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At least 1,153 records · Page 64Linked to original sources
PubMed1986

Effect of bipolar coagulation and occlusion clamping on the patency rate in microvascular anastomosis.

Even with extremely high output from a bipolar coagulator applied close to a vessel, the patency in the femoral arteries and veins of 20 rats was found to be excellent. Scanning electron microscopy and histopathologic studies showed minimal disturbances within the intima. When the extremely high pressure (350 gramforce/mm2) of the Ikuta microvascular occlusion clamp was applied to the rat femoral veins for 1 hour, only one out of six veins became nonpatent. Lower pressures had no detrimental effects on the other 33 veins. When high clamping pressure and high output bipolar coagulation were applied together in 20 femoral veins of the rat, patency rate was 80 percent. From the results, it appears that bipolar coagulation is extremely safe, while clamping can be harmful to patency in microvascular anastomoses.

Animals↗
PubMed1987

The euglycemic clamp in patients with thalassaemia intermedia.

In order to evaluate the influence of haemosiderosis on the glucose metabolism we studied tissue sensitivity to insulin and the metabolic clearance rate (M.C.R.) of this hormone by means of euglycemic clamp technique using an artificial endocrine pancreas in 8 patients with thalassaemia intermedia and 8 control subjects. During the steady-state of euglycemic-hyperinsulinemic clamp (40 mU/m2/min) plasma insulin values were significantly lower and the insulin M.C.R. was significantly higher in thalassaemic patients compared to the controls. To achieve a comparable steady-state insulin concentration to the controls, we performed for a second time the euglycemic clamp in the thalassaemic patients increasing the insulin infusion rate to 80/mU/m2/min. The insulin M.C.R., the M index and the M/IRIs-s ratio were significantly higher in the thalassaemic patients compared to the controls. These results are indicative of an increased tissue peripheral sensitivity to insulin as well as the metabolic clearance rate of this hormone.

Adult↗
PubMed1984

Biochemical determination of training effects using insulin clamp technique.

Tissue sensitivity to insulin was evaluated in 6 trained athletes and 9 untrained controls using the euglycemic insulin clamp technique. The amount of glucose metabolism is a measure of overall tissue sensitivity to insulin. During insulin clamp study comparable plasma glucose (80-90 mg/dl) and insulin (90-100 microU/ml) were achieved in both groups. The glucose infusion rate in the athletes (10.70 +/- 0.32 mg/kg/min) was significantly higher than in the controls (7.37 +/- 0.23 mg/kg/min). During euglycemic hyperinsulinemia plasma FFA decreased by 80% while glycerol showed only a 20% reduction in both groups. Urinary catecholamine concentrations in the athletes were similar to those in the controls, and these levels did not change during the insulin clamp period. From these results, it might be concluded that tissue sensitivity to exogenous insulin is 45% higher in trained athletes and that physical exercise can reduce the insulin requirements in insulin-dependent diabetic patients in addition to being beneficial to noninsulin-dependent diabetics.

Adolescent↗
PubMed1984

Can mixed venous blood be used to measure insulin action during the hyperinsulinemic clamp?

Mandatory use of arterialized venous blood for glucose measurement during insulin clamp studies can preclude its use in obese subjects. In order to assess the distortion produced by performing clamp studies with mixed venous blood, we have carried out the present study. Sixteen subjects (BMI range 21-53 kg/m2) whose glucose tolerance varied from normal to diabetic, had hyperinsulinemic clamps performed at plasma insulin concentrations approximately 250 microU/ml above basal, using arterialized venous (a.v.) blood for plasma glucose and insulin measurements. The test was repeated on the same subjects, but mixed venous (m.v.) blood was substituted for a.v. blood to determine plasma glucose and insulin concentrations. During the two studies, the mean (+/- SEM) steady state plasma glucose (a.v. = 110.6 +/- 8.8 mg/dl; m.v. = 111.3 +/- 9.8 mg/dl) and the mean incremental increases in insulin concentrations (a.v. = 263 +/- 20 microU/ml; m.v. = 273 +/- 21 microU/ml) were essentially identical. Furthermore, there was almost a perfect correlation between the individual values obtained for both SSPG (r = 0.97, p less than 0.001) and incremental insulin levels (r = 0.98, p less than 0.001) with the two methods. In addition, the amounts of glucose metabolized calculated from the two methods were very similar (a.v. = 4.96 +/- 0.67 mg/kg/min; m.v. = 5.25 +/- 0.76 mg/kg/min) and the two determinations of M were highly correlated (r = 0.97, p less than 0.001). Finally, the relative rank order of the M values obtained for the 16 subjects during the two studies were extremely comparable.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1981

[Comparative examinations of hysterectomy clamps (author's transl)].

