Julio L. Vergara - Academia.edu (original) (raw)

Papers by Julio L. Vergara

The Journal of Physiology, 1974

1. Single giant barnacle muscle fibres from Megabalanus psittacus (Darwin) were used to measure t... more 1. Single giant barnacle muscle fibres from Megabalanus psittacus (Darwin) were used to measure the Ca entry and the development of tension in the fibres under membrane potential control.2. Fibres bathing in 60 m m‐MgCl2 sea water, free of Ca, did not develop tension with sudden displacements of the membrane potential towards more positive values. This failure to develop tension with depolarizations was observed with and without the internal application of Ca buffers.3. Fibres bathing in artificial sea water with either 10, 20, 60 or 100 m m‐CaCl2 developed tension with depolarization even after 60 min of internal perfusion of the fibres with solution containing no Ca buffers. In this case the maximum tension recorded during a voltage clamp run decreased with time from nearly 2·5 to 0·2 kg/cm2. However, addition of 10 m m‐Tris‐EGTA (ethyleneglycol‐bis (β‐aminoethyl ether) N, N′ — tetraacetic acid) to the perfusing solution rapidly eliminated the development of tension; after 10 min ...

The Journal of Cell Biology, 1975

The transverse tubular system (TTS) of skeletal muscle fibers represents the morphological basis ... more The transverse tubular system (TTS) of skeletal muscle fibers represents the morphological basis for the inward spread of conduction of the electrical signal that triggers muscle contraction. A historical account of the main steps contributing to the elucidation of the structure and function of the TSS has been presented by Huxley (1971). While the localization of the TSS and its association with the sarcoplasmic reticulum (SR) is well documented; there is still a need further to develop our knowledge of the morphology of the connection between the TSS and the plasma membrane. It is generally believed that the TSS opens directly to the extracellular space and that there is continuity between its membrane and the sarcolemma. However, direct observation of such a connection has been clearly shown only for the myotome of fish (Franzini-Armstrong and Porter, 1964). In other muscle fibers, only indirect evidence of the connection has been provided by experiments showing penetration of ex...

Biophysical Journal, 2014

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1983

Ca2+-selective electrodes have been used to measure free intraceilular Ca 2+ concentrations in sq... more Ca2+-selective electrodes have been used to measure free intraceilular Ca 2+ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 ttm in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca 2+ down to about 3/tM in solutions containing 0.3 M K + and 0.025 M Na +. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca 2+ over Mg 2+, H +, K + and Na +. To calibrate them properly, a set of standard solutions were prepared using different Ca 2+ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca 2 + association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca 2+, the mean resting intracellular ionized calcium concentration was 0.106 pM (n = 15). The Ca2+-electrodes were used to investigate effects of different experimental procedures on the [Ca2+]i. The main conclusions are: (i) intact axons can extrude calcium ions at low ICa2+ h levels by a process independent of external Na+; (ii) poisoned axons can extrude calcium ions at high levels of [Ca2+]i by an external Na+-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.

Biophysical Journal, 2014

membrane capacitance measurement. By recording vesicle fusion with capacitance measurements at th... more membrane capacitance measurement. By recording vesicle fusion with capacitance measurements at this synapse, here we show that acute application of BDNF inhibits exocytosis. This effect is specific to BDNF because application of K252a, an inhibitor of the BDNF receptor, TkB, blocks the actions of BDNF on presynaptic function. Moreover, BDNF inhibits rapid and slow endocytosis, thus providing a regulatory mechanism for vesicle recycling. Furthermore, we recorded the presynaptic calcium current directly at the calyx nerve terminal and found for the first time that BDNF inhibits calcium current. It has been shown previously, that both exo-and endocytosis are regulated by calcium influx. Thus, our findings establish that neurotrophins regulate the function of the nerve terminal via presynaptic calcium channels. Further understanding of these mechanisms will advance our knowledge of synaptic transmission regulation, a process essential for our brain function.

Journal of General Physiology, 2006

The spatiotemporal characteristics of the Ca2+ release process in mouse skeletal muscle were inve... more The spatiotemporal characteristics of the Ca2+ release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 μM of the low-affinity Ca2+ indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca2+ chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca2+] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks (∼23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the t...

