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At least 1,081 records · Page 60Linked to original sources
PubMed1991

Separate mechanisms of long-term potentiation in two input systems to CA3 pyramidal neurons of rat hippocampal slices as revealed by the whole-cell patch-clamp technique.

Excitatory synaptic transmission from two input systems to hippocampal CA3 pyramidal neurons was investigated by the whole-cell patch-clamp technique for thin slice preparation, with special reference to long-term potentiation (LTP) in these systems. Excitatory postsynaptic currents (EPSCs) evoked by fimbrial stimulation consisted of two components; one was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the other was persistent at depolarized membrane potentials and blocked by D-2-amino-5-phosphonovalerate (D-AP5). The contribution of the D-AP5-sensitive component to EPSCs evoked by stimulation of mossy fibers was much less than that to fimbrial EPSCs. High-frequency stimulation of afferent fibers, under current-clamp conditions, elicited LTP. Bath application of D-AP5 blocked the induction of LTP in the fimbrial but not in the mossy fiber synapses. Induction of fimbrial LTP was completely blocked by 10 mM BAPTA applied intracellularly. In contrast, mossy fiber LTP was not blocked by 10 mM BAPTA. Furthermore, mossy fiber LTP, but not fimbrial LTP, was elicited by high-frequency stimulation under voltage-clamp (-80 mV) conditions. These results suggest that activation of NMDA receptors, increase in postsynaptic [Ca2+]i, and postsynaptic membrane depolarization are required for the induction of fimbrial but not for mossy fiber LTP.

2-Amino-5-phosphonovalerate↗
PubMed1993

A whole cell patch-clamp study on the pacemaker potential in dopaminergic neurons of rat substantia nigra compacta.

A whole-cell patch-clamp recording was obtained from dopamine (DA) neurons (n = 68) in the substantia nigra compacta (SNc) in in vitro slice preparations in order to study the underlying current for pacemaker-like slow depolarization (PLSD) which was considered as a basis for rhythmic firing of DA neurons. SNc DA neurons were identified immunohistochemically after recording. Results demonstrated that: (1) Under current clamped condition in the presence of TTX, DA neurons (n = 5) displayed the oscillation of membrane potential with high threshold spikes. An application of a hyperpolarizing and depolarizing current pulse (at the membrane potential where oscillation was no longer seen) induced a prominent anomalous rectification and pacemaker-like slow depolarization (PLSD), respectively. (2) Under voltage-clamped conditions in the presence of TTX, a command pulse positive to -50 mV from a holding potential of -80 mV induced a persistent Ca2+ current which was usually preceded by either a transient K+ (n = 7) or a transient Ca2+ (n = 4) current recorded with a patch pipette containing potassium gluconate (145 mM). (3) When outward currents were suppressed by 140 mM CsCl and 10 mM EGTA intercellularly applied through the patch pipette, a command pulse positive to -50 to -40 mV induced either a persistent Ca2+ current alone (n = 4) or a persistent Ca2+ current preceded by a transient Ca2+ current (n = 11). (4) The threshold for activation of the persistent Ca2+ current (Ip) was around -60 to -55 mV. The amplitude of Ip produced by a command pulse stepped to -50 mV from a holding potential of -80 mV was -78 +/- 42 pA (n = 23). (5) The threshold for activation of transient Ca2+ current (IT) was around -70 to -65 mV and inactivated completely at -70 to -65 mV (n = 11). The peak amplitude of IT evoked at -60 to -55 mV from a holding potential of more negative than -80 mV was 489 +/- 170 pA (n = 11). (6) The decay time constant of IT was 28 +/- 12 ms at -60 mV (n = 8) and that of IP was 2.35 +/- 1.37 s at -50 mV (n = 11) when recorded with a pipette containing 10 mM EGTA and 140 mM CsCl. (7) The decay of IP was apparently accelerated by decreasing the concentration of EGTA in the pipette solution from 10 to 1 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Amplifiers, Electronic↗
PubMed1993

Electrophysiological and morphological properties of granule cells: patch-clamp recordings of newborn rabbit olfactory bulb slices.

