Alkali metal cation-hexacyclen complexes: effects of alkali metal cation size on the structure and binding energy.

Threshold collision-induced dissociation (CID) of alkali metal cation-hexacyclen (ha18C6) complexes, M(+)(ha18C6), with xenon is studied using guided ion beam tandem mass spectrometry techniques. The alkali metal cations examined here include: Na(+), K(+), Rb(+), and Cs(+). In all cases, M(+) is the only product observed, corresponding to endothermic loss of the intact ha18C6 ligand. The cross-section thresholds are analyzed to extract zero and 298 K M(+)-ha18C6 bond dissociation energies (BDEs) after properly accounting for the effects of multiple M(+)(ha18C6)-Xe collisions, the kinetic and internal energy distributions of the M(+)(ha18C6) and Xe reactants, and the lifetimes for dissociation of the activated M(+)(ha18C6) complexes. Ab initio and density functional theory calculations are used to determine the structures of ha18C6 and the M(+)(ha18C6) complexes, provide molecular constants necessary for the thermodynamic analysis of the energy-resolved CID data, and theoretical estimates for the M(+)-ha18C6 BDEs. Calculations using a polarizable continuum model are also performed to examine solvent effects on the binding. In the absence of solvent, the M(+)-ha18C6 BDEs decrease as the size of the alkali metal cation increases, consistent with the noncovalent nature of the binding in these complexes. However, in the presence of solvent, the ha18C6 ligand exhibits selectivity for K(+) over the other alkali metal cations. The M(+)(ha18C6) structures and BDEs are compared to those previously reported for the analogous M(+)(18-crown-6) and M(+)(cyclen) complexes to examine the effects of the nature of the donor atom (N versus O) and the number donor atoms (six vs four) on the nature and strength of binding.

Alkali metal cation interactions with 15-crown-5 in the gas phase: revisited.

Quantitative interactions of the alkali metal cations with the cyclic 15-crown-5 polyether ligand (15C5) are studied. In this work, Rb(+)(15C5) and Cs(+)(15C5) complexes are formed using electrospray ionization and studied using threshold collision-induced dissociation with xenon in a guided ion beam tandem mass spectrometer. The energy-dependent cross sections thus obtained are interpreted to yield bond dissociation energies (BDEs) using an analysis that includes consideration of unimolecular decay rates, internal energy of the reactant ions, and multiple ion-neutral collisions. 0 K BDEs of 175.0 ± 9.7 and 159.4 ± 9.6 kJ/mol, respectively, are determined and exceed those previously measured [J. Am. Chem. Soc. 1999, 121, 417-423] by 68 and 57 kJ/mol, respectively, consistent with the hypothesis proposed there that excited conformers had been studied. Because the analysis techniques have advanced since this early study, we also reanalyze the published data for the Na(+)(15C5) and K(+)(15C5) systems to ensure a self-consistent interpretation of all four systems. Revised BDEs for these systems are 296.1 ± 15.5 and 215.6 ± 10.6 kJ/mol, respectively, which are within experimental uncertainty of the previously reported values. In addition, quantum chemical calculations are conducted at the B3LYP/def2-TZVPPD level of theory with theoretical BDEs in reasonable agreement with experiment. Computations are also used to explore features of the potential energy surfaces for isomerization of the M(+)(15C5) complexes.

Screening for visual impairment in elderly patients with hip fracture: validating a simple bedside test.

AIMS: 1. To assess the prevalence of visual impairment in those patients who sustain proximal hip fracture after a simple fall. 2. To test the validity of a simple screening test to identify patients with visual impairment. METHODS: Patients on the orthopaedic rehabilitation ward recuperating from proximal hip fracture were recruited. The nurse screener and examining Ophthalmologist independently assessed the patients' distance visual acuity and visual field to confrontation. In addition, the nurse screener assessed for the presence of cataract in the red reflex and the examining Ophthalmologist performed a dilated slit-lamp examination. On completion of the examination, the Ophthalmologist documented the cause(s) of any visual impairment found. RESULTS: A total of 89 patients were assessed. In all, 29 patients (33%) could be classified as visually impaired using the United States criteria and 52 patients (58%) had a distance visual acuity of 6/18 or worse in at least one eye. The test reliably identified those patients with visual impairment (sensitivity 94% (+/-5%), specificity 92% (+/-6%)), but was less reliable at identifying those patients with potentially remedial visual impairment (sensitivity 70% (+/-10%), specificity 92% (+/-6%)). CONCLUSION: The level of visual impairment in this group of patients is high and screening for visual impairment in the elderly with a history of falls is justified. We have demonstrated that a suitably trained member of the rehabilitation team can identify over 94% of those patients with impaired vision. We believe this simple test should now be incorporated into the assessment of all patients requiring rehabilitation after a proximal hip fracture.

