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G Isenberg

Publications and source records attributed to G Isenberg.

At least 37 records · Page 2Linked to original sources
PubMed2000

Prospective evaluation of an over-the-wire catheter US probe.

BACKGROUND: Pancreaticobiliary strictures identified at endoscopic retrograde cholangiopancreatography (ERCP) can be evaluated by intraductal ultrasonography (US). Two major difficulties are that sphincterotomy may be required and the stricture may not be traversable. We prospectively evaluated the ease and success of intraductal US using a new over-the-wire catheter US probe. METHODS: Biliary or pancreatic strictures discovered at ERCP were imaged with the new probe. Intraductal US performance times, image clarity, imaging depth and technical ease were measured. RESULTS: Twenty-one patients with a variety of inflammatory and malignant pancreaticobiliary lesions were studied. Thirteen of the 16 (81%) masses imaged by intraductal US were 10 mm or less in diameter. Sphincterotomy was not required. All strictures traversed by a guidewire were imaged. The sphincter of Oddi was successfully imaged in all patients with intact normal sphincters. Performance of intraductal US was rated as technically easy in all cases and image clarity was rated as good or very good in 15 of 21 (71%) cases. CONCLUSIONS: The new over-the-wire catheter US probe facilitates intraductal US. Sphincterotomy is avoided and strictures are successfully traversed. This probe makes it possible to image the sphincter of Oddi.

Adult↗
PubMed2000

Augmentation of SR Ca(2+) release by rapamycin and FK506 causes K(+)-channel activation and membrane hyperpolarization in bladder smooth muscle.

1. The immunosuppressants rapamycin and FK506 are known to relax smooth muscle despite facilitating Ca(2+) release through ryanodine-receptors of the sarcoplasmic reticulum (SR). The apparent contradiction was studied in isolated guinea-pig urinary bladder myocytes. 2. Modulation of spontaneous SR Ca(2+) release was monitored by means of spontaneous transient outward currents (or STOCs) in isolated smooth muscle cells voltage-clamped to -20 mV. Rapamycin (10 microM, n=18) significantly increased amplitude (50+/-12%, mean+/-s.d.), life time (77+/-19%), and time integral of STOCs (113+/-22%), and it reduced the interval between STOCs (20+/-7%). FK506 (20 microM, n=24) increased amplitude (15+/-7%), life time (50+/-7%), time integral (104+/-26%). Cyclosporin A (20 microM, n=18) had no significant effects on STOCs. 3. The basal cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) measured by Indo1-fluorescence was insensitive to rapamycin or FK506. Pretreatment with rapamycin (20 microM, 2 min) did not impair the SR Ca(2+) load as can be concluded from caffeine-induced Ca(2+)-transients. 4. As it was expected from the enhanced STOC activity, the non-clamped membrane was hyperpolarized by rapamycin (15+/-2 mV) or by FK506 (15+/-3 mV). 5. The data are consistent with the idea that rapamycin and FK506 augment spontaneous SR Ca(2+) release by removal of FK-binding proteins from the RyR-complex. Smooth muscle relaxation is interpreted as negative Ca(2+) feedback: augmented Ca(2+) activation of STOCs induces membrane hyperpolarization that reduces Ca(2+) influx through voltage gated channels.

Animals↗
PubMed1999

Fragments from alpha-actinin insert into reconstituted lipid bilayers.

Recent experiments have indicated that alpha-actinin interacts with phospholipid membranes. Using computer analysis methods we determined two possible lipid binding sites capable of membrane attachment/insertion, residues 281-300 and 720-739 of the primary amino acid sequence on smooth muscle alpha-actinin. Having expressed these regions as fusion proteins with schistosomal GST (glutathione S-transferase), we used differential scanning calorimetry (DSC) to investigate their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers. Calorimetric measurements showed that as fusion protein concentration increased, the main chain transition enthalpy decreased and chain melting temperatures shifted, which is indicative of partial protein insertion into the hydrophobic region of the lipid membranes. Centrifugation assay and subsequent SDS/Page chromatography confirmed this finding.

