PubMed Health⌕ Search

Biomedical subjects

G Isenberg

Publications and source records attributed to G Isenberg.

At least 55 records · Page 3Linked to original sources
PubMed1998

Peptide-specific antibodies localize the major lipid binding sites of talin dimers to oppositely arranged N-terminal 47 kDa subdomains.

Using ultrastructural analysis and labeling with polyclonal antibodies that recognize peptide sequences specific for phospholipid binding, we mapped the functional domain structure of intact platelet talin and its proteolytic fragments. The talin dimer, which is crucial for actin and lipid binding, is built of a backbone containing the 200 kDa rod portions, at both ends of which a 47 kDa globular domain is attached. Peptide-specific polyclonal antibodies were raised against three potential lipid binding sequences residing within the N-terminal 47 kDa domain (i.e. S19, amino acids 21-39; H18, amino acids 287-304; and H17, amino acids 385-406). Antibodies H17 and H18 localize these lipid binding sequences within the N-terminal 47 kDa globular talin subdomains opposed at the outer 200 kDa rod domains within talin dimers. Hence, we conclude that in its dimeric form, which is used in actin and lipid binding, talin is a dumbbell-shaped molecule built of two antiparallel subunits.

Amino Acid Sequence↗
PubMed1998

Use of guanylyl cyclase C for detecting micrometastases in lymph nodes of patients with colon cancer.

INTRODUCTION: Guanylyl cyclase C appears to be expressed only in colorectal cancer cells in extraintestinal tissues. Thus, guanylyl cyclase C may be useful as a marker to detect colorectal cancer micrometastases not detectable by histopathology in lymph nodes of patients. METHODS: Twelve patients with colon adenocarcinoma, Dukes Stages A through C2, and one patient with a tubulovillous adenoma were included in this study. Forty-two lymph nodes were collected from fresh surgical specimens, and each was examined by histopathology and reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers. Histopathology identified colon cancer cells in 6 of 16 lymph nodes from five Dukes Stage C patients but not in lymph nodes from the patient with a tubulovillous adenoma, the Dukes Stage A patient, or six Dukes Stage B patients. Reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers was performed on all 42 lymph nodes. RESULTS: Guanylyl cyclase C messenger RNA was not detected by reverse transcription followed by polymerase chain reaction in lymph nodes from the patient with the tubulovillous adenoma or the patient with Dukes Stage A colon carcinoma. Seven lymph nodes from Dukes Stage C patients revealed guanylyl cyclase C messenger RNA including six lymph nodes containing histopathologically confirmed metastases. Of significance, guanylyl cyclase C messenger RNA was detected in 6 of 21 lymph nodes from Dukes Stage B patients. Indeed, clinical staging of two patients could be upgraded from B to C using reverse transcription followed by polymerase chain reaction and guanylyl cyclase C-specific primers. CONCLUSION: Reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers might be useful to more accurately assess micrometastases in lymph nodes of colorectal cancer patients undergoing disease staging.

Adenocarcinoma↗
PubMed1998

Endoscopic ultrasound in restaging of esophageal cancer after neoadjuvant chemoradiation.

BACKGROUND: Endoscopic ultrasound (EUS) is established as the most accurate method currently available for determining the depth of primary cancer invasion (T stage). Standard EUS criteria may not be accurate in assessing depth of cancer invasion and nodal status after patients have received chemotherapy or radiotherapy. METHODS: We conducted a prospective study to determine whether EUS estimation of tumor size could be used to assess response to preoperative chemoradiation. Using EUS, TNM stage was assessed in 31 patients (22 men, 9 women; mean age 62 years) with cancer of esophagus or cardia (19 adenocarcinoma, 12 squamous cell cancer) before initiation of combined radiation and 5-fluorouracil/cisplatin (and/or carboplatinum) chemotherapy. The cross-sectional area of the tumor in the transverse plane at the location where the tumor had maximal thickness was calculated to estimate tumor size. EUS staging and measurement of maximal cross-sectional area were repeated at completion of chemoradiation just before surgery. Response to preoperative chemoradiation was defined as 50% reduction in maximal cross-sectional area. Surgical staging was compared between responders and nonresponders. RESULTS: Eight patients who did not undergo surgery were excluded from analysis. EUST stage in the remaining 23 patients before therapy was as follows: 3 T2, 16 T3, and 4 T4. After chemoradiation, EUS T staging was changed in 6 patients (3 T4 downstaged to T3, 2 T3 downstaged to T2, and 1 T3 downstaged to T1). At surgical pathological examination, 3 patients had no residual tumor in the esophagus (T0), 5 had T1, 3 had T2, 10 had T3, and 2 had T4 tumors. EUS T staging accuracy after adjuvant therapy was only 43%. Maximal cross-sectional area decreased from a mean of 5.5 +/- 2.4 to 1.6 +/- 0.9 cm2 in responders, whereas maximal cross-sectional area went from 7.0 +/- 3.0 to 5.4 +/- 2.2 cm2 in nonresponders (p = 0.009). Ten of thirteen patients with at least a 50% reduction in maximal cross-sectional area (responders) had T0, T1, or T2 tumors at surgery, whereas 9 of 10 nonresponders had T3 or T4 tumors at surgery (p = 0.001). CONCLUSIONS: (1) Standard EUS staging criteria are not accurate after neoadjuvant chemoradiation, (2) reduction in maximal cross-sectional area of tumor appears to be a more useful measure for assessing response of esophageal cancer to preoperative chemoradiation, and (3) responders have an increased likelihood of downstaging at surgery than nonresponders.