Granule cells were investigated by performing whole-cell patch-clamp recordings from thin slices of the olfactory bulb of newborn rabbits. Granule cells were intracellularly stained with Lucifer Yellow in their intact environment. During current-clamp measurements these neurones were characterized by their lack of action potentials upon depolarization. Evidence for a Ca2+ dependent K+ conductance was found. Two types of outward currents were identified in the whole cell mode during voltage clamp; a non-inactivating K+ current that shared some properties of the delayed rectifier K+ current and a non-inactivating K+ current were recorded. No fast inward current was registrated.

Action Potentials↗
PubMed1989

Postmenopausal osteoporosis: response of immunoextracted calcitonin to a calcium clamp.

Fourteen postmenopausal females with osteoporosis (PMO) and 14 healthy postmenopausal females (PMF) were studied by means of the calcium clamp-technique. Plasma was collected before start and during the 1 hour calcium clamp. Calcitonin (CT) was immunoextracted from plasma with antiserum No. 1 directed against the mid- and carboxyterminal portion of CT. Thereafter radioimmunoassay was performed on the extracts with antiserum No. 2 directed against the carboxyterminal of CT. The detection limit of the assay was 0.8 pg/tube. There was no difference between patients and controls in the basal CT values and CT was detectable in all samples after immunoextraction. Plasma CT increased significantly during the calcium clamp in osteoporotic patients as well as in controls and the CT response did not differ between the groups. The results do not support the hypothesis of a diminished CT secretion as a contributing factor to the development of osteoporosis.

Aged↗
PubMed2005

Development of a peptide nucleic acid polymerase chain reaction clamping assay for semiquantitative evaluation of genetically modified organism content in food.

In the present study a peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method was developed and applied to the detection of genetically modified organisms (GMO), to test PCR products for band identity and to obtain a semiquantitative evaluation of GMO content. The minimal concentration of PNA necessary to block the PCR was determined by comparing PCRs containing a constant amount of DNA in the presence of increasing concentration of target-specific PNA. The lowest PNA concentration at which specific inhibition took place, by the inhibition of primer extension and/or steric hindrance, was the most efficient condition. Optimization of PCR clamping by PNA was observed by testing five different PNAs with a minimum of 13 bp to a maximum of 15 bp, designed on the target sequence of Roundup Ready soybean. The results obtained on the DNA extracted from Roundup Ready soybean standard flour were verified also on DNA extracted from standard flours of maize GA21, Bt176, Bt11, and MON810. A correlation between the PNA concentration necessary for inducing PCR clamping and the percentage of the GMO target sequence in the sample was found.

DNA Primers↗
PubMed2005

DNA sliding clamps: just the right twist to load onto DNA.

Two recent papers illuminate a key step in DNA sliding clamp loading: one reveals the structure of the PCNA clamp wrapped around DNA--still open from being loaded--while the other finds that the clamp may assist this process by forming a right-handed helix upon opening.

DNA↗
PubMed2005

Specific amino acid residues in the beta sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli.

Escherichia coli dnaN159 strains encode a mutant form of the beta sliding clamp (beta159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the beta clamp with the alpha catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (beta-G174A), while the other contained D173A, G174A and H175A substitutions (beta-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Delta polB (pol II), Delta dinB (pol IV) and/or Delta umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the beta clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in beta159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.

Amino Acid Substitution↗
PubMed2006

Clubbing together on clamps: The key to translesion synthesis.

Translesion synthesis is an important mechanism by which cells replicate past DNA damage. The sliding clamp DNA polymerase processivity factors play a central role in this process. The clamps are dimeric in bacteria and trimeric in eukaryotes and archaea, raising the question of whether more than one polymerase can interact with the clamp simultaneously. Recently published data suggest that this is indeed the case.

DNA Replication↗
PubMed2006

How to clamp the main pulmonary artery during video-assisted thoracoscopic surgery lobectomy.

We sometimes run across difficulty in dissection of the pulmonary arteries due to dense pleural adhesions and bleeding from the pulmonary artery during the video-assisted thoracoscopic surgery (VATS) lobectomy. In these cases, conversion of the VATS approach to open thoracotomy is a requisite. The presence of an easy and safe technique for pulmonary artery clamping will make the switch of the surgical procedure unnecessary. We developed the new technique for pulmonary artery clamping using 1-0 silk suture. This may become one of the standard techniques for pulmonary artery clamping not only in VATS but also in open thoracotomy, as well.

Constriction↗
PubMed2004

Does the sequence of clamp application during open abdominal aortic aneurysm surgery influence distal embolisation?

