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J Requena

Publications and source records attributed to J Requena.

At least 19 recordsLinked to original sources
PubMed2004

Modification of the longevity-related degree of fatty acid unsaturation modulates oxidative damage to proteins and mitochondrial DNA in liver and brain.

Previous studies have shown that tissue fatty acid unsaturation correlates inversely with maximum longevity. However, it is unclear if this is related to the effects of fatty acid unsaturation only on lipids, or also on proteins and DNA, specially on mitochondrial DNA (mtDNA) oxidative damage. In this investigation the degree of fatty acid unsaturation of liver and brain was successfully manipulated in Wistar rats by chronic feeding with specially designed semipurified diets rich in saturated or unsaturated fats. The brain, an organ of special relevance for aging, was most profoundly affected by the increase in fatty acid unsaturation, and showed significant increases in malondialdehyde (MDA)-lysine, aminoadipic semialdehyde (a protein carbonyl), N(epsilon)-(carboxymethyl)lysine, and N(epsilon)-(carboxyethyl)lysine in proteins, as well as in 8-oxo,7,8-dihydro-2'-deoxyguanosine (8-oxodG) in mtDNA without changes in nuclear DNA (nDNA). In the liver 8-oxodG was also increased in mtDNA and not in nDNA. These DNA results are consistent with the presence of a high density of mitochondrial inner membranes (rich in lipids and in reactive oxygen species generation capacity) near mtDNA but not near nDNA. Among the protein markers analyzed, MDA-lysine was most consistent and responsive to fatty acid unsaturation, since it increased in both organs and showed the highest increase. These results, together with previous data from our laboratories, show that increasing the degree of fatty unsaturation of postmitotic tissues in vivo can raise not only lipid but also protein and mtDNA oxidative damage. This is mechanistically relevant in relation to the constitutively low tissue fatty acid unsaturation of long-lived animals.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals.

Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders.

Animals↗
PubMed1991

Intracellular ionized calcium changes in squid giant axons monitored by Fura-2 and aequorin.

Squid giant axons were injected simultaneously with Ca indicators Fura-2 and aequorin. Fura-2 was calibrated in situ by measuring fluorescence at 510 nm upon UV excitation at 340 nm, 360 nm, and 380 nm with a time-sharing multiple wavelength spectrofluorimeter. Limiting values for dye fluorescence were obtained by allowing a massive load of Ca to enter the axon with the aid of procedures such as prolonged depolarization in the presence of CN (for saturation) and by sequestration of all Ca present in the axoplasm accomplished with injection of EGTA into the axon (for a zero-Ca signal). The average intracellular Ca concentration obtained with Fura-2 was 184 nM. The sensitivity of Fura-2 to intracellular Ca is at least as great as that of aequorin, thus permitting its use in the characterization of Ca homeostasis mechanisms such as Na-Ca exchange. It was found, however, that for voltage-clamp experiments requiring an internal current electrode, Fura-2 is not a convenient Ca probe because electrode reactions in the axoplasm denature the dye, thereby restricting its use in characterization of Ca movements associated with electrically induced changes in membrane potential. A comparison of aequorin luminescence with Fura-2 fluorescence demonstrated that light output by aequorin is linear with intracellular Ca concentrations up to values of 750 nM, changing to a square law relationship from 750 nM up to 10 microM Ca.

Aequorin↗
PubMed1988

A comparison of the effect of experimental general anaesthetics on nerve impulse blockade and on a proteinaceous target site.

While it has been reported that general anaesthetics inhibit the enzyme luciferase and thus reduce the light output of the reaction with luciferin, we find that in squid giant axons injected with luciferin and luciferase, treatment with experimental general anaesthetics at concentrations sufficient to block axonal conduction leads to an increase in the light production by the reaction. This potentiation of the protein activity is best observed when luciferin concentration is above the apparent association constant. Our findings raise doubts regarding the suitability of luciferase as a model for the target region of general anesthetic action.

Action Potentials↗
PubMed1988

The interaction of homologous series of alkanols with sodium channels in nerve membrane vesicles.