Adenocarcinoma↗
PubMed1998

Interaction of cytoskeletal proteins with membrane lipids.

Rapid and significant progress has been made in understanding lipid/protein interactions involving cytoskeletal components and the plasma membrane. Covalent and noncovalent lipid modifications of cytoskeletal proteins mediate their interaction with lipid bilayers. The application of biophysical techniques such as differential scanning colorimetry, neutron reflection, electron spin resonance, CD spectroscopy, nuclear magnetic resonance, and hydrophobic photolabeling, allow various folding stages of proteins during electrostatic adsorption and hydrophobic insertion into lipid bilayers to be analyzed. Reconstitution of proteins into planar lipid films and liposomes help to understand the architecture of biological interfaces. During signaling events at plasma membrane interfaces, lipids are important for the regulation of catalytic protein functions. Protein/lipid interactions occur selectively and with a high degree of specificity and thus have to be considered as physiologically relevant processes with gaining impact on cell functions.

Amino Acid Sequence↗
PubMed1997

Examining F-actin interaction with intact talin and talin head and tail fragment using static and dynamic light scattering.

We examined the binding kinetics of intact talin and talin head and tail fragment with F-actin at pH 7.0 and at low ionic strength. We observed by a transient kinetic method a fast followed by a slower binding process for intact talin and talin tail fragment with filamentous actin. The latter can be attributed to F-actin cross-linking and/or bundling, which was observed in cosedimentation assays as well as by low shear viscometry and electron microscopy [Zhang, J., Robson, R. M., Schmidt, J. M. & Stromer, M. H. (1996) Biochem. Biophys. Res. Commun. 218, 530-537]. This finding is supported by dynamic light scattering measurements, indicating changes in internal actin filament dynamics due to cross-linking/bundling events with intact talin and talin tail fragment. No binding of the talin head fragment with F-actin was detected by either method.

Actins↗
PubMed1997

Viscoelasticity of actin-gelsolin networks in the presence of filamin.

Cross-linking of actin filaments by filamin by means of frequency-dependent rheology yields an increase in the filament's elasticity and stiffness. Higher cross-linker (filamin) ratios are required for mean actin-filament lengths of 5-6 microm than for random-length distribution of actin filaments. The loss modulus (i.e. the viscous portion) in the region of the internal-chain dynamics [G"(omega) approximately omega(alpha)] is influenced by the cross-linking of filaments, and with an increasing molar ratio of filamin/actin a reduction of alpha is observed. Rheological measurements reveal that actin networks are already formed at the polymerizing stage at a molar ratio of filamin/actin of less than 1:100, and electron micrographs show phase separation of actin/filament networks of low density and of actin/filament bundles.

Actins↗
PubMed1996

Probing phosphatidylinositolphosphates and adenosinenucleotides on talin nucleated actin polymerization.

We have investigated the binding of PI, PIP and PIP2 to talin and the effect of phosphoinositides and adenosinenucleotides on talin-induced actin polymerization. At physiological salt concentrations, talin coprecipitates with liposomes when containing phosphoinositides but not when containing PI. The nucleating effect of talin as reflected by a twofold increase of fluorescence during the polymerization of actin labelled with NBD is not inhibited by phosphoinositides. The polymerization of ADP-actin versus ATP-actin was investigated in the presence and absence of talin by NBD fluorescence. ADP-actin nucleation induced by talin is comparably efficient as with ATP-actin. These experimental findings in summary have implications when evaluating the role of talin during cell activation.

