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PubMed1996

Ligation of double-stranded and single-stranded [oligo(dT)] DNA by vaccinia virus DNA ligase.

Vaccinia virus DNA ligase has been expressed in Escherichia coli, purified, and biochemically characterized. The enzyme ligates double-stranded (ds) DNA substrates with either cohesive or blunt-end termini and the latter reaction is stimulated by PEG. Vaccinia virus DNA ligase can also ligate oligo(dT) when annealed to either a poly(dA) or a poly(rA) backbone and, remarkably, free oligo(dT). This ligation of a single-stranded (ss) substrate is unique among eukaryotic DNA ligases. The enzyme requires high ATP concentrations with a Km for the overall ligation of a ssDNA substrate of 0.8 mM. The salt, divalent cation, temperature, and pH requirements of the enzyme for the optimal ligation of ss and ds substrate are described.

Adenosine Triphosphate↗
PubMed1987

A rapid method for analyzing the ligation products of synthetic oligodeoxyribonucleotides.

A method is described for the rapid analysis of DNA ligation products in the assembly of synthetic genes and gene fragments. The method is based on the simultaneous analysis of multiple ligation reactions where a single but different DNA oligomer is radiolabelled per ligation reaction. After each ligation the reaction mixture is electrophoresed on a denaturing, as well as a non-denaturing, polyacrylamide gel allowing one to monitor the ligation reaction products. In addition, a unique method for generating single stranded DNA sizing standards up to approximately 300 nucleotides in length is described.

Base Sequence↗
PubMed1994

Pomeroy tubal ligation by laparoscopy and minilaparotomy.

Pomeroy tubal ligation is a widely-used method for surgical female sterilization. A laparoscopic technique of Pomeroy tubal ligation using endoloop sutures is compared with the conventional technique of Pomeroy tubal ligation by minilaparotomy. Forty-four women requiring sterilization were randomly selected for either laparoscopy (n = 24) or minilaparotomy (n = 20). Mean operative time in the laparoscopy group (18.90 +/- 5.56 with a range of 12-35 minutes) was not significantly different from that in the minilaparotomy (23.12 +/- 8.24 with a range of 15-45 minutes) group. In all women, tubal specimens confirmed tubal histology. Both laparoscopy and minilaparotomy procedures may be performed for Pomeroy tubal ligation with minor morbidity. In clinics with appropriate conditions, Pomeroy tubal ligation by laparoscopy may be preferable to minilaparotomy.

Adult↗
PubMed1998

CD40-ligation-mediated protection from apoptosis of a Fas-sensitive Hodgkin's-disease-derived cell line.

Modulation of Fas expression and function by CD40 ligation was investigated in the Fas-sensitive human Hodgkin's disease cell line HDLM2. The recombinant human trimeric soluble CD40L (sCD40L) protected this cell line from apoptosis induced by an agonistic Fas antibody at all concentrations tested. sCD40L also protected HDLM2 when added up to 2 h after Fas ligation. Apoptosis induced by a cell-permeable synthetic ceramide could not be prevented by sCD40L. Thus, CD40 ligation is likely to intervene in the early phases of the Fas signal transduction pathway. When CD40 ligation preceded Fas ligation, it rendered the cells refractory to Fas-induced apoptosis. sCD40L-mediated protection could not be attributed to reduction in surface Fas expression, increase in Bcl-2 levels or to increase in the levels of soluble Fas isoforms.

Apoptosis↗
PubMed1995

Study of heme Fe(III) ligated by OH- in cytochrome b-559 and its low temperature photochemistry in intact chloroplasts.