The potency of members of the homologous series of alkanols to block 22Na uptake through sodium channels stimulated by veratridine was studied in membrane vesicles obtained from lobster walking leg nerves. A cut-off was revealed at the level of 1-undecanol. However, secondary isomers of inactive primary homologues, such as 5-dodecanol and 5-tridecanol, were able to block ion flux. From the concentration required for an equipotent effect, it was calculated that the standard free energy for adsorption of primary alkanols is -725 cal/mol CH2. Furthermore, since the concentration required for an equipotent effect for primary isomer was found to be lower than that obtained for secondary isomers, it is concluded that the latter are less potent than the former. The similarity between this set of results and those obtained in intact frog sciatic nerve (J. Requena et al., J. Membrane Biol., 84:229-238, 1985) offers further support to the notion that the procedure employed to isolate the membrane vesicles does preserve the Na channels. However, the mechanism of alcohol inhibition of the Na channel in isolated membrane vesicles would seem to be somewhat different from that preferred in axons. While in vesicles the block needs to be thought in terms of a reduction in the number of conducting Na channel, in axons this is considered to be the less likely mode of action, mainly because under veratridine it is not possible to invoke a shift in the steady-state activation or inactivation.

Alcohols↗
PubMed1988

The potassium conductance of the resting squid axon and its blockage by clinical concentrations of general anaesthetics.

1. The effects of some neutral clinical and experimental general anaesthetics on the resting potential of normal squid axons and squid axons exposed to tetrodotoxin and 3,4-diaminopyridine have been studied. 2. Depolarizations of 1-4 mV were produced by all the anaesthetics at 'clinical' concentrations in the normal axon. Larger depolarizations (5-11 mV) were produced by the same anaesthetic concentrations in axons exposed to tetrodotoxin and 3,4-diaminopyridine. 3. The conductance of axons exposed to tetrodotoxin and either tetraethyl-ammonium or 3,4-diaminopyridine in zero Na+, 430 mM-K+ artificial sea water was examined by voltage clamp and AC bridge techniques. 4. The evidence that this conductance is due predominantly to K+ is discussed. 5. Pre-pulse protocols under voltage clamp have been used to show that part of this conductance arises from the incompletely blocked delayed rectifier. 6. Substantial reductions in this conductance are produced by anaesthetics at 'clinical' concentrations. 7. It is concluded that there is a component of the K+ conductance of the resting squid axon other than the Hodgkin-Huxley delayed rectifier which is extremely sensitive to anaesthetics and which to an appreciable extent determines the resting potential.

4-Aminopyridine↗
PubMed1986

Dependence of ionized and total Ca in squid axons on Nao-free or high-Ko conditions.

The level of intracellular Ca in squid axons (both ionized and total Ca) was studied as a function of the experimental variables [Na]i, [Na]o, pHi, cyanide, and depolarization. Ionized Ca was measured by following the light emission of aequorin while total Ca was measured by the atomic absorption analysis of samples of axoplasm. Aequorin glow is known to be increased either by the application of Nao-free solutions or by depolarization produced by external solutions containing greater than normal K concentrations. The present results show that if [Na]i is low, the depolarization that is brought about by solutions with elevated [K] leads to a resting light emission that is decreased rather than increased, as is the case when [Na]i is high. In axons where [Na]i is varied, a comparison of the increments in light emission produced by the application first of Na-free and then of high-K solutions shows that they have an identical dependence on [Na]i, with a half-activation of Ca entry produced by an [Na]i of 25-30 mM. Changes in pHi affect the aequorin signal produced by depolarization, with acidification reducing and alkanization increasing the response. Cyanide did not greatly affect the size of the signal resulting from either Nao removal or that from depolarization.

Animals↗
PubMed1985

Isomers of long-chain alkane derivatives and nervous impulse blockage.

The potency to block nervous impulse of members of normal aliphatic homologous series of primary and secondary isomers of functional derivatives of alkanes was tested in bundles of a few axons from sciatic nerves of the toad Bufo marinus. For the primary substituted functional derivates of pentane, the relative potency series was I greater than H approximately equal to Br greater than Cl greater than COOCH3 greater than F greater than CH2OH greater than COCH3 greater than OH approximately equal to NH2 greater than COOH. For the homologous series of primary alkanols, and from saturated Ringer's solutions, the time required to reversibly reduce the amplitude of the action potential to one-half its initial value were determined. The cut-off effect was detected at the level of dodecan-1-ol, while for the primary bromoalkanes it was bromooctane. However, solutions of the secondary isomer of the inactive primary homologues, such as tridecan-5-ol and tridecan-7-ol or 2-bromononane, were able to block nervous impulse conduction reversibly. From the concentration required for an equipotent effect it was calculated that the standard free energy for adsorption of primary alkanols was -705 cal mol- CH2. Similarly, for primary bromoalkanes a value of -733 cal mol-1 CH2 was obtained. The concentration required for an equipotent effect for primary isomer (either of alkanols or bromoalkanes) is lower than those obtained for the secondary isomers. Therefore, the latter are less potent than the former. Among secondary isomers the potency decreases as the functional group is moved away from the terminal carbon. The differential effect of structural isomers of long-chain alkane derivates around the point of cut-off cannot be explained in terms of differences in chemical properties, concentration in aqueous and membrane phases, or mean molecular volume. It is concluded that a volume related to that of the hydrophobic region of the agent and not its mean molecular volume should be responsible for an expansion of the target region.