Actins↗
PubMed1996

A large-conductance K+ channel that is inhibited by the cytoskeleton in the smooth muscle cell line DDT1 MF-2.

1. DDT1 MF-2 smooth muscle cells responded to the bath application of histamine or ATP with an increase in the cytosolic Ca2+ concentration ([Ca2+]c) and the whole-cell K+ current, IK(BA). 2. In cell-attached patches, histamine (100 microM) activated currents through a 200 pS K+ channel ('BKA' channel). In the absence of agonists, the BKA channel was activated by excision of the patch. Both histamine and patch excision increased the channel activity (NPo; where N is the number of channels per patch and Po is the open probability) by reducing the long closures between the bursts of openings. 3. In inside-out patches, the BKA channel had a conductance of 201 +/- 4 pS (symmetrical solutions of 150 mM KCl, 2 mM MgCl2 and 2 mM EGTA). Replacement of K+ in the patch electrode by Na+, Li+ or Cs+ prevented the flow of inward currents and reduced the outward K+ conductance to 113 pS. 4. NPo was insensitive to changes in [Ca2+]c from 10 nM to 1 microM. NPo was also not modified either by cytosolic Na+, ATP, GTP, GTP gamma S, dithiothreitol or TEA (10 mM) or by extracellular 4-aminopyridine (5 mM), glibenclamide (20 microM) or TEA (10 mM). The BKA channel was blocked by 5 mM intracellular BaCl2 or by 10 nM extracellular iberiotoxin. 5. In cell-attached patches, BKA channel activity could be induced by 1 microM cytochalasin B, applied either through the patch pipette or in the bath solution. The effects of cytochalasin B, of patch excision, or of histamine on NPo were not additive but saturative. 6. Whole DDT1 MF-2 cells had resting potentials of -10 mV, dominated by the chloride conductance; the resting potential changed to -82 mV when the K+ conductance was increased by cytochalasin B or by histamine. The effects of cytochalasin B and histamine on IK(BA) were not additive but saturative. 7. We discuss the hypothesis that the interaction between the cytoskeleton and the BKA channel promotes the long channel closures; depolymerization of F-actin may constitute a mechanism by which the agonists histamine or ATP disinhibit BKA channel activity.

Adenosine Triphosphate↗
PubMed1996

Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.

Animals↗
PubMed1996

Dissociation of subsarcolemmal from global cytosolic [Ca2+] in myocytes from guinea-pig coronary artery.

1. Changes in cytosolic Ca2+ concentration (delta[Ca2+]c) were measured by global indo-1 fluorescence and compared with changes in subsarcolemmal Ca2+ concentration (delta[Ca2+]sl) indicated by Ca(2+)-activated K+ currents (IK(Ca)). 2. At -50 mV holding potential, 10mM caffeine increased both IK(Ca) and [Ca2+]c without measurable delay. While IK(Ca) peaked within 0.3 +/- 0.16 s (mean +/- S.D.) and decayed to 50% within 0.4 +/- 0.2 s, delta[Ca2+]c peaked within 1.5 +/- 0.5 s and decayed to 50% within 5.2 +/- 1.0 s. The different time courses support the idea that [Ca2+]sl and [Ca2+]c deviate. 3. When 10 mM caffeine was applied 20 s after an initial 2 s caffeine application, IK(Ca) was suppressed to 22 +/- 5% and delta [Ca2+]c to 40 +/- 4%. During the following 1 min caffeine-free period, IK(Ca) recovered to 61 +/- 7% while delta [Ca2+]c remained at 40 +/- 3%. The differences between IK(Ca) and delta[Ca2+]c suggest that Ca2+ deprivation and Ca2+ refilling is faster in peripheral than in central sarcoplasmic reticulum (SR). 4. During the loading period of indo-1, a spontaneous delta[Ca2+]c of 30-80 nM appeared both at -50 mV and at more positive potentials. The amplitude of spontaneous delta[Ca2+]c increased with the amplitude, the frequency or the fusion of spontaneous transient outward currents (STOCs). 5. Block of sarcolemmal Ca2+ fluxes by 1 mM La3+ increased [Ca2+]c by 250 +/- 100 nM and suppressed the spontaneous delta[Ca2+]c. However, La3+ did not significantly retard the rate of decay of STOCs which may therefore be limited by Ca2+ diffusion into the cytosol and not by Ca2+ extrusion. 6. The dissociation of IK(Ca) (or STOCs) and delta[Ca2+]c may indicate a Ca2+ concentration gradient during Ca2+ release directed from the sarcolemma towards the centre of the cell, which later reverses direction.

