Advanced distal colonic lesions as predictors of advanced lesions in the proximal colon.
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Biopsy↗
Biomedical subjects
G Isenberg
Publications and source records attributed to G Isenberg.
Objective assessment of the contribution of each diagnostic test and of the ordering sequence in jaundice caused by pancreatobiliary carcinoma.
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Biliary Tract Neoplasms↗
Phospholipid binding of synthetic talin peptides provides evidence for an intrinsic membrane anchor of talin.
Talin, an actin-binding protein, is assumed to anchor at the membrane via an intrinsic amino acid sequence. Three N-terminal talin fragments, 21-39 (S19), 287-304 (H18), and 385-406 (H17) have been proposed as potential membrane anchors. The interaction of the corresponding synthetic peptides with lipid model systems was investigated with CD spectroscopy, isothermal titration calorimetry, and monolayer expansion measurements. The membrane model systems were neutral or negatively charged small unilamellar vesicles or monolayers with a lateral packing density of bilayers (32 mN/m). S19 partitions into charged monolayers/bilayers with a penetration area A(p) = 140 +/- 30 A(2) and a free energy of binding of DeltaG(0) = -5.7 kcal/mol, thereby forming a partially alpha-helical structure. H18 does not interact with lipid monolayers or bilayers. H17 penetrates into neutral and charged monolayers/bilayers with A(p) = 148 +/- 23 A(2) and A(p) = 160 +/- 15 A(2), respectively, forming an alpha-helix in the membrane-bound state. Membrane partitioning is mainly entropy-driven. Under physiological conditions the free energy of binding to negatively charged membranes is DeltaG(0) = -9. 4 kcal/mol with a hydrophobic contribution of DeltaG(h) = -7.8 kcal/mol, comparable to that of post-translationally attached membrane anchors, and an electrostatic contribution of DeltaG(h) = -1.6 kcal/mol. The latter becomes more negative with decreasing pH. We show that H17 provides the binding energy required for a membrane anchor.
Amino Acid Sequence↗
Endosonographic assessment of multimodality therapy predicts survival of esophageal carcinoma patients.
BACKGROUND: Standard endosonographic (EUS) staging criteria are unreliable for staging esophageal carcinoma after neoadjuvant therapy; however, measurement of tumor size reduction can identify patients who have achieved a pathologic response. In the current study the authors prospectively compared survival between patients classified as responders and those classified as nonresponders by EUS. METHODS: The maximal transverse cross-sectional area of the tumor was measured before and after neoadjuvant therapy in patients who were candidates for multimodality treatment. Response was defined as a > or = 50% reduction in tumor area. RESULTS: A total of 59 patients at 2 centers were followed for a median of 19 months. EUS assessed response in 34 patients (58%). Overall, responders had a median survival of 17.6 months compared with 14.5 months for nonresponders (P < 0.005). Survival was significantly longer in responders compared with nonresponders in the patient subgroup who underwent surgical resection (19.7 months vs. 14.6 months; P < 0. 005), the patient subgroup with adenocarcinoma (21.4 months vs. 10.8 months; P < 0.005), and the patient subgroup initially classified as having T3N1 disease (17.6 months vs. 14.1 months; P < 0.05). Survival was not found to differ significantly between responders and nonresponders in the subgroup of patients with squamous cell carcinoma. EUS response was the only clinical variable that was associated with survival time in a multivariate analysis (relative hazard = 0.27; P < 0.005). CONCLUSIONS: Patients with esophageal carcinoma who respond to neoadjuvant treatment as identified by EUS measurement of reduction in tumor size have a significantly better prognosis than nonresponders.
Adenocarcinoma↗
Cyclic mechanical strain decreases the DNA synthesis of vascular smooth muscle cells.
In vivo, smooth muscle cells of the vascular wall are rhythmically stretched by the arterial pulse. Here, we test the hypothesis that rhythmical stretch is important for suppressing the growth of vascular smooth muscle (vsm) cells. DNA-synthesis rate, cell number, metabolic activity, and cell death were compared between rhythmically stretched and non-stretched vsm cells from the rat embryonic aortic A10 cell line. Rhythmical stretch (0.5 Hz, 5% elongation, 48 h) did not induce vsm cell proliferation, that is the vsm cell number was constant. Cell damage or necrosis was excluded because the release of lactate dehydrogenase (LDH) was identical. The low rate of apoptosis (0.2%) was not different between stretched cells and control cells. Stretch significantly reduced the DNA-synthesis rate [measured as incorporation of 5-bromo-2'-deoxyuridine (BrdU)] in a time-dependent manner. BrdU incorporation was decreased by 32% after 24 h of cyclic stretching and was further diminished to 50% after 48 h of strain. Metabolic activity (measured by Wst-1 cleavage) was only modestly influenced. The stretch-induced decrease in DNA synthesis was independent of the extracellular matrix. No differences were detected when laminin- or pronectin-coated membranes were used instead of collagen-coated membranes. The effect of stretch was unlikely to be mediated by secretion of an unknown "factor", because vsm cells incubated with medium conditioned by stretched cells did not show a significant decrease in BrdU uptake. The results support the idea that rhythmical stretch is important to keep the rate of DNA synthesis and thereby the proliferation of vsm cells at a low level.
Analysis of Variance↗
Stretch-activated currents in ventricular myocytes: amplitude and arrhythmogenic effects increase with hypertrophy.
BACKGROUND: Mechanical dilation of the human ventricle is known to induce arrhythmias, the underlying ionic mechanisms, however, remain to be clarified. METHODS: Ventricular myocytes isolated from human, guinea-pig or rat hearts were stretched between the patch electrode and a glass stylus. RESULTS: Local stretch prolonged the action potential, depolarized the resting membrane and caused extra systoles. Under voltage-clamp conditions, stretch activated several ionic current components. The most prominent current was a stretch activated current (I(SAC)) through non-selective cation channels. I(SAC) followed a linear voltage-dependence, reversed polarity close to 0 mV and was suppressed by 5 microM Gd(3+). During stretch, I(SAC) became steady within 200 ms. I(SAC) did not inactivate and it completely disappeared upon relaxation. Stretch-sensitivity was evaluated from the slope of I(SAC) versus amplitude of stretch. Stretch sensitivity was 75 pA/microm in myocytes from young (3 month), 143 pA/microm in myocytes from old (15 months), and 306 pA/microm in hypertrophied myocytes from old (15 months) spontaneously hypertensive animals. Stretch sensitivity was 262 pA/microm in hypertrophied myocytes from human failing hearts, and it was 143 pA/microm in guinea-pig ventricular myocytes. CONCLUSIONS: Local stretch of adult single ventricular myocytes can induce arrhythmias that resemble surface-recordings from whole hearts. Stretch modulates multiple current components, I(SAC) being the current with the largest arrhythmogenic potential. Stretch-sensitivity of I(SAC) is higher in hypertrophied than in control myocytes as can be expected from the observation that hypertrophy and failure increase the risk of stretch-induced arrhythmias.
Action Potentials↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
Differences in the diagnostic criteria used by Japanese and Western pathologists to diagnose colorectal carcinoma.
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Adenocarcinoma↗
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↗
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↗