Dafna Singer-Lahat

Tel Aviv University, Tell Afif, Tel Aviv, Israel

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Publications (24)104.84 Total impact

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    ABSTRACT: It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.
    Diabetologia 03/2012; 55(6):1709-20. · 6.49 Impact Factor
  • Biophysical Journal - BIOPHYS J. 01/2011; 100(3).
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    ABSTRACT: Regulation of exocytosis by voltage-gated K(+) channels has classically been viewed as inhibition mediated by K(+) fluxes. We recently identified a new role for Kv2.1 in facilitating vesicle release from neuroendocrine cells, which is independent of K(+) flux. Here, we show that Kv2.1-induced facilitation of release is not restricted to neuroendocrine cells, but also occurs in the somatic-vesicle release from dorsal-root-ganglion neurons and is mediated by direct association of Kv2.1 with syntaxin. We further show in adrenal chromaffin cells that facilitation induced by both wild-type and non-conducting mutant Kv2.1 channels in response to long stimulation persists during successive stimulation, and can be attributed to an increased number of exocytotic events and not to changes in single-spike kinetics. Moreover, rigorous analysis of the pools of released vesicles reveals that Kv2.1 enhances the rate of vesicle recruitment during stimulation with high Ca(2+), without affecting the size of the readily releasable vesicle pool. These findings place a voltage-gated K(+) channel among the syntaxin-binding proteins that directly regulate pre-fusion steps in exocytosis.
    Journal of Cell Science 06/2010; 123(Pt 11):1940-7. · 5.88 Impact Factor
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    Lori Feinshreiber, Dafna Singer-Lahat, Uri Ashery, Ilana Lotan
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    ABSTRACT: Voltage-gated ion channels are well characterized for their function in excitability signals. Accumulating studies, however, have established an ion-independent function for the major classes of ion channels in cellular signaling. During the last few years we established a novel role for Kv2.1, a voltage-gated potassium (Kv) channel, classically known for its role of repolarizing the membrane potential, in facilitation of exocytosis. Kv2.1 induces facilitation of depolarization-induced release through its direct interaction with syntaxin, a protein component of the exocytotic machinery, independently of the potassium ion flow through the channel's pore. Here, we review our recent studies, further characterize the phenomena (using chromaffin cells and carbon fiber amperometry), and suggest plausible mechanisms that can underlie this facilitation of release.
    Annals of the New York Academy of Sciences 02/2009; 1152:87-92. · 4.38 Impact Factor
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    Dafna Singer-Lahat, Dodo Chikvashvili, Ilana Lotan
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    ABSTRACT: K(+) efflux through voltage-gated K(+) (Kv) channels can attenuate the release of neurotransmitters, neuropeptides and hormones by hyperpolarizing the membrane potential and attenuating Ca(2+) influx. Notably, direct interaction between Kv2.1 channels overexpressed in PC12 cells and syntaxin has recently been shown to facilitate dense core vesicle (DCV)-mediated release. Here, we focus on endogenous Kv2.1 channels and show that disruption of their interaction with native syntaxin after ATP-dependent priming of the vesicles by Kv2.1 syntaxin-binding peptides inhibits Ca(2+) -triggered exocytosis of DCVs from cracked PC12 cells in a specific and dose-dependent manner. The inhibition cannot simply be explained by the impairment of the interaction of syntaxin with its SNARE cognates. Thus, direct association between endogenous Kv2.1 and syntaxin enhances exocytosis and in combination with the Kv2.1 inhibitory effect to hyperpolarize the membrane potential, could contribute to the known activity dependence of DCV release in neuroendocrine cells and in dendrites where Kv2.1 commonly expresses and influences release.
    PLoS ONE 02/2008; 3(1):e1381. · 3.73 Impact Factor
  • D. Chikvashvili, D. Singer-Lahat, R. Nachman, I. Lotan
    European Neuropsychopharmacology - EUR NEUROPSYCHOPHARMACOL. 01/2008; 18.