Alcohols↗
PubMed1985

Ca2+ entry in squid axons during voltage-clamp pulses is mainly Na+/Ca2+ exchange.

Intact squid axons were injected with aequorin and bathed in 3 mM Ca seawater (a concentration close to that of squid blood). Sodium and potassium currents were pharmacologically blocked and repetitive voltage-clamp pulses of a duration of 1.5 ms were applied (to simulate the duration of an action potential) at amplitudes of +30 to +90 mV and at frequencies of 100/s. In a very fresh axon (low internal Na concentration) no detectable change in aequorin glow resulted from this treatment, whether the axons were in Na-containing or in Na-free seawater. In axons subjected to modest Na loading, repetitive voltage-clamp pulsing did not result in an increased aequorin glow when the pulses were delivered in Na seawater, whereas in Na-free seawater there was an easily measurable increase in aequorin light emission during repetitive pulsing. The increase in aequorin photons emitted per voltage-clamp pulse was e-fold for 22 mV of depolarization, and the process showed no signs of saturating at pulse amplitudes of +180 mV (i.e., at a membrane potential close to ECa). The aequorin light emission per voltage-clamp pulse increased linearly with pulse duration (at constant amplitude).

Action Potentials↗
PubMed1985

The influence of chemical agents on the level of ionized [Ca2+] in squid axons.

Squid giant axons injected with either aequorin or arsenazo III and bathed in 3 mM Ca (Na) seawater were transferred to 3 mM Ca (K) seawater and the response of the aequorin light or the change in the absorbance of arsenazo III was followed. These experimental conditions were chosen because they measure the change in the rate of Na/Ca exchange in introducing Ca into the axon upon depolarization; [Ca]o is too low to effect a channel-based system of Ca entry. This procedure was applied to axons treated with a variety of compounds that have been implicated as inhibitors of Na/Ca exchange. The result obtained was that the substances tested could be placed in three groups. (a) Substances that were without effect on Ca entry effected by Na/Ca exchange were: D600 at 10-100 microM, nitrendipine at 1-5 microM, Ba2+ and Mg2+ at concentrations of 10-50 mM, lidocaine at 0.1-10 mM, cyanide at 2 mM, adriamycin at a concentration of 3 microM, chloradenosine at 35 microM, 2,4-diaminopyridine at 1 mM, Cs+ at 45-90 mM, and tetrodotoxin at 10(-7). (b) Substances that had a significant inhibitory effect on Na/Ca exchange were: Mn2+, Cd2+, and La3+ at 1-50 mM, and quinidine at 50 microM. (c) There were also blocking agents and biochemical inhibitors whose action appeared to be the inhibition of nonmitochondrial Ca buffering in axoplasm rather than an inhibition of Na/Ca exchange. These were the general anesthetic l-octanol at 0.1 mM and 1 mM orthovanadate plus apyrase.

Aequorin↗
PubMed1984

A comparison of measurements of intracellular Ca by Ca electrode and optical indicators.

Squid giant axons were injected with aequorin or arsenazo III and impaled with a Ca-sensing electrode. The light output of aequorin or the spectrophotometer output when measuring arsenazo was compared with the voltage output of the electrode when the squid axon was depolarized with high-K solutions, when the seawater was made Na-free, or when the axon was tetanized for several minutes. The results from these treatments were that the optical response rose (as much as 50-fold) with all treatments known to increase Ca entry, while the electrode remained unaffected by these treatments. If axons previously subjected to Ca load are treated with electron-transport poisons such as CN, it is known that [Ca]i rises after a time necessary to deplete ATP stores. In such axons one expects a rise of [Ca]i in axoplasm which does not necessarily have to be uniform although the source of such Ca is the mitochondria and these are uniformly distributed in axoplasm. Under conditions of CN application, the optical signals from aequorin or arsenazo and Ca electrode output do rise together when [Ca]i is high, but there is a region of [Ca]i concentration where aequorin light output or arsenazo absorbance rises while electrode output does not. Axons not loaded with Ca but injected with apyrase and vanadate have mitochondria that still retain some Ca and this can be released by CN in a truly uniform manner. The results show that such a release (which is small) can be readily measured with aequorin, but again the Ca electrode is insensitive to such [Ca]i change.