BACKGROUND: Embolisation of atherosclerotic debris during abdominal aortic aneurysm (AAA) repair is responsible for significant peri-operative morbidity. Reports have suggested that preferential clamping of the distal vessel(s) before the proximal aorta may decrease the number of emboli passing distally and hence reduce complications. METHODS: Forty patients undergoing AAA repair were randomised to have either first clamping of the proximal aorta or the iliac vessels. Emboli passing through the Superficial Femoral Arteries were detected with a Transcranial Doppler ultrasound system. RESULTS: There was no difference between the two groups in the number of emboli detected (p=0.49) and no significant correlation between number of emboli and dissection time (r=0.0008). However, there was a significantly higher number of emboli in the patient sub-group that were current smokers (p=0.034). CONCLUSIONS: There appears to be no difference in the numbers of emboli detected when the first vascular clamp is applied to the proximal aorta or iliacs.

Aged↗
PubMed2004

First clinical experiences with an endovascular clamping system for neuroprotection during carotid stenting.

OBJECTIVES: This report evaluates the feasibility and efficacy of an endovascular blood flow blockage technique to prevent intracerebral embolization of plaque debris during carotid artery stenting. METHODS: Forty-two patients were enrolled in five clinical sites in Germany and Italy with either an asymptomatic internal carotid artery stenosis > or =75% (mean 87%) or a symptomatic stenosis > or =60% (mean 85%). Cerebral protection during the stenting procedure was achieved using an endovascular clamping technique, obtained by occlusion of the external and common carotid artery via two independently inflatable balloons integrated in the Mo.Ma system. Blood with particulate plaque debris was aspirated before flow was restored. The patient's clinical and the neurological status were assessed during intervention, at discharge, and at 3 months follow-up. RESULTS: Stenting was performed in all but one patient. The mean flow occlusion time was 10.6+/-6.5 min. Transient clamping intolerance was observed in five patients (12%). In two patients, neurological deficits persisted for 2 and 12 h, respectively. Two minor strokes (4,7%) occurred at 5 and 72 h after the procedure. No major strokes or deaths were observed at 3 months follow-up. CONCLUSIONS: This first clinical experience with the Mo.Ma device substantiates the feasibility of endovascular clamping in preventing cerebral embolization during carotid artery stenting.

Carotid Artery, Internal↗
PubMed2005

Differentiation of bacterial strains by thermal gradient gel electrophoresis using non-GC-clamped PCR primers for the 16S-23S rDNA intergenic spacer region.

The method for DNA fingerprinting of the 16S-23S rDNA intergenic spacer region was modified to increase resolution of bacterial strains by thermal gradient gel electrophoresis (TGGE) analysis. By utilizing the high melting temperature region of the tRNA gene located in the middle of the 16S-23S rDNA intergenic spacer region as an internal clamp for TGGE, multiple melting domain problems were solved. PCR primers lacking a stretch of GC-rich sequences (GC-clamp) amplified the intergenic spacer region more efficiently than GC-clamped primers. Therefore, PCR artifacts were avoided by using low, 17-cycle, PCR. The method was successfully applied to diverse bacterial species for strain differentiation by TGGE without requiring a special PCR primer set.

Bacterial Typing Techniques↗
PubMed2004

Intestinal injury after thoracic aortic cross-clamping in the pig.

BACKGROUND: The mucosal surface epithelium is an essential part of the functional intestinal barrier, but its structural response to ischemia/reperfusion is only partly characterized. The purpose of this study was to provide a detailed morphological evaluation of intestinal surface epithelium after aortic cross-clamping. MATERIAL AND METHODS: Pigs were subjected to thoracic aortic cross-clamping for 60 min and subsequent reperfusion for 120 min. Tissue blood flow and high-energy phosphates were measured with microspheres and HPLC, respectively. Urinary excretion of (14)C polyethylene glycol (MW 4000 Da) (PEG-4000), loaded into an intestinal loop, provided an index of intestinal permeability. RESULTS: Jejunal blood flow was restored at 10 min after aortic declamping. Denudation of the basement membrane of the intestinal villi tips, as a consequence of epithelial shedding, increased markedly during the initial 60 min of reperfusion (P = 0.002). During the following 45 min, the denuded basement membrane was partly covered with low cuboidal and squamous-shaped cells extending lamellipodia over a wavy basement membrane. Restoration of ATP at 60 min after aortic declamping correlated inversely to the extent of denuded basement membrane (r = 0.75, P = 0.032). Permeability of PEG-4000 increased markedly after aortic declamping and was linearly correlated to the area of denuded basement membrane (r = 0.87, P = 0.01). CONCLUSIONS: Reperfusion for 2 h after aortic cross-clamping is associated with initial aggravation of ischemia-induced injury in the porcine jejunum, but thereafter with restitution of the surface epithelium. Restoration of ATP may be important to avoid intestinal injury after ischemia. Increased permeability of a macromolecule in response to reperfusion is closely correlated to injury of the surface epithelium.