Actinin↗
PubMed1999

Twitch-potentiation increases calcium in peripheral more than in central mitochondria of guinea-pig ventricular myocytes.

1. The mitochondrial total calcium content ([Ca]mt) was studied with electron probe microanalysis (EPMA) in isolated guinea-pig ventricular myocytes in order to answer the question of whether electrical stimulation increases [Ca]mt in subsarcolemmal and central mitochondria to a different extent. 2. In unstimulated myocytes subsarcolemmal [Ca]mt was (mean +/- s.e.m.) 535 +/- 229 micromol (kg dry weight (DW))-1 and central [Ca]mt was 513 +/- 162 micromol (kg DW)-1. These values do not differ and correspond to approximately 180 micromol calcium per litre of mitochondria or 180 microM. 3. Contractions were potentiated to an optimum by stimulation with trains of 12 paired stimuli. After potentiation with 12 paired action potentials, cells were shock-frozen 120 ms after the start of the first action potential of the 13th pair. Subsarcolemmal [Ca]mt was 1.3 +/- 0.4 mmol (kg DW)-1 (433 microM) and central [Ca]mt was 227 +/- 104 micromol (kg DW)-1 (76 microM). The difference was significant. 4. After potentiation with 12 paired voltage-clamp pulses, cells were shock-frozen 120 ms after the start of the first pulse of the 13th pair. Subsarcolemmal [Ca]mt was 2.2 +/- 1.0 mmol (kg DW)-1 (733 microM) and central [Ca]mt was 630 +/- 180 micromol (kg DW)-1 (210 microM). After removal of extracellular K+, five paired voltage-clamp pulses increased subsarcolemmal [Ca]mt to 2.1 +/- 0.8 mmol (kg DW)-1 (700 microM), which was significantly higher than the central [Ca]mt of 389 +/- 88 micromol (kg DW) -1 or 130 microM. 5. In unstimulated cells, [Na] and [K] in subsarcolemmal and central mitochondria were not different. In potentiated myocytes, subsarcolemmal [Na]mt was 236 +/- 20 mmol (kg DW)-1 or 79 mM, which is significantly higher than the central [Na]mt of 50 +/- 5 mmol (kg DW)-1 or 16 mM. 6. The differences in [Ca]mt and [Na]mt are attributed to subsarcolemmal cytosolic microdomains of elevated [Ca2+] and [Na+] generated during contractile potentiation by transmembrane Ca2+ and Na+ fluxes.

Animals↗
PubMed1999

Fragments from actin binding protein (ABP-280; filamin) insert into reconstituted lipid layers.

Previous computer analyses suggested two possible lipid binding sites, residues 49-71 and 131-155, of the primary amino acid sequence on ABP-280 (filamin), which could facilitate membrane attachment/insertion. We expressed these regions as fusion proteins with schistosomal GST and investigated their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers by differential scanning calorimetry (DSC). Using vesicles of mixed DMPC/DMPG with increasing fusion protein concentrations, we established in calorimetric assays a decrease of the main chain transition enthalpy, DeltaH, and a shift in chain melting temperature. This is indicative of the insertion of these fragments into the hydrophobic region of lipid membranes. We confirmed these findings by the film balance technique using lipid monolayers (DMPG). The binding judged from both methods was of moderate affinity.

Amino Acid Sequence↗
PubMed1999

A new fluorescence-based, hydrophobic photolabeling technique for analyzing membrane-associated proteins.

We introduce a new, fluorescent and photoactivatable fatty acid derivative (SANU) for hydrophobic labelling of membrane-bound proteins. The technique allows fast and highly sensitive screening of hydrophobically inserting proteins analyzed by SDS-PAGE with a detection limit below 0.1 pmol. A reliable calculation of labelling efficiencies is achieved by simultaneous densitometry of fluorescence and protein staining. We have applied the new technique on the membrane inserting protein talin, G-actin, and, as a negative control, on RNase, which only binds electrostatically to negatively charged lipid interfaces. In several ways superior to radiolabelling, we can recommend this technique for all laboratories under any circumstances.