EPR properties of Cyt b-559 have been investigated in intact chloroplasts that are functionally competent in O2 evolution and in CO2 fixation. After chemical oxidation of Cyt b-559 by 10 mM 2,3-dicyano, 4,5-dichloro-p-benzoquinone (DDQ) the major part of Cyt b-559 is found to be present in the high spin Fe(III) form. Only a small fraction of low spin heme Fe(III) (less than 5%) was formed by chemical or light-induced oxidation. This fraction increased during aging of intact chloroplasts. A comparison with the EPR signal of Fe(III) in myoglobin (Mb) reveals that the structure of the high spin signal in intact chloroplasts is indicative for the presence of an axial OH- ligand at the heme Fe(III). This type of ligation comprised a considerable part (approximately 40%) of the total Cyt b-559 content. Removal of the Mn-cluster caused a change of the EPR parameters of OH- ligation. When in intact chloroplasts the heme Fe is chemically oxidized to Fe(III) ligated by OH-, this OH- ligation disappeared after a subsequent illumination at 80K by red light. Upon illumination at 140K this disappearance was accompanied by the formation of a high spin Fe(III) that is not ligated by OH-. These results are discussed in terms of removal of OH- from Fe(III) caused by structural changes or photooxidation at a complex of Cyt b-559 that could possibly also comprise the Mn-cluster. This photooxidation is assumed to be accompanied by the formation of a bound OH. radical. The possibility is discussed that this process is related to photosynthetic water oxidation.

Benzoquinones↗
PubMed1989

A novel DNA deletion-ligation reaction catalyzed in vitro by a developmentally controlled activity from Tetrahymena cells.

Developmentally controlled genomic deletion-ligations occur during ciliate macronuclear differentiation. We have identified a novel activity in Tetrahymena cell-free extracts that efficiently catalyzes a specific set of intramolecular DNA deletion-ligation reactions. When synthetic DNA oligonucleotide substrates were used, all the deletion-ligation products resembled those formed in vivo in that they resulted from deletions between pairs of short direct repeats. The reaction is ATP-dependent, salt-sensitive, and strongly influenced by the oligonucleotide substrate sequence. The deletion-ligation activity has an apparent size of 200-500 kd, no nuclease-sensitive component, and is highly enriched in cells developing new macronuclei. The temperature inactivation profile of the activity parallels the temperature lethality profile specific for Tetrahymena cells developing new macronuclei. We suggest that this deletion-ligation activity carries out the genomic deletions in developing macronuclei in vivo.

Animals↗
PubMed1993

Changes in enkephalin immunoreactivity of sympathetic ganglia and digestive tract of the cat after splanchnic nerve ligation.

The aim of the present study was to analyze changes in the enkephalin immunoreactivity of sympathetic prevertebral ganglia coeliac plexus and inferior mesenteric ganglion) and intestinal tract (myenteric plexus and external muscle layers) in cats 2 days after left thoracic splanchnic nerve ligation, using radioimmunoassay and immunohistochemical techniques. Specific polyclonal antibodies directed against methionine- and leucine-enkephalin were used. The nerve ligation led to a considerable increase in the enkephalin immunoreactivity in the cranial part of the ligated nerves. This finding confirms the presence, in the cat, of an enkephalin output originating from thoracic spinal structures which are probably enkephalin-containing preganglionic neurons. In prevertebral ganglia the nerve ligation induced a marked decrease in the enkephalin immunoreactivity, which was probably due to the interruption of thoracic enkephalin efferents projecting towards both the coeliac plexus and the inferior mesenteric ganglion. In the digestive tract, the nerve ligation depressed the methionine-enkephalin immunoreactivity only in the gastro-duodenal region, and had no effect on the ileo-colonic region. The results of the present study add to the growing evidence that the sympathetic nervous system is involved in regulating the enteric enkephalinergic innervation, which is probably involved in controlling the intestinal motility.

Animals↗
PubMed1990

Subunit hybridization studies of partially ligated cyanomethemoglobins using a cryogenic method. Evidence for three allosteric states.

Reaction of tetrameric hemoglobin with ligands at the four heme sites yields nine species that have structurally unique combinations of ligated and unligated subunits. Using hemoglobins where the ligated subunits contain cyanomethemoglobin, Smith and Ackers studied the dimer-tetramer assembly reactions in all nine of the partially ligated species (F. R. Smith and G. K. Ackers, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 5347). They found a third assembly free energy in addition to those of unligated hemoglobin and fully ligated cyanomethemoglobin. The observed distribution of the three assembly free energies among the ten species was found to be incompatible with the two-state mechanism of allosteric control (J. Monod, J. Wyman and J. P. Changeaux, J. Mol. Biol. 12 (1965) 81). The results indicated a mechanism of 'combinatorial switching' in which the binding free energies per site change with configuration of occupied sites and not just their number. In this study, we have confirmed the existence of three assembly free energies among the ten ligation species using a cryogenic method (M. Perrella and L. Rossi-Bernardi, Methods Enzymol. 76 (1981) 133). For one of the species we find a different free energy assignment from that reported by Smith and Ackers; for all other species we observe the same assignments as in earlier work. The revised distribution also requires a 'combinatorial' mechanism of allosteric switching among the three states.