Adenosine Diphosphate↗
PubMed2005

Suprarenal aortic clamping and reperfusion decreases medullary and cortical blood flow by decreased endogenous renal nitric oxide and PGE2 synthesis.

OBJECTIVE: This study examined the hypothesis that clamping the aorta above the superior mesenteric artery (SMA) followed by suprarenal aortic clamping and reperfusion (SRACR) decreases microvascular blood flow by loss of endogenous medullary and cortical nitric oxide (NO) and prostaglandin (PG) E(2) synthesis. STUDY DESIGN: Anesthetized male Sprague-Dawley rats (350 g) had either microdialysis probes or laser Doppler fibers inserted into the renal cortex to a depth of 2 mm and into the renal medulla at 4 mm. Laser Doppler blood flow was continuously monitored (data reported as percentage of change compared to basal), and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 microL/min with lactated Ringer solution. Dialysate fluid was collected at basal time zero, following 30 minutes of suprarenal aortic clamping (ischemia) followed by 60 minutes of reperfusion and compared to a sham operation. Both groups were treated with saline carrier, indomethacin (INDO) (10 mg/kg, a cyclooxygenase [COX] inhibitor), N(G)-nitro-L-arginine methyl ester (L-NAME) (20 mg/kg, a NO synthase [NOS] inhibitor), or L-arginine (200 mg/kg, an NO precursor). Dialysate was analyzed for total NO (muM) and PGE(2) (pg/mL) synthesis. The renal cortex and medulla were analyzed for inducible NOS (iNOS) and COX-2 content by Western blot. All data are reported as mean +/- SEM, N > 5 and analyzed by analysis of variance. RESULTS: SRACR caused a marked decrease in medullary and cortical blood flow with a concomitant decrease in endogenous medullary and cortical NO synthesis. Treatment with L-NAME further decreased blood flow and NO synthesis in the medulla and cortex. L-Arginine restored medullary and cortical NO synthesis and blood flow in the cortex but not the medulla. SRACR did not alter renal medullary or cortical PGE(2); however, addition of INDO, COX inhibitor, caused a concomitant decrease in medullary and cortical PGE(2) synthesis and blood flow. CONCLUSIONS: NO is an important endogenous renal vasodilator that, when maintained can help preserve cortical blood flow following SRACR. These data also suggest that avoidance of COX-2 inhibitors can help maintain endogenous renal cortical and medullary PGE(2) synthesis and thus contribute to maintaining normal blood flow. CLINICAL RELEVANCE: This study is the first to combine in vivo physiologic assays to simultaneously identify clinically relevant intrarenal vasodilators (cortical and medullary) that are required to maintain microvascular blood flow. Identification of endogenous renal cortical and medullary vasodilators responsible for maintaining renal microvascular blood flow will allow development of treatment strategies to preserve these vasodilators following SRACR. Successful preservation of endogenous intrarenal vasodilators will help maintain renal microvascular blood flow and renal function in the treatment of complex aortic pathology that requires SRACR.

Analysis of Variance↗
PubMed2006

Increased incidence of cerebral clamping ischemia during early contralateral carotid endarterectomy.