Conventional and recently developed hysterectomy clamps were examined with respect to sure, nonslip grip and minimum resulting tissue trauma. Slippage was appraised by means of the tenacity testing device, tissue damage on the basis of macroscopic, histologic, and scanning-electron-microscope findings. The new clamp (Zeppelin) comes closest to meeting the demands set for sure grip and gentleness to tissue as compared to the conventional clamps (Wertheim, Rogers, Masterson) and best satisfies requirements for the use in hysterectomies today.

Female↗
PubMed2000

A new method of intrathecal PO2, PCO2, and pH measurements for continuous monitoring of spinal cord ischemia during thoracic aortic clamping in pigs.

BACKGROUND: Impaired spinal cord circulation during thoracic aortic clamping may result in paraplegia. Reliable and fast responding methods for intraoperative monitoring are needed to facilitate the evaluation of protective measures and efficiency of revascularization. METHODS: In 11 pigs, a multiparameter PO2, PCO2, and pH sensor (Paratrend 7, Biomedical Sensors Ltd, United Kingdom) was introduced into the intrathecal space for continuous monitoring of cerebrospinal fluid (CSF) oxygenation during thoracic aortic cross-clamping (AXC) distal to the left subclavian artery. A laser-Doppler probe was inserted into the epidural space for simultaneous measurements of spinal cord flux. Registrations were made before and 30 minutes after clamping and 30 and 60 minutes after declamping. The same measuring points were used for systemic hemodynamic and metabolic data acquisition. RESULTS: The mean CSF PO2 readings of 41 mm Hg (5.5 kPa) at baseline decreased within 3 minutes to 5 mm Hg (0.7 kPa) during AXC (P < .01). Spinal cord flux measurement responded immediately in the same way to AXC. Both methods indicated normalization of circulation during declamping. Significant (P < .01) changes were also observed in the CSF metabolic parameters PCO2 and pH. CONCLUSIONS: In this experimental model of spinal ischemia by AXC, online monitoring of intrathecal PO2, PCO2, and pH showed significant changes and correlated well with epidural laser-Doppler flowmetry (P < .01).

Animals↗
PubMed2002

Posttranslational modification of the umuD-encoded subunit of Escherichia coli DNA polymerase V regulates its interactions with the beta processivity clamp.

The Escherichia coli umuDC (pol V) gene products participate in both a DNA damage checkpoint control and translesion DNA synthesis. Interactions of the two umuD gene products, the 139-aa UmuD and the 115-aa UmuD' proteins, with components of the replicative DNA polymerase (pol III), are important for determining which biological role the umuDC gene products will play. Here we report our biochemical characterizations of the interactions of UmuD and UmuD' with the pol III beta processivity clamp. These analyses demonstrate that UmuD possesses a higher affinity for beta than does UmuD' because of the N-terminal arm of UmuD (residues 1-39), much of which is missing in UmuD'. Furthermore, we have identified specific amino acid residues of UmuD that crosslink to beta with p-azidoiodoacetanilide, defining the domain of UmuD important for the interaction. We have recently proposed a model for the solution structure of UmuD(2) in which the N-terminal arm of each protomer makes extensive contacts with the C-terminal globular domain of its intradimer partner, masking part of each surface. Taken together, our findings suggest that UmuD(2) has a higher affinity for the beta-clamp than does UmuD'(2) because of the structures of its N-terminal arms. Viewed in this way, posttranslational modification of UmuD, which entails the removal of its N-terminal 24 residues to yield UmuD', acts in part to attenuate the affinity of the umuD gene product for the beta-clamp. Implications of these structure-function analyses for the checkpoint and translesion DNA synthesis functions of the umuDC gene products are discussed.

Acetanilides↗
PubMed2003

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein.

In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3' end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the "lips" region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp.

Amino Acid Substitution↗
PubMed2002

A genetically clamped renin transgene for the induction of hypertension.