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    ABSTRACT: Kv channels inhibit release indirectly by hyperpolarizing membrane potential, but the significance of Kv channel interaction with the secretory apparatus is not known. The Kv2.1 channel is commonly expressed in the soma and dendrites of neurons, where it could influence the release of neuropeptides and neurotrophins, and in neuroendocrine cells, where it could influence hormone release. Here we show that Kv2.1 channels increase dense-core vesicle (DCV)-mediated release after elevation of cytoplasmic Ca2+. This facilitation occurs even after disruption of pore function and cannot be explained by changes in membrane potential and cytoplasmic Ca2+. However, triggering release increases channel binding to syntaxin, a secretory apparatus protein. Disrupting this interaction with competing peptides or by deleting the syntaxin association domain of the channel at the C terminus blocks facilitation of release. Thus, direct association of Kv2.1 with syntaxin promotes exocytosis. The dual functioning of the Kv channel to influence release, through its pore to hyperpolarize the membrane potential and through its C-terminal association with syntaxin to directly facilitate release, reinforces the requirements for repetitive firing for exocytosis of DCVs in neuroendocrine cells and in dendrites.
    Journal of Neuroscience 03/2007; 27(7):1651-8. · 6.91 Impact Factor
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    ABSTRACT: Previously we suggested that interaction between voltage-gated K+ channels and protein components of the exocytotic machinery regulated transmitter release. This study concerns the interaction between the Kv2.1 channel, the prevalent delayed rectifier K+ channel in neuroendocrine and endocrine cells, and syntaxin 1A and SNAP-25. We recently showed in islet beta-cells that the Kv2.1 K+ current is modulated by syntaxin 1A and SNAP-25. Here we demonstrate, using co-immunoprecipitation and immunocytochemistry analyses, the existence of a physical interaction in neuroendocrine cells between Kv2.1 and syntaxin 1A. Furthermore, using concomitant co-immunoprecipitation from plasma membranes and two-electrode voltage clamp analyses in Xenopus oocytes combined with in vitro binding analysis, we characterized the effects of these interactions on the Kv2.1 channel gating pertaining to the assembly/disassembly of the syntaxin 1A/SNAP-25 (target (t)-SNARE) complex. Syntaxin 1A alone binds strongly to Kv2.1 and shifts both activation and inactivation to hyperpolarized potentials. SNAP-25 alone binds weakly to Kv2.1 and probably has no effect by itself. Expression of SNAP-25 together with syntaxin 1A results in the formation of t-SNARE complexes, with consequent elimination of the effects of syntaxin 1A alone on both activation and inactivation. Moreover, inactivation is shifted to the opposite direction, toward depolarized potentials, and its extent and rate are attenuated. Based on these results we suggest that exocytosis in neuroendocrine cells is tuned by the dynamic coupling of the Kv2.1 channel gating to the assembly status of the t-SNARE complex.
    Journal of Biological Chemistry 10/2003; 278(36):34320-30. · 4.65 Impact Factor
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    ABSTRACT: Recently we suggested that direct interactions between voltage-gated K(+) channels and proteins of the exocytotic machinery, such as those observed between the Kv1.1/Kvbeta channel, syntaxin 1A, and SNAP-25 may be involved in neurotransmitter release. Furthermore, we demonstrated that the direct interaction with syntaxin 1A enhances the fast inactivation of Kv1.1/Kvbeta1.1 in oocytes. Here we show that G-protein betagamma subunits play a crucial role in the enhancement of inactivation by syntaxin 1A. The effect caused by overexpression of syntaxin 1A is eliminated in the presence of chelators of endogenous betagamma subunits in the whole cell and at the plasma membrane. Conversely, enhancement of inactivation caused by overexpression of beta(1)gamma(2) subunits is eliminated upon knock-down of endogenous syntaxin or its scavenging at the plasma membrane. We further show that the N terminus of Kv1.1 binds brain synaptosomal and recombinant syntaxin 1A and concomitantly binds beta(1)gamma(2); the binding of beta(1)gamma(2) enhances that of syntaxin 1A. Taken together, we suggest a mechanism whereby syntaxin and G protein betagamma subunits interact concomitantly with a Kv channel to regulate its inactivation.
