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Biomedical subjects

I Lucas

Publications and source records attributed to I Lucas.

At least 19 recordsLinked to original sources
PubMed2001

Topoisomerase II can unlink replicating DNA by precatenane removal.

We have analysed the role of topoisomerase II (topo II) in plasmid DNA replication in Xenopus egg extracts, using specific inhibitors and two-dimensional gel electrophoresis of replication products. Topo II is dispensable for nuclear assembly and complete replication of plasmid DNA but is required for plasmid unlinking. Extensive unlinking can occur in the absence of mitosis. Replication intermediates generated in the absence of topo II activity have an increased positive superhelical stress (+DeltaLk), suggesting a deficiency in precatenane removal. The geometry of replication intermediates cut by poisoning topo II with etoposide and purified by virtue of their covalent attachment to topo II subunits demonstrates that topo II acts behind the forks at all stages of elongation. These results provide direct evidence for unlinking replicating DNA by precatenane removal and reveal a division of labour between topo I and topo II in this eukaryotic system. We discuss the role of chromatin structure in driving DNA unlinking during S phase.

Animals↗
PubMed2001

Importance of validation of immunoassays for intact proinsulin.

The aim of this study was to compare results obtained from two commercially available immunoassay kits for intact proinsulin. Fasting and post-prandial samples were obtained from both healthy subjects and patients with type 2 diabetes mellitus and assays were carried out according to the manufacturers' instructions. Coefficient of variation of the duplicates in both assays was acceptable with the MLT Intact Proinsulin assay giving slightly better overall precision. Regression analysis indicated a good correlation between the assays (r=0.97), however, a procedure better designed to compare analytical methods demonstrated a considerable lack of agreement for some samples. Dilution of samples in the Dako assay greatly affected the results when compared to samples assayed undiluted, whereas in the MLT assay, dilution of samples produced the expected results. Repeat comparison, assaying samples neat in the MLT assay and diluted 1:5 in the Dako assay, resulted in a considerable improvement in the agreement between the Dako and MLT assays. This study underlines the importance of the use of validation procedures which demonstrate quantitative analytical recoveries from a variety of specimens over the working range of the assay method in question.

Case-Control Studies↗
PubMed2000

Hemicatenanes form upon inhibition of DNA replication.

Plasmid DNA incubated in interphase Xenopus egg extracts is normally assembled into chromatin and then into synthetic nuclei which undergo one round of regulated replication. During a study of restriction endonuclease cut plasmid replication intermediates (RIs) by the Brewer-Fangman 2D gel electrophoresis technique, we have observed the formation of a strong spike of X-shaped DNA molecules in extracts that otherwise yield very little or no RIs. Formation of these joint molecules is also efficiently induced in replication-competent extracts upon inhibition of replication fork progression by aphidicolin. Although their electrophoretic properties are quite similar to those of Holliday junctions, 2D gels of doubly cut plasmids show that these junctions can link two plasmid molecules at any pair of DNA sequences, with no regard for sequence homology at the branch points. Neutral-neutral-alkaline 3D gels show that the junctions only contain single strands of parental size and no recombinant strands. A hemicatenane, in which one strand of a duplex is wound around one strand of another duplex, is the most likely structure to account for these observations. The mechanism of formation of these novel joint DNA molecules and their biological implications are discussed.

Animals↗
PubMed2000

Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos.

Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.3-42.2 kbp) in Xenopus egg extracts by two-dimensional gel electrophoresis and electron microscopy. We confirm that replication initiates without sequence specificity on all plasmids. We demonstrate for the first time that multiple initiation events occur on large plasmids, but not on small (<10 kb) plasmids, at average intervals of approximately 10 kb. Origin interference may prevent multiple initiation events on small plasmids. Multiple initiation events are neither synchronous nor regularly spaced. Bubble density is higher on later than on earlier replication intermediates, showing that initiation frequency increases throughout S phase, speeding up replication of late intermediates. We suggest that potential origins are abundant and randomly distributed, but that the increase of initiation frequency during S phase, and possibly origin interference, regulate origin activation to ensure rapid completion of replication.

Animals↗
PubMed1998

Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis.

Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent. Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs. Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells.

Animals↗
PubMed1997

Role of nuclear architecture in the initiation of eukaryotic DNA replication.

The eukaryotic genome is compacted in the cell nucleus, in a way that allows its faithful and ordered replication each cell cycle. Chromatin is organized into topologically constrained loops that are anchored to the nuclear matrix by specific attachment regions (SARs). Chromatin loops were proposed to correspond to replication units. In particular, it has been suggested that replication origins coincide with SARs. Critical examination of these hypotheses has long been hampered by the elusive nature of higher eukaryotic DNA replication origins and termini. In recent years, however, a number of loci have been mapped for both SARs and replication units, and studies on the nuclear localization of replicating DNA and replication proteins have begun. We review these data and argue that they question this model. We then try to delineate other aspects of chromosome compartmentalization and cell-cycle remodeling which might be responsible for the specification and activation of metazoan DNA replication origins.

Animals↗
PubMed1996

[The POEMS syndrome, apropos of 2 cases and review of the literature].