Experimental analysis of the effects of individual components of complex mammalian systems is frequently impeded by compensatory adjustments that animals make to achieve homeostasis. We here introduce a genetic procedure for eliminating this type of impediment, by using as an example the development and testing of a transgene for "genetically clamping" the expression of renin, the major homeostatically responding component of the renin-angiotensin system, one of the most important regulators of blood pressure. To obtain a renin transgene whose expression is genetically clamped at a constant level, we have used single-copy chosen-site gene targeting to insert into a liver-specific locus a single copy of a modified mouse renin transgene driven by a liver-specific promoter/enhancer. The resulting transgene expresses renin ectopically at a constant high level in the liver and leads to elevated plasma levels of prorenin and active renin. The transgenic mice display high blood pressure, enhanced thirst, high urine output, proteinuria, and kidney damage. Treatment with the angiotensin II type I receptor antagonist, losartan, reduces the hypertension, albuminuria, and kidney damage, but does not affect expression of the transgene. This genetically clamped renin transgene can be used in models in which hypertension and its complications need to be investigated in a high prorenin/renin environment that is not subject to homeostatic compensations by the animal when other factors are changed.

Animals↗
PubMed1984

Serotonin increases intracellular Ca2+ transients in voltage-clamped sensory neurons of Aplysia californica.

Noxious stimulation of the tail of Aplysia californica produces behavioral sensitization; it enhances several related defensive reflexes. This reflex enhancement involves heterosynaptic facilitation of transmitter release from sensory neurons of the reflex. The facilitation is stimulated by serotonin (5-HT) and involves suppression of a 5-HT-sensitive K+ current (the S current). Suppression of the S current broadens the action potential of the sensory neurons and is thought to enhance transmitter release by prolonging entry of Ca2+ in the presynaptic terminals. We now report a component of enhanced Ca2+ accumulation that is independent of changes in spike shape. We have measured intracellular free Ca2+ transients during long depolarizing steps in voltage-clamped sensory neuron cell bodies injected with the Ca2+-sensitive dye arsenazo III. The free Ca2+ transients elicited by a range of depolarizing voltage-clamp steps increase in amplitude by 75% following application of 5-HT. Since it is observed under voltage-clamp conditions, this increase in the free Ca2+ transients is not merely secondary to the changes in K+ current but must reflect an additional mechanism, an intrinsic change in the handling of Ca2+ by the cell. We have not yet determined whether this change in Ca2+ handling reflects an increase in Ca2+ influx, a reduction in intracellular Ca2+ uptake, or a release of Ca2+ from intracellular stores. Regardless of the underlying mechanism, however, it seems possible that the enhancement of Ca2+ accumulation and the reduction in K+ current act synergistically in producing short-term presynaptic facilitation. Alternatively, this additional modulation of Ca2+ by 5-HT might contribute to processes such as classical conditioning or long-term sensitization that may depend on Ca2+.

Animals↗
PubMed1989

Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes.

Denaturing gradient gel electrophoresis (DGGE) can be used to distinguish two DNA molecules that differ by as little as a single-base substitution. This method detects approximately 50% of all possible single-base changes in DNA fragments ranging from 50 to approximately 1000 base pairs. To increase the number of single-base changes that can be distinguished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human beta-globin genes. We show that this GC-clamp allows the detection of mutations, including the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human beta-globin gene, that were previously indistinguishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from less than 5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.

Animals↗
PubMed1993

Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target.

Triple helices can be formed on single-stranded oligopurine target sequences by composite oligonucleotides consisting of two oligonucleotides covalently linked by either a hexaethylene glycol linker or an oligonucleotide sequence. The first oligomer forms Watson-Crick base pairs with the target, while the second oligomer engages in Hoogsteen base pairing, thereby acting as a molecular clamp. The triple-helical complex formed by such an oligonucleotide clamp, or "oligonucleotide-loop-oligonucleotide" (OLO), is more stable than either the corresponding trimolecular triple helix or the double helix formed upon binding of the oligopyrimidine complement to the same oligopurine target. Attaching a psoralen derivative to the 5' end of the OLO allowed us to photoinduce a covalent linkage to the target sequence. The psoralen moiety became covalently linked to all three portions of the triplex, thereby making the oligonucleotide clamp irreversible. These crosslinking reactions introduced strong stop signals during DNA replication, as shown on a plasmid containing a portion of the HIV proviral sequence of human immunodeficiency virus. A 16-mer oligopurine sequence corresponding to the "polypurine tract" of human immunodeficiency virus was chosen as a target for a psoralen-OLO conjugate. Three different stop signals for DNA polymerase were observed, corresponding to different sites of polymerase arrest on its template. Even in the absence of photoinduced crosslinking, the psoralen-OLO conjugate was able to arrest DNA replication. The formation of triple-helical structures on single-stranded targets may provide an alternative to the antisense strategy for the control of gene expression.

Base Composition↗
PubMed2000

ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C.