Freezing↗
PubMed1983

A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs.

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.

Alkaline Phosphatase↗
PubMed1993

In-vivo assessment of DNA ligation efficiency and fidelity in cells from patients with Fanconi's anemia and other cancer-prone hereditary disorders.

We developed a host cell DNA ligation assay, in which we transfected linearized plasmid pZ189 into human lymphoblasts or fibroblasts in order to assess the efficiency and accuracy of DNA ligation within these host cells. We used cell lines from patients with Fanconi's anemia and other chromosome breakage or instability syndromes (Bloom's syndrome, ataxia telangiectasia, Werner's syndrome). With the Fanconi's anemia lymphoblast line GM8010 we did not find a reduced, but a slightly hypermutable DNA ligation. Mutation analysis revealed a unique 7.9-12.5-fold increase in insertions or complex mutations. With cells from the other chromosome breakage/instability syndromes we also found a hypermutable and/or reduced DNA ligation. An impaired DNA ligation might be a common molecular mechanism of genetic instability in these disorders.

Cell Line↗
PubMed1991

The mode of action of 1-beta-D-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells.

Mammalian cells permeabilised by treatment with saponin are capable of UV excision repair. We have developed an assay system which permits measurement of the later stages of repair, i.e. repair synthesis and ligation. Incomplete repair sites are accumulated in UV-irradiated cells by incubating them with DNA synthesis inhibitors hydroxyurea and aphidicolin. On removal of the inhibitors at the time of permeabilisation, these incomplete sites, detected as DNA breaks, are rapidly ligated in a reaction that requires deoxyribonucleoside triphosphates and is blocked by aphidicolin. Thus ligation is possible only after a significant amount of DNA synthesis. We have used the assay to clarify the mode of inhibition of DNA repair by 1-beta-D-arabinofuranosylcytosine (ara C), another DNA polymerase inhibitor. It is well known that incomplete repair sites accumulated in whole cells with ara C are ligated at a slow rate, if at all. The hypothesis that ara C blocks or reduces further polymerisation after its incorporation into repair patches is disproved by our demonstration that, in permeable cells, the accumulated DNA breaks are ligated very rapidly. The likely explanation of the action of ara C is that, once phosphorylated, it remains in the cell as ara CTP and continues to inhibit polymerisation through competition with dCTP; in permeable cells, it readily leaks out.

Aphidicolin↗
PubMedDate not supplied

Epidural anesthesia for postpartum tubal ligation using epidural catheters placed during labor.

STUDY OBJECTIVES: To evaluate the success of epidural anesthesia for postpartum tubal ligation using epidural catheters placed during labor and to determine whether patient characteristics, timing of surgery, or technical factors (e.g., length of epidural catheter inserted into the epidural space) influenced the success of subsequent epidural anesthesia. DESIGN: Retrospective study. SETTING: University hospital labor and delivery suite. PATIENTS: 90 consecutive women scheduled for postpartum tubal ligation using epidural catheters placed during labor. INTERVENTIONS: Epidural catheters were reinjected with 1.5% to 2% lidocaine with epinephrine 5 micrograms/ml or 2% to 3% 2-chloroprocaine immediately before surgery. MEASUREMENTS AND MAIN RESULTS: 74% of the women received satisfactory intraoperative anesthesia using in situ epidural catheters. Reinjecting the catheter within 4 hours of delivery was associated with a greater frequency of successful epidural anesthesia for tubal ligation (95% vs. 67%; p = 0.029). There was no significant difference between the two groups in the length of catheter inserted into the epidural space. CONCLUSIONS: Although other factors may influence the timing of postpartum tubal ligation after delivery, the success of epidural anesthesia for tubal ligation using in situ epidural catheters is greater if surgery is performed shortly after delivery.

Adult↗
PubMed1995

Labor epidural catheter reactivation or spinal anesthesia for delayed postpartum tubal ligation: a cost comparison.