We describe two cases of POEMS syndrome, both with polyneuropathy, monoclonal gammopathy of the IgG lambda type, thickening of the skin with Raynaud phenomenon, multiple osteosclerotic lesions and hypothyroidism. One of them, also had papilledema, elevated cerebrospinal fluid protein, intracranial hypertension and phrenic nerve palsy; the other one had ascites and hepatosplenomegaly. Phrenic nerve palsy associated to this syndrome has not been described previously.

Ascites↗
PubMed1981

Renal clearance of methotrexate in man during high-dose oral and intravenous infusion therapy.

The renal excretion and clearance of methotrexate (MTX) following high-dose (800 mg) therapy followed by folinic acid rescue was studied in 12 patients (2 female, 10 male): the mean age was 49.3 +/- 5.5 (SE), weight 68.6 +/- 3.9 (SE) and body surface area 1.8 +/- 0.1 m2. Plasma and urine were collected over 154 h at intervals of 2-24 h, and the collection times, volume, and pH of urine samples recorded. Total MTX concentrations in urine and plasma were measured by the highly specific competitive protein-binding assay method. Plasma and urinary creatinine levels were measured on an SMA-12 autoanalyser. The renal clearance of MTX was calculated for each urine collection period. Following oral administration, clearance values during the first 6 h were high at 257 +/- 8.3 (ml/Min), followed by a trough in clearance of 27.9 +/- 4.2 (ml/min) in the 20- to 30-h period. This was followed by a secondary rise of MTX renal clearance to 180.4 +/- 14.6 ml/min during the 68- to 84-h period and again to 84.9 +/- 17.1 ml/min between 84 and 112 h. In the last two periods it rose to 209 +/- 57.9 ml/min. Similar fluctuations were seen following IV administration. The changes in clearance were statistically significant at the p less than 0.005 level. It is suggested that high concentrations of MTX in the renal tubules result in inhibition of carrier protein synthesis, leading to a fall in active tubular secretion. When MTX concentrations fall the tubular cell recovers and a secondary rise in renal clearance occurs, leading to cyclical changes in MTX elimination.

Administration, Oral↗
PubMed1979

Comparison of intravenous and oral high-dose methotrexate in treatment of solid tumours.

An outpatient regimen of oral high-dose methotrexate was studied in 14 patients with solid tumours over 12 months. Detailed pharmacokinetic analysis in five patients showed high oral bioavailability (mean +/- SE of mean 87.6 +/- 1.5%), indicating that with this regimen oral methotrexate was well absorbed and the first-pass effect low. Oral administration resulted in peak plasma methotrexate concentrations of 8.4 +/- 0.5 mumol/l (382 +/- 23 microgram/100 ml) and was almost as effective as intravenous administration, which achieved peak concentrations of 9.9 +/- 0.4 mumol/l (450 +/- 18 microgram/100 ml). In all 14 patients the clinical response to oral treatment was comparable to that reported to intravenous administration of high-dose methotrexate used in combination with other cytotoxic drugs. The disease-free interval in cases of adult sarcoma was 7.4 +/- 1.3 months and the relapse rate 29%. Out of four patients with small-cell carcinoma, two showed an objective response to oral treatment. We suggest that oral high-dose methotrexate given in divided doses is a rational alternative to expensive intravenous high-dose methotrexate regimens, but further clinical evaluation is necessary.

Administration, Oral↗
PubMed1979

Ocular side effects with 5-fluorouracil.

The concentrations of 5-fluorouracil in tears and plasma were studied in eight patients receiving the drug for carcinoma of the colon. The drug was measured by a gas liquid chromatographic method and found to be present only in the tears of patients with excessive lacrimation. In three patients with watery eyes the peak concentration in tears (16-23.8 micrograms/ml) occurred 15 minutes after IV administration and this corresponded with the end of the distribution (alpha) phase in the plasma when the plasma levels ranged from 18-26 micrograms/ml. In five patients without eye symptoms the drug was undetectable in the lacrimal fluid even though they had similar plasma levels (15-25 micrograms/ml) of 5-fluorouracil. The volumes of distribution and plasma clearance rates were similar in the two groups [being 0.2-0.52 (mean = 0.33) 1/kg and 612-978 (mean = 850) ml/min respectively in patients with excessive lacrimation and 0.13-0.79 (mean = 0.36) 1/kg and 435-1138 (mean = 831) ml/min respectively in the five patients without symptoms.]. It appears that 5-fluorouracil produces irritation of the lacrimal apparatus in about 30% of patients on the drug and in association with this appears in the tears where it may be responsible for the reported irritation and fibrosis of the tear duct.

Chromatography, Gas↗
PubMed1979

[A clinical report. Atypical subacute thyroiditis].

We present a patient 52 years old with a symtomatology of three month's evolution with: rapid weight loss, weakness in legs fasciculated tremor increased by any physical effort, nervousness and anxiety. The isotopic exploration of thyroid in basal conditions and after estimulation with TSH shows a very small captation of iodide. The determination of T 4 shows a notable increase (24 U. U. Normal range is 7,5 to 10,5 U. U.). The clinical symtomatology biochemical and gammagraphic datum support the diagnostic of subacute thyroiditis and also the posterior evolution of symtomatology in spite of the absence of odynophagia and the increase of syze of the thyroid. We comment on the particularity of this clinical entity that could be confused with thireotoxicosis because of its atypical character of presentation.

Humans↗