OBJECTIVES: The aim of our study was to assess the influence of previous contralateral carotid endarterectomy (CEA) and of the timing of the procedures on cerebral clamping ischemia during the second operation in patients undergoing staged bilateral CEA. METHODS: We reviewed the 251 patients who presented with bilateral carotid stenosis of > or =70% at the time of the first admission and underwent staged bilateral CEA between January 2001 and December 2004. Surgery was performed under locoregional anesthesia. Cerebral perfusion was monitored with mental status and contralateral motor function evaluation in awake patients. Selective carotid shunting was performed for patients who manifested neurologic deficits. Univariate and multivariate analyses were performed for the variables of interest. RESULTS: Twenty-two patients (8.8%) required carotid shunting during the first procedure and 28 (11.1%) during the second one. Nine of the latter also had shunts during the first CEA, whereas 19 tolerated cross-clamping during the first operation. Among the patients who underwent contralateral CEA < or =30 days, 23 of 146 required carotid shunting; between 31 and 60 days, 4 of 73; and after 61 days, 1 of 32 (P = .023; univariate analysis). The chi2 for trend was statistically significant (P = .009). Patients operated on the second side < or =30 days had a nearly fourfold risk of shunting during the second procedure compared with patients operated on > or =31 days. The highest risk was observed in patients with a shunt during the first operation who underwent the second CEA < or =30 days. Multivariate analysis also identified the time intervals between CEAs and the need of shunting during the first procedure as independent risk factors (P = .042 and P < .001). CONCLUSIONS: These data show an increased incidence of cerebral clamping ischemia during contralateral endarterectomy performed < or =30 days; whereas after longer intervals between CEAs, the need for shunting is significantly reduced.

Aged↗
PubMed2004

The cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer.

The human cytomegalovirus DNA polymerase consists of a catalytic subunit, UL54, and a presumed processivity factor, UL44. We have solved the crystal structure of residues 1-290 of UL44 to 1.85 A resolution by multiwavelength anomalous dispersion. The structure reveals a dimer of UL44 in the shape of a C clamp. Each monomer of UL44 shares its overall fold with other processivity factors, including herpes simplex virus UL42, which is a monomer that binds DNA directly, and the sliding clamp, PCNA, which is a trimer that surrounds DNA, although these proteins share no obvious sequence homology. Analytical ultracentrifugation and gel filtration measurements demonstrated that UL44 also forms a dimer in solution, and substitution of large hydrophobic residues along the homodimer interface with alanine disrupted dimerization and decreased DNA binding. UL44 represents a hybrid processivity factor as it binds DNA directly like UL42, but forms a C clamp that may surround DNA like PCNA.

Binding Sites↗
PubMed2005

Refined suture techniques to prevent bleeding from accidental slippage of vascular clamps during living donor hepatectomy.

Accidental slippage of vascular clamps during living donor hepatectomy can induce brisk bleeding and even imperil the donor. After practicing more than 1000 cases of living donor hepatectomy, the investigators realized that specialized suture techniques were important to secure the vascular closure to prevent unnecessary bleeding. For secure division of intrahepatic vein branches, we devised a continuous penetration suture method in which the orders of procedures were changed to clamping-closure-cut sequence. For secure division of the main and accessory hepatic vein branches from the inferior vena cava, we applied stay sutures at each corner and midpoint of the hepatic vein stump so as not to permit its slippage. After application of these methods, we did not experience any episode of accidental clamp slippage. We are sure that these suture techniques are beneficial to prevent unnecessary bleeding during living donor hepatectomy and to make surgeons feel at ease during the living donor operation.

Blood Loss, Surgical↗
PubMed1996

The effects of supraceliac aortic clamping on the anticoagulant effects of heparin.

BACKGROUND: Coagulopathy is frequently encountered during supraceliac aortic clamping. Patients are routinely heparinized during these operations. This experiment was undertaken to evaluate the effect of supraceliac aortic cross-clamping (XC) on heparin pharmacodynamics. MATERIAL AND METHODS: Fourteen mongrel dogs were randomly divided into three groups: Group 1 (X; n = 6), 90 minutes aortic XC; Group 2 (SH; n = 4), sham operation and heparin, 100 mg/kg intravenously; and Group 3 (XH; n = 4), heparin, 100 mg/kg intravenously 3 minutes before XC. Prothrombin time (PT) and partial prothrombin time (PTT) were measured serially. Heparin concentrations were estimated via automated titration with protamine sulfate. RESULTS: After XC, PTT is increased during reperfusion. Heparin administration (XH) before aortic XC increases PTT above those with X or SH. Anticoagulation was potentiated and prolonged by XC. There was no difference in heparin concentration or PT. CONCLUSION: Supraceliac aortic clamping significantly affects heparin pharmacodynamics and may contribute to coagulopathy.

Animals↗