Animals↗
PubMed1996

Buffering of plasmalemmal Ca2+ current by sarcoplasmic reticulum of guinea pig urinary bladder myocytes.

The effects of cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), on cytosolic Ca2+ concentration ([Ca2+]c) and membrane currents were studied in isolated urinary bladder myocytes to test the hypothesis that the sarcoplasmic reticulum (SR) buffers Ca2+, which enters the myocyte at a slow to moderate rate. Inhibition of SERCA by CPA was demonstrated by the following modifications of the caffeine-induced [Ca2+]c transients: 1) CPA prolonged the 90% decay time from peak to resting [Ca2+]c from 2.2 +/- 0.3 to 8.3 +/- 0.92 s (n = 5), 2) CPA abolished the "undershoot" of the [Ca2+]c transient that follows the washout of caffeine, and 3) CPA prevented caffeine from inducing a second [Ca2+]c transient. CPA reversibly increased resting [Ca2+]c. Starting from a control [Ca2+]c of 137 +/- 10 nM, 19 of 24 cells responded with a monotonic increase in [Ca2+]c to a steady [Ca2+]c of 238 +/- 10 nM, whereas 5 of 24 cells responded with a transient rise of [Ca2+]c to 472 nM (within 2.8 +/- 0.5 s) followed by a decay to a steady [Ca2+]c of 161 +/- 10 nM. The CPA-mediated rise in [Ca2+]c was augmented by increasing extracellular Ca2+ concentration ([Ca2+]o), suggesting a "leakage pathway" for Ca2+ influx that is unmasked by SERCA blockade. CPA reduced [Ca2+]c transients and Ca(2+)-activated K+ currents (IK,Ca), induced by depolarizing clamp steps from -60 to 0 mV, compatible with suppression of SR Ca2+ release on depletion of SR Ca2+. To reduce the contribution due to Ca(2+)-induced Ca2+ release, the cells were depolarized with a slow ramplike command (-60 to 0 mV, 15 mV/s). In 12 of these 40 cells, CPA increased [Ca2+]c and IK,Ca signals. If spontaneous transient outward currents were present, they were suppressed by CPA. CPA reduced the peak L-type Ca2+ channel current apparently through increased Ca2+ inactivation of voltage-gated Ca2+ channels. The current could be restored to control by elevating [Ca2+]o from 2.5 to 5 mM. Under these conditions, CPA increased the ramp-induced [Ca2+]c transients from 105.1 +/- 22 to 162.0 +/- 31 nM (n = 9, P < 0.05). These results suggest that Ca2+ sequestration by the SR can buffer part of the Ca2+ influx during slow depolarizations.

Animals↗
PubMed1996

Effect of membrane potential on the initiation of acetylcholine-induced Ca2+ transients in isolated guinea pig coronary myocytes.

The muscarinic stimulation of single voltage-clamped coronary arterial smooth muscle cells of the guinea pig was used to evaluate the effect of membrane potential on the inositol 1,4,5-tris-phosphate (IP3)-mediated changes of ionized [Ca2+] in the cytoplasm (Ca2+ transient) measured with indo 1. When applied at the membrane potential of -50 mV, 10 micromol/L acetylcholine (ACh) induced a [Ca2+]i increase after the mean latency of 2.6+/-0.9 s. The latency was reduced to 1.1 +/- 0.3 s when the same dose was applied at a holding potential of +50 mV. In paired experiments in the same cells, the latency of response at +50 mV was reduced by a factor of 2.2 +/- 0.3 compared with the response at -50 mV. Supramaximal [ACh] (100 micromol/L) induced Ca2+ transients with a 0.4 +/- 0.1-s latency, which was independent of membrane potential. When applied repetitively at -50 mV, ACh induced Ca2+ transients with a progressively reduced amplitude and slower rate of rise. Depolarization to +50 mV accelerated the rate of rise of the Ca2+ transient by a factor of 3.4 +/- 0.4 without affecting the amplitude. The modulation of the initiation of Ca2+ transient by a 100-mV depolarization can be explained by an approximately threefold increase in the rate of IP3 accumulation.

