Modified continuous femoral three-in-one block for postoperative pain after total knee arthroplasty.

UNLABELLED: We prospectively studied the continuous "modified" femoral three-in-one block for postoperative pain after total knee arthroplasty. Sixty-two patients undergoing elective knee arthroplasty under spinal anesthesia with bupivacaine (B) and fentanyl were randomized to receive 0.2% B, 0.1% B, or placebo at 10 mL/h for 48 h after an initial bolus of 30 mL of the same solution via the femoral block catheter. The catheters were inserted under the fascia iliaca using a "double pop" technique and a peripheral nerve stimulator and were advanced 15-20 cm cranially. Venous plasma levels of B, desbutylbupivacaine, and 4-hydroxy B were measured daily for 3 days. All patients received patient-controlled analgesia with morphine and indomethacin suppositories for 48 h. Using computed tomography, we evaluated the catheter location for 20 patients. The catheter tips, located superior to the upper third of the sacroiliac joint in the psoas sheath, were labeled as ideally located. The group receiving 0.2% B had a larger block success rate, smaller morphine consumption in the immediate postoperative period (15 vs 22 mg) and during the first postoperative day (9 vs 18 mg), and achieved a greater range of motion in the immediate postoperative period (91 degrees +/- 10 degrees vs 80 degrees + 13 degrees ). Visual analog scores for pain during both rest and activity were low but similar between the groups. Forty percent of the catheters evaluated were ideally located. Ideal location and use of 0.2% B resulted in 100% success of blockade of all three nerves. The S1 root was blocked in up to 76% of patients. The plasma levels of B, 4-hydroxy B, and desbutylbupivacaine were below the toxic range during the infusion. We conclude that continuous fascia iliaca block with 0.2% B results in opioid-sparing and improved range of motion during the immediate postoperative period. Larger doses of bupivacaine may safely be used in the immediate postoperative period if needed. IMPLICATIONS: Continuous fascia iliaca block with 0.2% bupivacaine reduces opioid requirements and improves range of motion in the immediate postoperative period compared with a placebo and 0.1% bupivacaine. Plasma levels are below the toxic range with this dose. Only 40% of the catheters are positioned in the ideal location. With the smaller dose of bupivacaine, the success rate with this block is small.

Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase II beta.

Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug-induced DNA breakage were observed. These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.

DNA topoisomerase II is the molecular target of bisdioxopiperazine derivatives ICRF-159 and ICRF-193 in Saccharomyces cerevisiae.

Bisdioxopiperazines such as ICRF-159 and ICRF-193 have been shown to inhibit DNA topoisomerase II. To determine the molecular target of these compounds in vivo, we utilized a yeast genetic system in which the topoisomerase II activity can be modulated. To reduce topoisomerase II activity, we used top2-1 mutant yeast cells that have normal DNA topoisomerase II activity at 25 degrees C but greatly reduced enzyme activity at 30 degrees C, a temperature that is semipermissive for growth. At 25 degrees C top2-1 cells are as sensitive to the ICRF compounds as the wild-type strain; at 30 degrees C the cells became hypersensitive to these agents. In contrast, top2-1 strains become very resistant to the class of topoisomerase II inhibitors such as amsacrine and etoposide, which stabilize the covalent enzyme-DNA intermediate of the enzyme reaction. Overexpression of topoisomerase II from a plasmid-born TOP2 gene results in lower susceptibility to ICRF compounds and higher susceptibility to amsacrine than the parental strain exhibits. These results support the hypothesis that the main cellular target of ICRF compounds is DNA topoisomerase II, and that these compounds, unlike amsacrine and etoposide, inhibit topoisomerase II activity without stabilizing an enzyme-DNA covalent complex.

Mechanistic studies of amsacrine-resistant derivatives of DNA topoisomerase II. Implications in resistance to multiple antitumor drugs targeting the enzyme.

Wild-type yeast DNA topoisomerase II and three of its amsacrine-resistant derivatives L475A/L480P, L475A/R476G, and A642G, named according to amino acid changes at the codons specified, were overexpressed and purified. Because cells expressing several mutant enzymes missing portions of the carboxyl-terminal domain of the wild-type enzyme were previously found to exhibit amsacrine resistance, a carboxyl-terminal truncation protein Top2(1-1166), which lacks the last 263 amino acids of the wild-type enzyme, was also overexpressed and purified. These purified enzymes were used in the measurement of the turnover numbers of the DNA-dependent hydrolysis of ATP, the transport of one DNA segment through another, and the effects of amsacrine, teniposide or Ca(II) on the formation of the enzyme-DNA covalent intermediate. The results of these studies indicate that mutations leading to cellular resistance to amsacrine may occur by several different mechanisms, including reduction of the nuclear concentration and attenuation of the intrinsic catalytic steps of the enzyme. The significance of this underpinning mechanistic diversity of drug resistance and its relation to the simultaneous development of cellular resistance to chemically distinct drugs that target DNA topoisomerase II are discussed.