Journal of Neuroscience Methods, 2014

h i g h l i g h t s • We provide instructions for building a low-cost epifluorescence upright mic... more h i g h l i g h t s • We provide instructions for building a low-cost epifluorescence upright microscope. • The design of a microscope using catalog-available parts reduces the time and price of assembling. • This microscope is suitable for visualized recording and fluorescence detection. • This open-box microscope allows researcher to modify it for a wide variety of experimental setups.

The Journal of General Physiology, 1982

The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced indiv... more The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced individually into cut single frog skeletal muscle fibers from which calcium transients have been elicited either by action potential stimulation or by voltage-clamp pulses of up to 50 ms in duration. Calcium transients recorded with both dyes at selected wavelengths have similar characteristics when elicited by action potentials. Longer voltage-clamp pulse stimulation reveals differences in the late phases of the optical signals obtained with the two dyes. The effects of different tension blocking methods on Ca transients were compared experimentally. Internal application of EGTA at concentrations up to 3 mM was demonstrated to be efficient in blocking movement artifacts without affecting Ca transients. Higher EGTA concentrations affect the Ca signals' characteristics. Differential effects of internally applied EGTA on tension development as opposed to calcium transients suggest that diff...

Biophysical Journal, 1983

The equilibrium interactions of the metallochromic indicator arsenazo III with calcium at physiol... more The equilibrium interactions of the metallochromic indicator arsenazo III with calcium at physiological ionic strength and pH were investigated spectrophotometrically and with the aid of a calcium electrode. Evidence suggests the formation of more than one dye-calcium complex. The analysis of data obtained over a 10,000-fold range of dye concentrations concludes that at the concentrations used for in vitro biochemical studies (10-100 ,M), arsenazo III absorbance changes in response to calcium binding primarily involve the formation of a complex involving two dye molecules and two calcium ions. At millimolar dye concentrations, typical of physiological calcium transient determinations in situ, a second complex involving two arsenazo III molecules and one calcium ion is additionally formed. A third complex, involving one arsenazo III molecule and one calcium ion, is formed at very low dye concentrations. The results reported here suggest that equilibrium calibrations of the dye with calcium cannot be used directly to satisfactorily relate transient absorbance changes in physiological preparations to calcium concentration changes since several stoichiometrically distinct complexes with different absorbances could be formed at different rates. The results of this study do not permit the elucidation of a unique kinetic scheme of arsenazo III complexation with calcium; for this, in vitro kinetic analysis is required. Results of similar analysis of the dye interaction with magnesium are also reported, and these appear compatible with a much simpler model of complexation.

The Journal of the Acoustical Society of America, 2013

ABSTRACT The goal of this study is to develop a structurally based constitutive model to characte... more ABSTRACT The goal of this study is to develop a structurally based constitutive model to characterize the structure-function relationship of the vocal folds. Compared to phenomenological models, structurally based constitutive models allow direct prediction of changes in the mechanical behavior of the vocal folds as a result of aging or pathological conditions. The first significant step in developing such a mathematical model is to characterize the structural arrangement and load-bearing behavior of the collagen and elastin fibers, the two most mechanically significant structural proteins in the vocal fold. A micro horizontal uniaxial tensile system has been designed and coupled with the non-invasive multi-photon microscopy method. The load-bearing or recruitment behavior of collagen was characterized by simultaneously measuring the waviness of the collagen fibers and stress of the cover layer at different strain conditions. The structural arrangement of the collagen and elastin fibers in the different layers of the lamina propria were also quantified. The results of this study will directly elucidate the specific contributions of the elastin and collagen fibers to the vocal fold mechanical behavior under uniaxial tension. [Work supported by NIH.].

The Journal of Neuroscience, 1997

The understanding of neurotransmitter release at vertebrate synapses has been hampered by the pau... more The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonic Xenopus neuromuscular junctions and simultaneous pre-and postsynaptic patch-clamp current-recording procedures to identify the major presynaptic conductances underlying the initiation of neurotransmitter release. Step depolarizations and action potential waveforms elicited Na and K currents along with Ca and Ca-activated K (K Ca) currents. The onset of K Ca current preceded the peak of the action potential. The predominantly-CgTX GVIA-sensitive Ca current occurred primarily during the falling phase, but there was also significant Ca 2ϩ entry during the rising phase of the action potential. The postsynaptic current began a mean of 0.7 msec after the time of maximum rate of rise of the Ca current.-CgTX also blocked K Ca currents and transmitter release during an action potential, suggesting that Ca and K Ca channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments, K Ca channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation of K Ca channels or the diffusion/removal of Ca 2ϩ from sites of neurotransmitter release after an action potential.