Animals↗
PubMed1999

A multicenter U.S. experience with EUS-guided fine-needle aspiration using the Olympus GF-UM30P echoendoscope: safety and effectiveness.

BACKGROUND: The aim of this study was to determine the safety, efficacy, and accuracy of endoscopic ultrasound (EUS)-guided fine-needle aspiration using the GF-UM30P echoendoscope. METHODS: GF-UM30P-guided EUS-guided fine-needle aspiration results from 3 EUS referral centers were prospectively recorded. Successful sampling required that the needle tip be seen within the lesion on at least 1 pass. Aspirates were considered adequate if they were diagnostic for cancer, contained suspicious or atypical cells, or were adequately cellular for interpretation but nondiagnostic. RESULTS: EUS-guided fine-needle aspiration was attempted on 162 lesions in 152 patients with no complications. Sampling was successful in 150 of 162 (93%) attempts (mean lesion size 2.5 +/- 1.2 cm (range 0.7 to 6.0 cm). Aspirates were adequately cellular in 138 of 162 (85%) attempts (43% diagnostic, 15% suspicious and/or atypical cells, 27% adequate cellularity but nondiagnostic). Sampling failed in 12 of 162 (7%) attempts. Ten of 12 (83%) failures and 11 of 12 (92%) inadequate aspirates occurred when lesions measured less than 2 cm. The sensitivity for malignancy was 93% if only successfully sampled lesions with surgically confirmed negative results were included. However, it was 68% if all attempts were included and when unconfirmed high/moderate suspicion negative results were counted as false negatives and low suspicion negative results as true negatives. CONCLUSIONS: The GF-UM30P may be clinically useful for EUS-guided fine-needle aspiration if a curved linear array instrument is unavailable.

Biopsy, Needle↗
PubMed1999

Prospective comparative evaluation of video US endoscope.

BACKGROUND: Limitations in the technical capabilities of the fiberoptic echoendoscope often necessitate concurrent diagnostic endoscopy at the time of an endosonographic (EUS) examination. Our aim was to determine whether a new video echoendoscope would allow the performance of EUS without diagnostic endoscopy. METHODS: EUS examinations on all patients at two centers were initiated with a fiberoptic echoendoscope (period of 5 weeks) or a video echoendoscope (period of 6 weeks). Note was made of need for concurrent diagnostic endoscopy. The endosonographer also rated the mechanical, optical, and sonographic performance of the video instrument using a 9-point comparative scale (1 = much worse, 5 = equal to, and 9 = much better than fiberoptic echoendoscope). RESULTS: A total of 103 patients including 40 and 28 with mural lesions and 22 and 15 with retroperitoneal lesions were examined with the fiberoptic and video echoendoscopes, respectively. Mean values for parameters rating the video echoendoscope's mechanical performance ranged between 5.0 and 5.9, optical performance ranged between 6.6 and 7.5, and sonographic performance ranged between 4.6 and 4.9. Concurrent diagnostic endoscopy was required less frequently when patients with mural lesions (18% vs. 70%, p < 0.005) or patients with retroperitoneal lesions (0% vs. 14%, p = no significance) were examined with the video echoendoscope compared with the fiberoptic echoendoscope. CONCLUSIONS: Moderately improved optics and slightly better mechanical characteristics of the new video echoendoscope allow the performance of EUS without concurrent diagnostic endoscopy in the majority of patients.

Adult↗
PubMed1999

The effect of intact talin and talin tail fragment on actin filament dynamics and structure depends on pH and ionic strength.