STUDY OBJECTIVES: To evaluate the costs and resource consumption associated with utilizing epidural catheters placed during labor versus spinal anesthesia for postpartum tubal ligation. To examine maternal demographics, anesthetic management variables, and time interval from delivery until surgery for association with epidural catheter reactivation success rate. DESIGN: Retrospective study. SETTING: University hospital labor and delivery center. PATIENTS: 120 consecutive postpartum patients with tubal ligations performed between June 1991 and December 1993. INTERVENTIONS: Postpartum women scheduled for tubal ligation with labor epidural catheters in place either had local anesthetic injected via the epidural catheter (n = 45) or had the catheter removed without reinjection and spinal anesthetic administered (n = 20). Patients with inadequate epidural anesthesia went on to receive spinal anesthesia. Women without a labor epidural catheter received spinal anesthesia (n = 55). MEASUREMENTS AND MAIN RESULTS: Adequate anesthesia for tubal ligation was achieved in 78% of women after reinjection of their epidural catheter. Operating room (OR) and anesthesia times were highest when epidural catheter reactivation was unsuccessful, intermediate when epidural catheter reactivation was successful, and lowest with initial spinal anesthesia (p < 0.05). The longer OR and anesthesia provider times associated with epidural catheter reactivation increased patient charges on average of $176 compared with the initial use of spinal anesthesia. CONCLUSIONS: Spinal anesthesia for postpartum tubal ligation was associated with lower anesthesia professional fees and OR charges compared with attempted reactivation of epidural catheters placed during labor. Anesthesiologists should weigh the cost advantages of spinal anesthesia against the small, but increased probability of headache after dural puncture.

Analgesics, Opioid↗
PubMed2005

The effect of spermatic vessel ligation on testicular nitric oxide levels and germ cell-specific apoptosis in rat testis.

Management of high testis may vary but the most popular method in surgical treatment is the Fowler-Stephens maneuver. The aim of the present study was to investigate the effects of spermatic vessel ligation on testicular nitric oxide (NO) levels, expression of inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) and germ cell-specific apoptosis in both ipsilateral and contralateral testes in rats. Twenty-eight animals were randomly allocated into four groups (n=7 each). The spermatic vessels were ligated as a simulation of the Fowler-Stephens maneuver. The groups of animals were sacrificed at 2 h (group 1), 4 h (group 2) and 24 h (group 3) after ligation, respectively. Sham-operated animals served as controls (group 4). Biochemical assessment of testicular NO levels was performed by the Griess method. iNOS and eNOS expression and apoptosis were studied in ipsilateral and contralateral testes. Testicular NO levels at 24 h after the simulated Fowler-Stephens maneuver were found to be significantly increased in both ipsilateral and contralateral testes when compared with the sham-operated group. eNOS expression was clearly increased in ipsilateral testes, whereas moderate expression was detected in the contralateral seminiferous tubules at 24 h after ligation. Mild focal iNOS immunostaining was also observed in seminiferous tubules of the ipsilateral testis at 24 h after the simulated Fowler-Stephens maneuver. Apoptosis was dramatically increased in ipsilateral testes; however, it was only detected in single cells in the contralateral side at 24 h after ligation. In conclusion, the simulated Fowler-Stephens maneuver induces testicular nitric oxide synthesis and germ cell-specific apoptosis in the ipsilateral testis. These results suggest that high levels of NO induce apoptosis and may impair spermatogenesis thus explaining the unsuccessful outcome of the Fowler-Stephens maneuver.

Animals↗
PubMed2004

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism.

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.

DNA↗
PubMed2004

An o-nitrobenzyl scaffold for peptide ligation: synthesis and applications.

Chemical ligation approaches facilitate the chemoselective assembly of unprotected peptides in aqueous solution. Here, two photolabile auxiliaries are described that enlarge the applicability of native chemical ligation to non-cysteine targets. The auxiliaries, designed to allow reaction with thioester peptides, generate an amide bond between the two initial fragments. The o-nitrobenzyl tertiary benzylamide that is formed at the ligation junction can be transformed into a native amide group under mild photolysis conditions. The veratryl auxiliary was found to be excessively labile during peptide purification and ligation. However, the auxiliary based on the o-nitrobenzyl group shows all the necessary properties for a general application in routine peptide and protein synthesis. In addition, the auxiliary linked to the N-terminus can be efficiently photolyzed, suggesting a new approach for the generation of photocaged amines. Synthesis, solid phase introduction onto peptide chains, ligation properties and photolysis in water are described, and a careful study of compatibility of the method with potentially fragile peptide side chains is reported.