American Journal of Physiology-Cell Physiology, 2000

The Na+/Ca2+ exchanger participates in Ca2+ homeostasis in a variety of cells and has a key role ... more The Na+/Ca2+ exchanger participates in Ca2+ homeostasis in a variety of cells and has a key role in cardiac muscle physiology. We studied in this work the exchanger of amphibian skeletal muscle, using both isolated inside-out transverse tubule vesicles and single muscle fibers. In vesicles, increasing extravesicular (intracellular) Na+ concentration cooperatively stimulated Ca2+ efflux (reverse mode), with the Hill number equal to 2.8. In contrast to the stimulation of the cardiac exchanger, increasing extravesicular (cytoplasmic) Ca2+ concentration ([Ca2+]) inhibited this reverse activity with an IC50 of 91 nM. Exchanger-mediated currents were measured at 15°C in single fibers voltage clamped at −90 mV. Photolysis of a cytoplasmic caged Ca2+ compound activated an inward current (forward mode) of 23 ± 10 nA ( n = 3), with an average current density of 0.6 μA/μF. External Na+ withdrawal generated an outward current (reverse mode) with an average current density of 0.36 ± 0.17 μA/μF (...

Physiology, 1987

There is a temperature-dependent lag between depolarization of transverse tubules by the action p... more There is a temperature-dependent lag between depolarization of transverse tubules by the action potential and onset of calcium release from the sacromplasmic reticulum that reveals the occurrence of a chemical step in excitation-contraction coupling. Recent studies suggest that in vertebrate muscle inositol 1,4,5-trisphosphate may act as a chemical link in this process.

Skeletal muscle, Jun 6, 2017

Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a ... more Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II trans...

<p>Panel A: Image of a VL fiber from a healthy volunteer obtained with a 20× objective. The... more <p>Panel A: Image of a VL fiber from a healthy volunteer obtained with a 20× objective. The inner part of the grease seals delimiting the experimental pool can be seen at both sides of the image. Panel B: The area delimited by the square in A was imaged with a 100× immersion objective. A sharp banding pattern and smooth border can be seen. Panels C and D shows a VL fiber from a HF patient at the same magnifications as in A and B, respectively. The diameter and sarcomere length were 62.0 and 3.0 µm for the fiber in A, and 80 and 2.8 µm for the fiber in B, respectively. The insets in panels B and D show intensity profiles measured across ∼4 sarcomeres. The profiles span ∼16 mm along the directions indicated by the lines superimposed in the corresponding images. The dots and lines represent the intensity values and Gaussian fits to the data, respectively. The scale in A corresponds to 100 µm in A and C, and 20 µm in B and D.</p

Skeletal muscle, 2016

Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wa... more Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca(2+) release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca(2+) handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low a...

Excitation-Contraction Coupling in Skeletal, Cardiac, and Smooth Muscle, 1992

1. Adv Exp Med Biol. 1992;311:227-36. Imaging of calcium transients during excitation-contraction... more 1. Adv Exp Med Biol. 1992;311:227-36. Imaging of calcium transients during excitation-contraction coupling in skeletal muscle fibers. Vergara J, DiFranco M. Department of Physiology, University of California Los Angeles. PMID: 1529756 [PubMed - indexed for MEDLINE]. ...

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1993

Quantitative data are presented on the composition of the major phospholipids in isolated giant b... more Quantitative data are presented on the composition of the major phospholipids in isolated giant barnacle muscle fibers. It is shown, using internal perfusion techniques, that the high specific activity of labeling of polyphosphoinositides in vivo is attained by the activities of specific kinases. Electrical stimulation causes a reduction in the specific activity of labeling of PtdInsP,. This phospholipid, which is the immediate precursor for the release of InsP3, is found at a significant concentration (40 nmol/g wet weight) in single barnacle muscle fibers, sufficient to support a role as precursor of second messengers. The rapid catabolization of PtdInsPz in the absence of external Ca*' suggests that E-C coupling in barnacle muscle may be associated with a voltage-dependent, CaZC-independent, activation of the breakdown of polyphosphoinositides.