We employed quasi-elastic light scattering and electron microscopy to investigate the influence of intact talin and talin tail fragment on actin filament dynamics and network structure. Using these methods, we confirm previous reports that intact talin induces cross-linking as well as filament shortening on actin networks. We now show that the effect of intact talin as well as talin tail fragment on actin networks is controlled by pH and ionic strength. At pH 7.5, actin filament dynamics in the presence of intact talin and talin tail fragment are characterized by a rapid decay of the dynamic structure factor and by a square root power law for the stretched exponential decay which is in contrast with the theory for pure actin solutions. At pH 6 and low ionic strength, intact talin cross-links actin filaments more tightly than talin tail fragment. Talin head fragment showed no effect on actin networks, indicating that the actin binding sites reside probably exclusively within the tail domain.

Actins↗
PubMed1998

Ca2+ influx through carbachol-activated non-selective cation channels in guinea-pig gastric myocytes.

1. Ca2+ microfluorometry (100 microM K5 fura-2) and the voltage-clamp technique were combined to study the effect of carbachol (CCh, 50 microM) in inducing currents (ICCh) through non-selective cation channels (NSCCCh) and increments in global cytosolic Ca2+ concentration (Delta[Ca2+]c). 2. In Na+-containing bath solution, ICCh fell from an initial phasic to a subsequent small (5 %) tonic component; Delta[Ca2+]c fell to zero. Tonic ICCh and [Ca2+]c became prominent after substitution of extracellular 140 mM Na+ by 140 mM Cs+. Tonic ICCh and Delta[Ca2+]c were insensitive to intracellular heparin (3 mg ml-1) and ryanodine (4 microM), i.e. they did not depend on Ca2+ release from sarcoplasmic reticulum (SR). 3. Single channel currents of NSCCCh could be resolved in whole-cell recordings. Substitution of Na+ by Cs+ increased NSCCCh activity by one order of magnitude and slope conductance from 22 to 30 pS. Extracellular quinidine (3 microM) reversibly blocked the NSCCCh activity. 4. Both tonic ICCh and tonic Delta[Ca2+]c (a) followed a similar time course of activation, desensitization and facilitation, (b) were reversibly blocked by 3 microM quinidine, and (c) persisted upon block of SR Ca2+ release. 5. A Ca2+ fractional current of tonic ICCh (fCa) of 0.009 was calculated by comparing the ratio Delta[Ca2+]c (corrected for simultaneous Ca2+ redistribution) over ICCh with depolarization-induced *Delta[Ca2+]c (Delta[Ca2+]c calculated from ICa induced by a 400 ms depolarization from -60 to 0 mV at 2 mM [Ca2+]o, 145 mM [Cs+]o) over ICa. fCa was 0.023 at [Ca2+]o = 4 mM. 6. With 110 mM extracellular CaCl2 and 145 mM intracellular CsCl, ICCh reversed at +19.5 mV suggesting a permeability ratio PCa/PCs of 2.8. 7. We conclude that Ca2+ influx through NSCCCh under physiological [Ca2+]o could induce Delta[Ca2+]c. The fCa was, however, much smaller than the one calculated from the reversal potential.

Algorithms↗
PubMed1998

Analysis of the F-actin binding fragments of vinculin using stopped-flow and dynamic light-scattering measurements.

Using amino acids 884-1066 and 884-1012 expressed from chicken vinculin as fusion proteins with schistosomal glutathione S-transferase, we determined the binding kinetics of the protein fragments with F-actin. We established by the stopped-flow method a two-step binding process: an initial rapid reaction followed by a slower process. The latter is attributed to F-actin cross-linking and/or bundling, which was previously detected by viscometry and electron microscopy [Johnson, R. P. & Craig, S. W. (1995) Nature 373, 261-264]. This is also supported by dynamic light-scattering measurements, indicating dramatic changes in the internal actin filament dynamics, i.e. in bending undulations due to thermal noise. The similar size of the binding reaction for both fusion proteins with F-actin indicates that the F-actin binding site(s) on vinculin are located between residues 884-1012. No binding of pure glutathione S-transferase or its fusion protein with vinculin peptide 1012-1066 with F-actin was detected by either method.

Actins↗
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↗