Aequorin↗
PubMed1981

The "late" Ca channel in squid axons.

Squid giant axons were injected with aequorin and then treated with seawater containing 50 mM Ca and 100-465 mM K+. Measurements of light production suggested a phasic entry of Ca as well as an enhanced steady-state aequorin glow. After a test K+ depolarization, the aequorin-injected axon was stimulated for 30 min in Li seawater that was Ca-free, a procedure known to reduce [Na]i to about one-half the normal concentration. Reapplication of the elevated K+ test solution now showed that the Ca entry was virtually abolished by this stimulation in Li. A subsequent stimulation of the axon in Na seawater for 30 min resulted in recovery of the response to depolarization by high K+ noted in a normal fresh axon. In axons first tested for a high K+ response and then stimulated in Na seawater for 30 min (where [Na]i increases approximately 30%), there was approximately eight fold enhancement in this response to a test polarization. Axons depolarized with 465 mM K seawater in the absence of external Ca for several minutes were still capable of producing a large phasic entry of Ca when [Ca]0 was made 50 mM, which suggests that it is Ca entry itself rather than membrane depolarization that produced inactivation. Responses to stimulation at 60 pulses/s in Na seawater containing 50 mM Ca are at best only 5% of those measured with high K solutions. The response to repetitive stimulation is not measurable if [Ca]o is made 1 mM, whereas the response to steady depolarization is scarcely affected.

Animals↗
PubMed1980

A dynamic X-ray diffraction study of anaesthesia action. Changes in myelin structure and electrical activity recorded simultaneously from frog sciatic nerves treated with n-alkanes.

Changes induced in the structure and electrical activity of myelin were recorded simultaneously from frog sciatic nerves treated with n-alkanes. The results suggest that the effect of n-alkanes seems to be two-fold: (a) there is an initial reversible phase, in which a significant modification of the X-ray diffraction patterns, concomitant with the continuous fall of the action potential, is observed; (b) there is a final phase which is irreversible. This occurs some time after the complete abolition of the electrical activity. At this stage, further changes of the X-ray diffraction patterns are detected, the most significant of them being in the n-pentane-treated myelin, and consist of an increase in the membrane bilayer thickness.

Action Potentials↗
PubMed1980

Some effects of aliphatic hydrocarbons on the electrical capacity and ionic currents of the squid giant axon membrane.

1. The electrical properties of squid giant axons were examined by means of admittance bridges at frequencies from 0.5 to 300 kHz. A simple equivalent circuit was used to estimate the membrane capacity. 2. The calculated membrane capacities decreased monotonically over the whole frequency range. 3. At 100 kHz and higher frequencies the membrane capacity was independent of potential. 4. At frequencies greater than 20 kHz, exposure of the axons to saturated or 0.9 saturated solutions of n-pentane (275-306 micrometer) reduced the capacity per unit area by 0.1-0.15 micro F cm-2. 5. At 1 kHz the effect of the saturated pentane solutions depended on the membrane potential. In axons having potentials between -60 and zero mV the pentane solutions lowered the capacity, whereas for potentials between -160 and -60 mV they produced little or no change. 6. Saturated solutions of n-hexane, n-heptane and n-octane exhibited qualitatively similar, but quantitatively smaller influences on the membrane capacity, the changes declining as the chain length increased. 7. Under voltage clamp, the peak inward and steady-state outward currents were partially suppressed by the hydrocarbons. Saturated solutions of n-pentane usually reduced the former (reversibly) by 60-80% and the latter by 20-40%. Solutions of n-hexane, n-heptane and n-octane appeared to have successively less effect. Except in deteriorating axons, none of the hydrocarbons produced any consistent changes in the passive membrane resistance, the resting potential or in the reversal potential of the transient inward current. 8. Both the changes in the clamp currents and in the membrane capacity were largely, though not usually completely, reversible. In the hydrocarbon solution the axons deteriorated more rapidly than normal. 9. The responses of axons of Doryteuthis plei to the hydrocarbons were very similar to those of Loligo forbesi with the exception that for the former all observed changes were some five times faster. 10. The time courses of the peak inward and steady-state outward currents on exposure of the axons to n-pentane resembled the time course of the change in membrane capacity at 100 kHz. 11. The simplest interpretation of the high frequency capacity results is suggested to be that, as for lipid bilayers, the membranes become thicker through adsorption of the hydrocarbon.

Alkanes↗