    Journal of Biological Chemistry 10/2002; 277(38):34909-17. · 4.65 Impact Factor
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    ABSTRACT: Presynaptic voltage-gated K(+) (Kv) channels play a physiological role in the regulation of transmitter release by virtue of their ability to shape presynaptic action potentials. However, the possibility of a direct interaction of these channels with the exocytotic apparatus has never been examined. We report the existence of a physical interaction in brain synaptosomes between Kvalpha1.1 and Kvbeta subunits with syntaxin 1A, occurring, at least partially, within the context of a macromolecular complex containing syntaxin, synaptotagmin, and SNAP-25. The interaction was altered after stimulation of neurotransmitter release. The interaction with syntaxin was further characterized in Xenopus oocytes by both overexpression and antisense knock-down of syntaxin. Direct physical interaction of syntaxin with the channel protein resulted in an increase in the extent of fast inactivation of the Kv1.1/Kvbeta1.1 channel. Syntaxin also affected the channel amplitude in a biphasic manner, depending on its concentration. At low syntaxin concentrations there was a significant increase in amplitudes, with no detectable change in cell-surface channel expression. At higher concentrations, however, the amplitudes decreased, probably because of a concomitant decrease in cell-surface channel expression, consistent with the role of syntaxin in regulation of vesicle trafficking. The observed physical and functional interactions between syntaxin 1A and a Kv channel may play a role in synaptic efficacy and neuronal excitability.
    Journal of Neuroscience 04/2001; 21(6):1964-74. · 6.91 Impact Factor
  • D Singer-Lahat, N Dascal, L Mittelman, S Peleg, I Lotan
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    ABSTRACT: We describe the preparation of a Xenopus oocyte plasma membrane patch attached to a cover-slip with its intracellular face exposed to the bath solution. The proteins attached to the plasma membrane were visualized by confocal microscopy after fluorescence labelling. Since cortical microfilament elements were detected in these plasma membrane preparations we termed the patches plasma membrane-cortex patches. The way these patches are formed and the low concentration of proteins needed for cytochemical detection make the membrane-cortex patches similar to electrophysiological membrane patches and therefore allow the cytochemical study of ion channels to be correlated with electrophysiological experiments. Furthermore, the described patch is similar to manually isolated plasma membranes used for biochemical analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Cytochemical analysis of membrane-cortex patches also enables the detection of the two-dimensional pattern of organization of membrane proteins (clustered or non-clustered forms). In addition, patch preparations enable cytochemical study of the relative localization of membrane proteins. The methodology enables integration of electrophysiological, biochemical and cytochemical studies of ion channels, giving a comprehensive perspective on ion channel function.
    Pflügers Archiv - European Journal of Physiology 09/2000; 440(4):627-33. · 4.87 Impact Factor
  • D Singer-Lahat, N Dascal, I Lotan
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    ABSTRACT: Modulation of fast-inactivating voltage-gated K+ channels can produce plastic changes in neuronal signaling. Previously, we showed that the voltage-dependent K+ channel composed of brain Kv1.1 and Kvbeta1.1 subunits (alpha(beta) channel) gives rise to a current that has a fast-inactivating and a sustained component; the proportion of the fast-inactivating component could be decreased by dephosphorylation of a basally phosphorylated Ser-446 on the alpha subunit. To account for our results we suggested a model that assumes a bimodal gating of the alpha(beta) channel. In this study, using single-channel analysis, we confirm this model. Two modes of gating were identified: (1) an inactivating mode characterized by low open probability and single openings early in the voltage step, and (2) a non-inactivating gating mode with bursts of openings. These two modes were non-randomly distributed, with spontaneous shifts between them. Each mode is characterized by a different set of open time constants (tau) and mean open times (t(0)). The non-inactivating mode is similar to the gating mode of a homomultimeric alpha channel. The phosphorylation-deficient alphaS446Abeta channel has the same two gating modes. Furthermore, alkaline phosphatase promoted the transition to the non-inactivating mode. This is the first report of modal behavior of a fast-inactivating K+ channel; furthermore, it substantiates the notion that direct phosphorylation is one mechanism that regulates the equilibrium between the two modes and thereby regulates the extent of macroscopic fast inactivation of a brain K+ channel.