The Journal of Physiology, 1974

1. Single giant barnacle muscle fibres from Megabalanus psittacus (Darwin) were used to measure t... more 1. Single giant barnacle muscle fibres from Megabalanus psittacus (Darwin) were used to measure the Ca entry and the development of tension in the fibres under membrane potential control.2. Fibres bathing in 60 m m‐MgCl2 sea water, free of Ca, did not develop tension with sudden displacements of the membrane potential towards more positive values. This failure to develop tension with depolarizations was observed with and without the internal application of Ca buffers.3. Fibres bathing in artificial sea water with either 10, 20, 60 or 100 m m‐CaCl2 developed tension with depolarization even after 60 min of internal perfusion of the fibres with solution containing no Ca buffers. In this case the maximum tension recorded during a voltage clamp run decreased with time from nearly 2·5 to 0·2 kg/cm2. However, addition of 10 m m‐Tris‐EGTA (ethyleneglycol‐bis (β‐aminoethyl ether) N, N′ — tetraacetic acid) to the perfusing solution rapidly eliminated the development of tension; after 10 min ...

The Journal of Cell Biology, 1975

The transverse tubular system (TTS) of skeletal muscle fibers represents the morphological basis ... more The transverse tubular system (TTS) of skeletal muscle fibers represents the morphological basis for the inward spread of conduction of the electrical signal that triggers muscle contraction. A historical account of the main steps contributing to the elucidation of the structure and function of the TSS has been presented by Huxley (1971). While the localization of the TSS and its association with the sarcoplasmic reticulum (SR) is well documented; there is still a need further to develop our knowledge of the morphology of the connection between the TSS and the plasma membrane. It is generally believed that the TSS opens directly to the extracellular space and that there is continuity between its membrane and the sarcolemma. However, direct observation of such a connection has been clearly shown only for the myotome of fish (Franzini-Armstrong and Porter, 1964). In other muscle fibers, only indirect evidence of the connection has been provided by experiments showing penetration of ex...

Biophysical Journal, 2014

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1983

Ca2+-selective electrodes have been used to measure free intraceilular Ca 2+ concentrations in sq... more Ca2+-selective electrodes have been used to measure free intraceilular Ca 2+ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 ttm in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca 2+ down to about 3/tM in solutions containing 0.3 M K + and 0.025 M Na +. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca 2+ over Mg 2+, H +, K + and Na +. To calibrate them properly, a set of standard solutions were prepared using different Ca 2+ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca 2 + association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca 2+, the mean resting intracellular ionized calcium concentration was 0.106 pM (n = 15). The Ca2+-electrodes were used to investigate effects of different experimental procedures on the [Ca2+]i. The main conclusions are: (i) intact axons can extrude calcium ions at low ICa2+ h levels by a process independent of external Na+; (ii) poisoned axons can extrude calcium ions at high levels of [Ca2+]i by an external Na+-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.

Biophysical Journal, 2014

membrane capacitance measurement. By recording vesicle fusion with capacitance measurements at th... more membrane capacitance measurement. By recording vesicle fusion with capacitance measurements at this synapse, here we show that acute application of BDNF inhibits exocytosis. This effect is specific to BDNF because application of K252a, an inhibitor of the BDNF receptor, TkB, blocks the actions of BDNF on presynaptic function. Moreover, BDNF inhibits rapid and slow endocytosis, thus providing a regulatory mechanism for vesicle recycling. Furthermore, we recorded the presynaptic calcium current directly at the calyx nerve terminal and found for the first time that BDNF inhibits calcium current. It has been shown previously, that both exo-and endocytosis are regulated by calcium influx. Thus, our findings establish that neurotrophins regulate the function of the nerve terminal via presynaptic calcium channels. Further understanding of these mechanisms will advance our knowledge of synaptic transmission regulation, a process essential for our brain function.

Journal of General Physiology, 2006

The spatiotemporal characteristics of the Ca2+ release process in mouse skeletal muscle were inve... more The spatiotemporal characteristics of the Ca2+ release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 μM of the low-affinity Ca2+ indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca2+ chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca2+] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks (∼23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the t...