The eukaryotic DNA sliding clamp that keeps DNA polymerase engaged at a replication fork, called proliferating cell nuclear antigen (PCNA), is loaded onto the 3' ends of primer DNA through its interaction with a heteropentameric protein complex called replication factor C (RFC). The ATPase activity of RFC is necessary for formation of a functional PCNA clamp. In the present study, the sensitivity of RFC to partial proteolysis is used to show that addition of ATP, ATPgammaS, or ADP induces different structural changes in RFC. Direct observation by electron microscopy reveals that RFC has a closed two-finger structure called the U form in the absence of ATP. This is converted into a more open C form on addition of ATP. In contrast, the structural changes induced by ATPgammaS or ADP are limited. These results suggest that RFC adapts on opened configuration intermediately after ATP hydrolysis. We further observe that PCNA is held between the two fingers of RFC and propose that the RFC structure change we observe during ATP hydrolysis causes the attached PCNA to form its active ring-like clamp on DNA.

Adenosine Triphosphate↗
PubMed1996

Protein-protein and protein-DNA interactions at the bacteriophage T4 DNA replication fork. Characterization of a fluorescently labeled DNA polymerase sliding clamp.

The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine 5'-O-(3-thiophosphate), events in holoenzyme assembly were assigned as either dependent or independent of ATP hydrolysis. A holoenzyme assembly mechanism is proposed in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner not requiring ATP hydrolysis. Upon ATP hydrolysis, the 44/62 complex triggers a conformational change in the 45 protein that may be attributed to the clamp loading onto DNA.

Adenosine Triphosphate↗
PubMed1998

Pre-steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia coli DNA polymerase III onto DNA.

The beta protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the gamma complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of beta onto DNA. Two beta dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), have been used to show that ATP binding is required for gamma complex and beta to associate with DNA, but that a gamma complex-catalyzed ATP hydrolysis is required for gamma complex to release the beta.DNA complex and complete the reaction. In the presence of ATP and gamma complex, the beta mutants associate with DNA as efficiently as wild type beta. However, completion of the reaction is much slower with the beta mutants because of decreased ATP hydrolysis by the gamma complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPgammaS in reactions using wild type beta. Thus, the assembly of beta around DNA is coupled tightly to the ATPase activity of the gamma complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.

Adenosine Triphosphate↗
PubMed1998

Abortive initiation of transcription at a hybrid promoter. An analysis of the sliding clamp activator of bacteriophage T4 late transcription, and a comparison of the sigma70 and T4 gp55 promoter recognition proteins.

Bacteriophage T4 late promoters are transcribed by an RNA polymerase holoenzyme comprising the Escherichia coli core, E, the phage gene 55-encoded promoter recognition subunit, gp55, and the gene 33-encoded co-activator, gp33. Transcriptional initiation is activated by the T4 gene 45-encoded sliding clamp, which is loaded on to DNA at enhancer-like sites by its clamp-loader. Correct initiation of transcription at late promoters in basal mode requires only RNA polymerase core and gp55 (E.gp55). Dinucleotide-primed abortive initiation of basal and activated T4 late transcription has been compared. Only the trinucleotide non-productive transcript is made at a high rate; all other short transcripts are made at rates of less than one molecule per productive transcript. Gp45 increases abortive trinucleotide synthesis along with productive transcription, although the proportion of productive transcripts is also elevated. Nevertheless, this increase accounts for only a small part of the activation of T4 late transcription that is generated by its activator and co-activator. The pattern of production of short transcripts differs subtly between basal and enhanced transcription, indicating that linking the RNA polymerase with its sliding clamp activator only generates minor changes in the transition from abortive to productive RNA chain elongation. The T4 late promoter is converted to a strong sigma70 promoter by inserting an appropriate -35 promoter element. A direct comparison at such a hybrid promoter shows sigma70 and gp55 generating qualitatively and quantitative different patterns of abortive initiation at the same start site.

Bacteriophage T4↗
PubMed2000

Steady-state and rapid kinetic analysis of topoisomerase II trapped as the closed-clamp intermediate by ICRF-193.

DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme. Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC(50) of 6.5 +/- 1 microM for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP. When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP. By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP. These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.

Adenosine Triphosphatases↗
PubMed2001

ATP utilization by yeast replication factor C. III. The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for DNA recognition and clamp loading.

The conserved lysine in the Walker A motif of the ATP-binding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid. Complexes of replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis. All of the mutant RFC complexes were capable of interacting with PCNA. Complexes containing a rfc1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis. In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity. In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding. A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations. Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine --> arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations.

Adenosine Triphosphate↗