    Pflügers Archiv - European Journal of Physiology 01/2000; 439(1-2):18-26. · 4.87 Impact Factor
  • D. Singer-Lahat, N. Dascal, L. Mittelman, S. Peleg, I. Lotan
    Pflugers Archiv-european Journal of Physiology - PFLUGERS ARCH-EUR J PHYSIOL. 01/2000; 440(4).
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    ABSTRACT: Modulation of A-type voltage-gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed-rectifier Kv1.1 channel can be converted to A-type upon association with Kvbeta1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gbeta1gamma2 subunits concomitantly with the channel (composed of Kv1.1 and Kvbeta1.1 subunits), but not after the channel's expression in the plasma membrane, increases the extent of conversion to A-type. Conversely, scavenging endogenous Gbetagamma by co-expression of the C-terminal fragment of the beta-adrenergic receptor kinase reduces the extent of conversion to A-type. The effect of Gbetagamma co-expression is occluded by treatment with dihydrocytochalasin B, a microfilament-disrupting agent shown previously by us to enhance the extent of conversion to A-type, and by overexpression of Kvbeta1.1. Gbeta1gamma2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvbeta1.1. Co-expression of Gbeta1gamma2 causes co-immunoprecipitation with Kv1.1 of more Kvbeta1.1 subunits. Thus, we suggest that Gbeta1gamma2 directly affects the interaction between Kv1.1 and Kvbeta1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A-type conversion.
    The EMBO Journal 04/1999; 18(5):1245-56. · 9.82 Impact Factor
  • D. Singer-Lahat, N. Dascal, I. Lotan
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    ABSTRACT: Modulation of fast-inactivating voltage-gated K+ channels can produce plastic changes in neuronal signaling. Previously, we showed that the voltage-dependent K+ channel composed of brain Kv1.1 and Kv&#351.1 subunits (&#33# channel) gives rise to a current that has a fast-inactivating and a sustained component; the proportion of the fast-inactivating component could be decreased by dephosphorylation of a basally phosphorylated Ser-446 on the ! subunit. To account for our results we suggested a model that assumes a bimodal gating of the &#33# channel. In this study, using single-channel analysis, we confirm this model. Two modes of gating were identified: (1) an inactivating mode characterized by low open probability and single openings early in the voltage step, and (2) a non-inactivating gating mode with bursts of openings. These two modes were non-randomly distributed, with spontaneous shifts between them. Each mode is characterized by a different set of open time constants (&#70) and mean open times (to). The non-inactivating mode is similar to the gating mode of a homomultimeric ! channel. The phosphorylation-deficient &#33S446A# channel has the same two gating modes. Furthermore, alkaline phosphatase promoted the transition to the non-inactivating mode. This is the first report of modal behavior of a fast-inactivating K+ channel; furthermore, it substantiates the notion that direct phosphorylation is one mechanism that regulates the equilibrium between the two modes and thereby regulates the extent of macroscopic fast inactivation of a brain K+ channel.
    Pflügers Archiv - European Journal of Physiology 01/1999; 439(1):18-26. · 4.87 Impact Factor
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    ABSTRACT: Kv1.1/Kvbeta1.1 (alphabeta) K+ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the alpha subunit at Ser-446, which impairs the interaction of the channel with microfilaments. The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of alpha, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C terminus of alpha. Truncation of the last six amino acids resulted in alphabeta channels with an extent of inactivation up to 2.5-fold larger and its further enhancement by cytochalasins being reduced 2-fold. The wild-type channels exhibited strong inactivation, which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the wild-type channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of proteins, we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phosphorylation of the channel, it regulates the extent of inactivation conferred by the beta subunit.