Journal of Neuroscience Methods, 2014

h i g h l i g h t s • We provide instructions for building a low-cost epifluorescence upright mic... more h i g h l i g h t s • We provide instructions for building a low-cost epifluorescence upright microscope. • The design of a microscope using catalog-available parts reduces the time and price of assembling. • This microscope is suitable for visualized recording and fluorescence detection. • This open-box microscope allows researcher to modify it for a wide variety of experimental setups.

The Journal of General Physiology, 1982

The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced indiv... more The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced individually into cut single frog skeletal muscle fibers from which calcium transients have been elicited either by action potential stimulation or by voltage-clamp pulses of up to 50 ms in duration. Calcium transients recorded with both dyes at selected wavelengths have similar characteristics when elicited by action potentials. Longer voltage-clamp pulse stimulation reveals differences in the late phases of the optical signals obtained with the two dyes. The effects of different tension blocking methods on Ca transients were compared experimentally. Internal application of EGTA at concentrations up to 3 mM was demonstrated to be efficient in blocking movement artifacts without affecting Ca transients. Higher EGTA concentrations affect the Ca signals' characteristics. Differential effects of internally applied EGTA on tension development as opposed to calcium transients suggest that diff...

Biophysical Journal, 1983

The equilibrium interactions of the metallochromic indicator arsenazo III with calcium at physiol... more The equilibrium interactions of the metallochromic indicator arsenazo III with calcium at physiological ionic strength and pH were investigated spectrophotometrically and with the aid of a calcium electrode. Evidence suggests the formation of more than one dye-calcium complex. The analysis of data obtained over a 10,000-fold range of dye concentrations concludes that at the concentrations used for in vitro biochemical studies (10-100 ,M), arsenazo III absorbance changes in response to calcium binding primarily involve the formation of a complex involving two dye molecules and two calcium ions. At millimolar dye concentrations, typical of physiological calcium transient determinations in situ, a second complex involving two arsenazo III molecules and one calcium ion is additionally formed. A third complex, involving one arsenazo III molecule and one calcium ion, is formed at very low dye concentrations. The results reported here suggest that equilibrium calibrations of the dye with calcium cannot be used directly to satisfactorily relate transient absorbance changes in physiological preparations to calcium concentration changes since several stoichiometrically distinct complexes with different absorbances could be formed at different rates. The results of this study do not permit the elucidation of a unique kinetic scheme of arsenazo III complexation with calcium; for this, in vitro kinetic analysis is required. Results of similar analysis of the dye interaction with magnesium are also reported, and these appear compatible with a much simpler model of complexation.

The Journal of the Acoustical Society of America, 2013

ABSTRACT The goal of this study is to develop a structurally based constitutive model to characte... more ABSTRACT The goal of this study is to develop a structurally based constitutive model to characterize the structure-function relationship of the vocal folds. Compared to phenomenological models, structurally based constitutive models allow direct prediction of changes in the mechanical behavior of the vocal folds as a result of aging or pathological conditions. The first significant step in developing such a mathematical model is to characterize the structural arrangement and load-bearing behavior of the collagen and elastin fibers, the two most mechanically significant structural proteins in the vocal fold. A micro horizontal uniaxial tensile system has been designed and coupled with the non-invasive multi-photon microscopy method. The load-bearing or recruitment behavior of collagen was characterized by simultaneously measuring the waviness of the collagen fibers and stress of the cover layer at different strain conditions. The structural arrangement of the collagen and elastin fibers in the different layers of the lamina propria were also quantified. The results of this study will directly elucidate the specific contributions of the elastin and collagen fibers to the vocal fold mechanical behavior under uniaxial tension. [Work supported by NIH.].

The Journal of Neuroscience, 1997

The understanding of neurotransmitter release at vertebrate synapses has been hampered by the pau... more The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonic Xenopus neuromuscular junctions and simultaneous pre-and postsynaptic patch-clamp current-recording procedures to identify the major presynaptic conductances underlying the initiation of neurotransmitter release. Step depolarizations and action potential waveforms elicited Na and K currents along with Ca and Ca-activated K (K Ca) currents. The onset of K Ca current preceded the peak of the action potential. The predominantly-CgTX GVIA-sensitive Ca current occurred primarily during the falling phase, but there was also significant Ca 2ϩ entry during the rising phase of the action potential. The postsynaptic current began a mean of 0.7 msec after the time of maximum rate of rise of the Ca current.-CgTX also blocked K Ca currents and transmitter release during an action potential, suggesting that Ca and K Ca channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments, K Ca channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation of K Ca channels or the diffusion/removal of Ca 2ϩ from sites of neurotransmitter release after an action potential.