Analysis of yeast DNA topoisomerase II mutants resistant to the antitumor drug amsacrine.

Site-directed mutagenesis of regions within a plasmid-borne yeast TOP2 gene encoding DNA topoisomerase II and hydroxylamine mutagenesis of the entire plasmid were carried out, and the mutagenized plasmid DNA pools were used separately to transform yeast with a temperature-sensitive top2-4 mutation in the chromosomal TOP2 locus. By selecting transformants that grow in the presence of the antitumor drug amsacrine at 35 degrees C, a nonpermissive temperature for the top2-4 allele, plasmid-borne top2 mutants expressing amsacrine-resistant and physiologically functional DNA topoisomerase II were readily obtained. The causality between amsacrine resistance and the presence of these mutations in yeast DNA topoisomerase II has been firmly established, and this causality in turn shows that, in yeast at least, DNA topoisomerase II is the only significant cellular target of amsacrine. Three classes of such mutants have been identified: one involves single or multiple changes in a sequence PLRGK-MLN located at positions 474-481 of yeast DNA topoisomerase II, a highly conserved motif in all type II DNA topoisomerases; a second involving a single mutation changing Ala642 to threonine or glycine; and a third involving deletions of portions of the carboxy-terminal domain of the yeast enzyme. The nature of drug resistance of these different classes of mutants is discussed. The approaches used in this work should be readily applicable to yeast cells expressing heterologous DNA topoisomerases such as human DNA topoisomerase II alpha. Other DNA topoisomerase II-targeting drugs can also be studied in such a system.

Use of yeast in the study of anticancer drugs targeting DNA topoisomerases: expression of a functional recombinant human DNA topoisomerase II alpha in yeast.

A plasmid was constructed for the expression of human DNA topoisomerase II alpha in yeast from a galactose-inducible promoter of the yeast GAL1 gene. Expression of a recombinant human enzyme, in which the first 28 of the 1531 codons of human DNA topoisomerase II alpha were replaced by the first five codons of yeast DNA topoisomerase II, was shown to rescue the lethal phenotype of thermal sensitive yeast DNA topoisomerase II mutants at 35 degrees C. Purification of the human enzyme overexpressed in yeast yielded a single polypeptide with an apparent mass of 170 kDa, and the properties of the purified recombinant enzyme were found to be the same as those reported for human DNA topoisomerase II alpha purified from HeLa cells. Studies with the anticancer drug amsacrine indicated that the human enzyme, either inside yeast cells or in its purified form, is a target of the drug; inhibition of the purified enzyme by teniposide (VM-26) and merbarone was also demonstrated. These studies demonstrate that yeast strains expressing human DNA topoisomerase II alpha provide a convenient system for studying drugs targeting the enzyme; unlike mammalian systems, potential complications due to the presence of human DNA topoisomerase II beta can be eliminated in this system. Overexpression of human DNA topoisomerase II alpha in yeast also provides a convenient source of the enzyme for in vitro studies.

Effects of afternoon injections of melatonin in hypothyroid male Syrian hamsters.

Male Syrian hamsters were kept under either 14 h light/10 h dark (lights on at 06.30 h) or 2 h light/22 h dark (lights on at 14.30 h) photoperiods. Groups of hamsters under each photoperiod were rendered hypothyroid by addition of 0.4% thiourea to the drinking water. These hamsters received, in addition, either a daily evening injection of saline or a daily injection of 25 micrograms melatonin in saline. Groups of intact controls and pinealectomized control hamsters were also maintained under the two photoperiodic conditions. After 10 weeks under the different conditions the hamsters were killed by decapitation, and serum samples assayed for thyroxin, thyroid-stimulating hormone (TSH), and prolactin (PRL). Pituitary extracts were assayed for TSH and PRL. Hypothyroidism in hamsters receiving thiourea was confirmed by radio-immunoassay data showing low serum thyroxin and greatly elevated serum TSH concentrations. Melatonin injections resulted in significant depression of serum TSH in thiourea-treated hamsters under short photoperiod compared to saline-injected controls. Both melatonin injections and short photoperiod resulted in a significant reduction of pituitary TSH in hamsters on thiourea compared to values obtained from similarly treated animals under the 14 h light/10 h dark photoperiod. Hypothalamic concentrations of thyrotropin-releasing hormone (TRH) were significantly elevated by melatonin injections and by short photoperiodic conditions, but not by thiourea administration. The short photoperiod resulted in testicular involution which was completely reversed by pinealectomy and partially reversed (to 53% of controls) by thiourea treatment. Involution of gonads was complete in thiourea-treated animals under short photoperiod, if they received melatonin injections.(ABSTRACT TRUNCATED AT 250 WORDS)