    Journal of Biological Chemistry 06/1997; 272(22):14021-4. · 4.65 Impact Factor
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    ABSTRACT: Kv1.1/Kvβ1.1 (αβ) K+ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the α subunit at Ser-446, which impairs the interaction of the channel with microfilaments. The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of α, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C terminus of α. Truncation of the last six amino acids resulted in αβ channels with an extent of inactivation up to 2.5-fold larger and its further enhancement by cytochalasins being reduced 2-fold. The wild-type channels exhibited strong inactivation, which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the wild-type channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of proteins, we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phosphorylation of the channel, it regulates the extent of inactivation conferred by the β subunit.
    Journal of Biological Chemistry 05/1997; 272(22):14021-14024. · 4.65 Impact Factor
  • Neuroscience Letters - NEUROSCI LETT. 01/1997; 237.
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    ABSTRACT: Voltage-gated K+ channels isolated from mammalian brain are composed of alpha and beta subunits. Interaction between coexpressed Kv1.1 (alpha) and Kvbeta1.1 (beta) subunits confers rapid inactivation on the delayed rectifier-type current that is observed when alpha subunits are expressed alone. Integrating electrophysiological and biochemical analyses, we show that the inactivation of the alphabeta current is not complete even when alpha is saturated with beta, and the alphabeta current has an inherent sustained component, indistinguishable from a pure alpha current. We further show that basal and protein kinase A-induced phosphorylations at Ser-446 of the alpha protein increase the extent, but not the rate, of inactivation of the alphabeta channel, without affecting the association between alpha and beta. In addition, the extent of inactivation is increased by agents that lead to microfilament depolymerization. The effects of phosphorylation and of microfilament depolymerization are not additive. Taken together, we suggest that phosphorylation, via a mechanism that involves the interaction of the alphabeta channel with microfilaments, enhances the extent of inactivation of the channel. Furthermore, phosphorylation at Ser-446 also increases current amplitudes of the alphabeta channel as was shown before for the alpha channel. Thus, phosphorylation enhances in concert inactivation and current amplitudes, thereby leading to a substantial increase in A-type activity.
    Journal of Biological Chemistry 12/1996; 271(46):29321-8. · 4.65 Impact Factor
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    ABSTRACT: Enhancement of cardiac L-type Ca2+ channel activity by norepinephrine via phosphorylation by protein kinase A (PKA) underlines the positive inotropic effect of this transmitter and is a classical example of an ion channel modulation. However, it is not clear whether the channel protein itself (and which subunit) is a substrate for PKA. We have expressed various combinations of the cardiac Ca2+ channel subunits in Xenopus oocytes by injecting subunit mR-NAs. Expression of beta or alpha 2/delta + beta subunits potentiated the native (endogenous) Ca2+ channel currents in the oocyte (similar to T or N but not L-type). This potentiated endogenous current was enhanced by intracellular injection of cAMP or of the catalytic subunit of PKA, and this effect was reversed by the injection of a PKA inhibitor suggesting the presence of basal phosphatase activity. When a cardiac channel of alpha 1 + beta, alpha 1 + alpha 2/delta or alpha 1 + alpha 2/delta + beta composition was expressed at levels high enough that the contribution of the endogenous current became negligible, cAMP and PKA failed to increase the Ca2+ channel current, whereas PKA inhibitors and the catalytic subunit of protein phosphatase 1 reduced the amplitude of the current. Reduction of the current by PKA inhibitors was observed regardless of the presence of the beta subunit, suggesting a major role for the alpha 1 subunit in this process. These results suggest that, like in the heart, when expressed in Xenopus oocytes, the cardiac L-type Ca2+ channels are phosphorylated in basal state and dephosphorylation reduces their activity. However, unlike the situation in the heart, the activity of the channel cannot be enhanced by PKA-catalyzed phosphorylation, suggesting that the channel is already fully phosphorylated in its basal state.
    Receptors and Channels 02/1994; 2(3):215-26.