American Journal of Physiology-Cell Physiology, 2000

The Na+/Ca2+ exchanger participates in Ca2+ homeostasis in a variety of cells and has a key role ... more The Na+/Ca2+ exchanger participates in Ca2+ homeostasis in a variety of cells and has a key role in cardiac muscle physiology. We studied in this work the exchanger of amphibian skeletal muscle, using both isolated inside-out transverse tubule vesicles and single muscle fibers. In vesicles, increasing extravesicular (intracellular) Na+ concentration cooperatively stimulated Ca2+ efflux (reverse mode), with the Hill number equal to 2.8. In contrast to the stimulation of the cardiac exchanger, increasing extravesicular (cytoplasmic) Ca2+ concentration ([Ca2+]) inhibited this reverse activity with an IC50 of 91 nM. Exchanger-mediated currents were measured at 15°C in single fibers voltage clamped at −90 mV. Photolysis of a cytoplasmic caged Ca2+ compound activated an inward current (forward mode) of 23 ± 10 nA ( n = 3), with an average current density of 0.6 μA/μF. External Na+ withdrawal generated an outward current (reverse mode) with an average current density of 0.36 ± 0.17 μA/μF (...

Physiology, 1987

There is a temperature-dependent lag between depolarization of transverse tubules by the action p... more There is a temperature-dependent lag between depolarization of transverse tubules by the action potential and onset of calcium release from the sacromplasmic reticulum that reveals the occurrence of a chemical step in excitation-contraction coupling. Recent studies suggest that in vertebrate muscle inositol 1,4,5-trisphosphate may act as a chemical link in this process.

Skeletal muscle, Jun 6, 2017

Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a ... more Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II trans...

<p>Panel A: Image of a VL fiber from a healthy volunteer obtained with a 20× objective. The... more <p>Panel A: Image of a VL fiber from a healthy volunteer obtained with a 20× objective. The inner part of the grease seals delimiting the experimental pool can be seen at both sides of the image. Panel B: The area delimited by the square in A was imaged with a 100× immersion objective. A sharp banding pattern and smooth border can be seen. Panels C and D shows a VL fiber from a HF patient at the same magnifications as in A and B, respectively. The diameter and sarcomere length were 62.0 and 3.0 µm for the fiber in A, and 80 and 2.8 µm for the fiber in B, respectively. The insets in panels B and D show intensity profiles measured across ∼4 sarcomeres. The profiles span ∼16 mm along the directions indicated by the lines superimposed in the corresponding images. The dots and lines represent the intensity values and Gaussian fits to the data, respectively. The scale in A corresponds to 100 µm in A and C, and 20 µm in B and D.</p

Skeletal muscle, 2016

Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wa... more Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca(2+) release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca(2+) handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low a...

Excitation-Contraction Coupling in Skeletal, Cardiac, and Smooth Muscle, 1992

1. Adv Exp Med Biol. 1992;311:227-36. Imaging of calcium transients during excitation-contraction... more 1. Adv Exp Med Biol. 1992;311:227-36. Imaging of calcium transients during excitation-contraction coupling in skeletal muscle fibers. Vergara J, DiFranco M. Department of Physiology, University of California Los Angeles. PMID: 1529756 [PubMed - indexed for MEDLINE]. ...

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1993

Quantitative data are presented on the composition of the major phospholipids in isolated giant b... more Quantitative data are presented on the composition of the major phospholipids in isolated giant barnacle muscle fibers. It is shown, using internal perfusion techniques, that the high specific activity of labeling of polyphosphoinositides in vivo is attained by the activities of specific kinases. Electrical stimulation causes a reduction in the specific activity of labeling of PtdInsP,. This phospholipid, which is the immediate precursor for the release of InsP3, is found at a significant concentration (40 nmol/g wet weight) in single barnacle muscle fibers, sufficient to support a role as precursor of second messengers. The rapid catabolization of PtdInsPz in the absence of external Ca*' suggests that E-C coupling in barnacle muscle may be associated with a voltage-dependent, CaZC-independent, activation of the breakdown of polyphosphoinositides.