Nitrobenzenes↗
PubMed2003

Role of opioid and nitric oxide systems in the nonadrenergic noncholinergic-mediated relaxation of corpus cavernosum in bile duct-ligated rats.

Changes in nonadrenergic noncholinergic (NANC)-mediated relaxation of the anococcygeus muscle have been demonstrated in cholestasis. Cholestasis is also associated with accumulation of endogenous opioid peptides and nitric oxide (NO) overproduction. This study was therefore undertaken to investigate the effect of cholestasis on the NANC-mediated relaxation of corpus cavernosum in bile duct-ligated rats and to examine the possible roles of the opioid system and nitric oxide in the cholestasis-associated alterations of corpus relaxation. Bile duct-ligated and sham-operated rats were treated for 2 weeks with either normal saline, N (omega)-nitro L-arginine methylester (L-NAME) (3 mg/kg/day, i.p.) or naltrexone (20 mg/kg/day, i.p.). On the 14th day, the strips of corpus cavernosum were mounted under tension in a standard oxygenated organ bath with guanethidine sulfate (5 microM) and atropine sulfate (1 microM) (to produce adrenergic and cholinergic blockade). The strips were precontracted with phenylephrine hydrochloride (7.5 microM) and electrical field stimulation was applied at different frequencies to obtain NANC-mediated frequency-dependent relaxant responses. The results showed that the amplitudes of relaxation responses at each frequency in bile duct-ligated rats were greater than the responses of sham-operated animals. This increase in relaxation responses in bile duct-ligated rats was inhibited by chronic L-NAME administration for 2 weeks so it seemed that it might be due to the nitric oxide overproduction in cholestatic states. Chronic administration of naltrexone for 2 weeks to bile duct-ligated rats had the same inhibitory effect on the relaxation responses. Our results demonstrated that in cholestasis, there was an increase in NANC-mediated relaxation of corpus cavernosum and both opioid and nitric oxide systems were involved in this increase.

Animals↗
PubMed2000

Bile duct ligation promotes covalent drug-protein adduct formation in plasma but not in liver of rats given zomepirac.

Acyl glucuronides are reactive electrophilic metabolites of carboxylate drugs, capable of undergoing hydrolysis, rearrangement and covalent binding reactions with proteins in vivo. Such covalent drug-protein adducts may be prerequisites for certain idiosyncratic immune and toxic responses in susceptible individuals. The present study examined the effect of experimental cholestasis on the extent and pattern of formation of protein adducts in plasma and liver of rats given the non-steroidal antiinflammatory drug (NSAID) zomepirac (ZP). Groups of intact, bile-exteriorized and bile duct-ligated rats given a 50 mg/kg i.v. dose of ZP were studied for 24 hr. In intact rats, only 1.4% of the dose was recovered as the sum of ZP, ZP acyl glucuronide (ZAG) and its rearrangement isomers (iso-ZAG) in urine in 24 hr. In bile-exteriorized animals, 0.5% of the dose was recovered in urine in 24 hr, with 31.6% of the dose being recovered in bile (2.7% as ZP, 20.0% as ZAG and 8.9% as iso-ZAG). In the bile duct-ligated group, recovery of dose in 24 hr urine totalled 17.5% (1.7% as ZP, 6.7% as ZAG and 9.1% as iso-ZAG). ZAG and iso-ZAG were measurable in plasma only in the bile duct-ligated group, and covalent binding of ZP to plasma proteins was much higher (5-6 fold) than in intact or bile-exteriorized rats. Total adduct concentrations in liver were not significantly different among the three groups. Immunoblotting using a polyclonal ZP antiserum confirmed that serum albumin was a major target protein in plasma. The major ZP-modified bands in the livers of intact and bile-exteriorized rats were at about 110, 140 and 200 kDa. However, the bands at 110 and 140 kDa were much lower in the livers of bile duct-ligated rats. The results show that about 30% of ZP doses are normally excreted as ZAG and its isomers in bile, with only minor excretion in urine. Bile duct ligation shunts the glucuronide into blood (and urine), strongly promoting adduct formation with plasma proteins, and alters the pattern but not the total quantity of drug-modified proteins formed in the liver.

Animals↗