Acetylcholine↗
PubMed1995

Calcium-induced calcium release in rat sensory neurons.

1. In isolated dorsal root ganglion cells (DRG neurons), changes in the concentration of global cytosolic Ca2+ (delta [Ca2+]c) were measured by the fluorescence of K5-indo-1. Depolarizations from -60 to 0 mV (500 ms) and Ca2+ influx through Ca2+ channels (ICa) increased [Ca2+]c by 480 +/- 113 nM, the peak occurring 542 +/- 76 ms (mean +/- S.E.M.) after repolarization. 2. Ryanodine (10 microM) reduced depolarization-induced delta [Ca2+]c by up to 80% and blocked delta [Ca2+]c induced by 20 mM caffeine. 3. Peak delta [Ca2+]c and peak ICa followed a similar bell-shaped voltage dependence. Removal of extracellular Ca2+ abolished depolarization-induced delta [Ca2+]c; its elevation from 2 to 8 mM increased peak ICa by 30% and delta [Ca2+]c by 108%. 4. Ca2+ influx at 0 mV was graded by pulse durations between 20 and 500 ms. Up to 200 ms, delta [Ca2+]c increased linearly with Ca2+ influx. Depolarizations longer than 200 ms induced a supralinear increase in delta [Ca2+]c that was abolished by caffeine (20 mM). 5. The supralinear increase in delta [Ca2+]c and the caffeine-induced delta [Ca2+]c were measured only in thirteen of nineteen DRG neurons; in the other six of nineteen cells both properties were absent. The results suggest that Ca(2+)-induced Ca2+ release (CICR) is expressed differently in different populations of DRG neurons. 6. A single action potential did not significantly increase [Ca2+]c. Trains of stimuli (20 Hz) induced delta [Ca2+]c that linearly increased with the number of action potentials. Delta [Ca2+]c due to 100 action potentials had a significant ryanodine-sensitive component. 7. It is discussed that CICR can contribute to the depolarization-induced [Ca2+]c, provided the Ca2+ influx lasts for a certain minimum period of time.

Action Potentials↗
PubMed1995

Ca2+ activation and Ca2+ inactivation of canine reconstituted cardiac sarcoplasmic reticulum Ca(2+)-release channels.

1. Calcium-release channels (ryanodine receptors) of canine cardiac sarcoplasmic reticulum (SR) were incorporated into lipid bilayer membranes at the tip of a patch pipette. Using symmetrical 150 mM KCl solutions, [Ca2+] > 0.3 microM activated single channels of 627 pS conductance. The kinetics of Ca(2+)-mediated channel activation, deactivation and inactivation were studied by stepwise changes in pCa (-log[Ca2+]) and analysis of current means. 2. Steps of [Ca2+] activated the channel open probability (Po) along a time course which could be fitted by a single exponential. The activation time constant was dependent on [Ca2+], which decreased from 4.9 ms at pCa 6.5 to 0.2 ms at pCa 3. Subsequent rapid reduction in [Ca2+] decreased Po along a mono-exponential deactivation time course, the time constant of which was independent of the [Ca2+] during the preceding activation period. Further analysis yielded the rate constants kon of 2 x 10(8) (M s)-1 and koff of 2 x 10(2) s-1, an apparent dissociation constant (KD) of 1 microM, and a Hill coefficient of 1.05. 3. The open probability increased with [Ca2+], reaching a peak at about pCa 5.5. At pCa < or = 5.5, Po decreased time dependently, the time constants decreasing along with [Ca2+] from 1 s at 3 microM to 0.2 s at 1 mM. During the 0.5 s period at 3 microM Ca2+, Po fell by 13% due to an extension of the closed times. At 1 mM Ca2+, Po 'inactivated' by 72%, which was due mostly to long closures. These differences suggest that the Ca(2+)-mediated decay of Po was dependent on Ca2+ binding to an intermediate (KD, 3 microM) and a low affinity site (KD, 360 microM). On the return of pCa from 3 to > 8, the channels briefly re-opened. 4. A 'refractory' behaviour of the channel was not observed for 20 ms steps between < 10 nM and < 10 microM [Ca2+] (25 Hz). For steps between 10 nM and 1 mM, however, such behaviour was marked by infrequent and irregular channel openings. 5. The results are described by a three Ca2+ binding site model and compared with the literature.

Animals↗