) and Eriodaphne (known as a... more The genus Persea consists of two subgenera, Persea (known as avocados) and Eriodaphne (known as aguacatillos, avocado-like species). The present study aimed to determine whether the genus Persea is a monophyletic group and whether the division into two subgenera is an artificial one. In conjunction with these goals, a hypothesis of the phylogenetic relationships among Persea species is proposed. Our results suggest that Persea is not a monophyletic group. Two clades of Persea can be recognized. The results suggest that Eriodaphne and Persea should be considered to be independent genera. Various leaf and floral characters contributed for separation into groups. The definition of Eriodaphne is based on fruit color, sessile glands at the base of the stamens, vein prominence and leaf shape. Persea is more closely related to genera Nectandra and Ocotea than Eriodaphne. The 11 species included in Persea are recognized as species. The group is defined by fruit flavor, mature leaf color, number of tertiary divergent veins, and pubescent bracts in the inflorescence. Within this group, a clade of six species in which P. guatemalensis is included is recovered by seed shape, venation pattern relief, and number of fruits per cluster. Moreover, the seed shape supports the separation of P. floccosa and P. zentmyerii. A molecular character analysis is necessary to support the Persea clades proposed herein. Nevertheless the phylogenetic relationships revealed by this study provide new bases for the selection and conservation of the species Persea.
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