Fear of falling, falls efficacy, and health outcomes in older people following hip fracture.

PURPOSE: This study sought to determine whether fear of falling and falls efficacy independently contribute to the prediction of health outcomes after a fall, controlling for length of stay in hospital, prefall activity problems, and history of falls. METHOD: Eighty-two older people (> or = 65 years) admitted to hospital as a result of a fall, with proximal femoral fracture, were interviewed to assess variables of interest. At two months after initial interview, participants (n = 57) were re-interviewed in their own home, and their functional limitation and further fall events were assessed. Regression analyses were carried out to determine the ability of the variables assessed in hospital to predict functional limitation and further falls post discharge. RESULTS: Perceived risk of falling and falls efficacy did not explain variance in functional limitation when added to a model containing biomedical factors. In the prediction of further falls, addition of falls efficacy and worry over further falls to a model containing biomedical factors resulted in a statistically reliable improvement, although falls efficacy was not independently associated with outcome. CONCLUSIONS: Assessing worry over further falls in hospital may help to identify older people with hip fracture at risk of poor health outcomes.

Schedule-dependent response of neuroblastoma cell lines to combinations of etoposide and cisplatin.

The growth inhibitory effects of cisplatin and etoposide on neuroblastoma cell lines were investigated in several scheduled combinations. Results were analyzed using median effect and combination index analyses. In all schedules in which cisplatin was administered prior to etoposide a synergistic effect was observed. Conversely, an antagonistic effect was seen in all schedules where etoposide was administered before cisplatin.

Deacetylase activity associates with topoisomerase II and is necessary for etoposide-induced apoptosis.

DNA topoisomerase II (topo II) is a ubiquitous nuclear enzyme that is involved in DNA replication, transcription, chromosome segregation, and apoptosis. Here we show by immunoprecipitation, pull down with glutathione S-transferase fusion proteins, and yeast two-hybrid analysis that both topo IIalpha and -beta physically interact with the histone deacetylase HDAC1. The in vitro DNA decatenation activity of recombinant topo IIalpha and -beta is inhibited by association with catalytically inactive, recombinant HDAC1. We provide evidence for the in vivo significance of the topo II-HDAC1 association, showing that inhibition of HDAC activity with trichostatin A suppresses apoptosis induced by the topo II poison etoposide, but not by the topoisomerase I inhibitor camptothecin. We suggest that chromatin remodeling by an HDAC-containing complex facilitates both topo II-catalyzed DNA rearrangement and etoposide-induced DNA damage in vivo.

Quantitation of DNA topoisomerase IIalpha and beta in human leukaemia cells by immunoblotting.

DNA topoisomerase II (topo II) is an essential nuclear enzyme and is the target for etoposide, which is used in the therapy of childhood acute lymphoblastic leukaemia (ALL). Topo II exists as two isoforms referred to as topo IIalpha and topo IIbeta. To determine whether cellular levels of topo IIalpha and beta are an important factor in determining drug sensitivity/resistance requires accurate, precise measurements of the two isoforms. We have developed a quantitative Western blotting method to accurately measure the absolute amounts of human topo IIalpha and beta, using recombinant human topo IIalpha and beta as standards. This quantitative method has been used to assess the efficiency of several commonly used topo II extraction protocols. The extractable amount of topo IIalpha and beta was found to be salt-dependent. However extraction using the optimal salt concentration was found to be as efficient as extraction with DNase I/Rnase A digestion and SDS solubilisation. Using the optimum extraction procedure and the quantitative immunoblotting method, topo IIalpha and beta was quantified in cell lines, peripheral blood lymphocytes and in lymphoblasts from children with newly diagnosed ALL.

An investigation into the formation of N- [2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and 6-[2-(dimethylamino)ethylamino]- 3-hydroxy-7H-indeno[2, 1-C]quinolin-7-one dihydrochloride (TAS-103) stabilised DNA topoisomerase I and II cleavable complexes in human leukaemia cells.

The antitumour agents DACA (XR5000; N-[2-(dimethylamino)ethyl]acridine-4-carboxamide) and TAS-103 (6-[2-(dimethylamino)ethylamino]-3-hydroxy-7H-indeno[2, 1-c]quinolin-7-one dihydrochloride) have been shown to inhibit two essential nuclear enzymes in vitro, DNA topoisomerase I and DNA topoisomerase (topo) II. To examine whether DACA or TAS-103 stabilise topo I, topo IIalpha, and topo IIbeta cleavable complexes in human leukaemia CCRF-CEM cells, the TARDIS assay (trapped in agarose DNA immunostaining) was used. This assay can reveal drug-stabilised topo-DNA complexes formed in situ in individual cells. The results showed that both DACA and TAS-103 can stabilise topo IIalpha cleavable complexes in these cells. Topo IIbeta cleavable complexes were also formed, but only at high concentrations of DACA and TAS-103. The effect on topo I was less clear, with TAS-103 showing only low levels of cleavable complex formation and DACA having no detectable effect under these assay conditions. This is in contrast to the purified enzyme cleavable complex assay, where both DACA and TAS-103 poisoned topo I. Although both DACA and TAS-103 show a preference for topo IIalpha in whole cells using the TARDIS assay, the formation of low levels of topo I or topo IIbeta cleavable complexes may still play a role in cell death.

Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the closed enzyme clamp of human topoisomerase IIalpha.

Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation at mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp. To investigate this unusual mode of action, we have used yeast to select plasmid-borne human topoisomerase IIalpha alleles resistant to ICRF-193. Mutations in topoisomerase IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA. These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193. We suggest that the inefficient trapping of the A648P closed clamp results either from the observed increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain. These results provide evidence for at least two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase IIalpha.

Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development.

To investigate the role of sox genes in vertebrate development, we have isolated sox11 from zebrafish (Danio rerio). Two distinct classes of sox11-related cDNAs were identified, sox11a and sox11b. The predicted protein sequences shared 75% identity. In a gene phylogeny, both sox11a and sox11b cluster with human, mouse, chick, and Xenopus Sox11, indicating that zebrafish, like Xenopus, has two orthologues of tetrapod Sox11. The work reported here investigates the evolutionary origin of these two gene duplicates and the consequences of their duplication for development. The sox11a and sox11b genes map to linkage groups 17 and 20, respectively, together with other loci whose orthologues are syntenic with human SOX11, suggesting that during the fish lineage, a large chromosome region sharing conserved syntenies with mammals has become duplicated. Studies in mouse and chick have shown that Sox11 is expressed in the central nervous system during development. Expression patterns of zebrafish sox11a and sox11b confirm that they are expressed in the developing nervous system, including the forebrain, midbrain, hindbrain, eyes, and ears from an early stage. Other sites of expression include the fin buds and somites. The two sox genes, sox11a and sox11b, are expressed in both overlapping and distinct sites. Their expression patterns suggest that sox11a and sox11b may share the developmental domains of the single Sox11 gene present in mouse and chick. For example, zebrafish sox11a is expressed in the anterior somites, and zebrafish sox11b is expressed in the posterior somites, but the single Sox11 gene of mouse is expressed in all the somites. Thus, the zebrafish duplicate genes appear to have reciprocally lost expression domains present in the sox11 gene of the last common ancestor of tetrapods and zebrafish. This splitting of the roles of Sox11 between two paralogues suggests that regulatory elements governing the expression of the sox11 gene in the common ancestor of zebrafish and tetrapods may have been reciprocally mutated in the zebrafish gene duplicates. This is consistent with duplicate gene evolution via a duplication-degeneration-complementation process.

Human topoisomerase IIalpha and IIbeta interact with the C-terminal region of p53.

The p53 tumor suppressor protein is a critical regulator of cell cycle progression and apoptosis following exposure of cells to DNA damaging agents such as ionizing radiation or anticancer drugs. An important group of anticancer drugs, including compounds such as etoposide and doxorubicin (Adriamycin), interacts with DNA topoisomerase II (topo II), causing the accumulation of enzyme-DNA adducts that ultimately lead to double-strand breaks and cell death via apoptosis. Human topo IIbeta has previously been shown to interact with p53, and we have extended this analysis to show that both topo IIalpha and IIbeta interact with p53 in vivo and in vitro. Furthermore, we show that the regulatory C-terminal basic region of p53 (residues 364-393) is necessary and sufficient for interaction with DNA topo II.

Camptothecin-stabilised topoisomerase I-DNA complexes in leukaemia cells visualised and quantified in situ by the TARDIS assay (trapped in agarose DNA immunostaining).

We have shown that the TARDIS assay (trapped in agarose DNA immunostaining) can be used to detect DNA-topoisomerase I (topo I) cleavable complexes in situ in individual cells following treatment with topo I-targeting drugs. This assay is a modification of the assay for DNA-topoisomerase II (topo II) cleavable complexes (Willmore et al., Mol Pharmacol 53: 78-85, 1998). Drug-stabilised topo I-DNA complexes were detected in situ by topo I-specific primary antibodies and then visualised using fluorescein isothiocyanate conjugated second antibodies. Immunofluorescence was then quantified using a cooled slow-scan coupled device camera and image analysis procedures. Camptothecin (CPT) was shown to stabilise topo I-DNA cleavable complexes in whole cells in a dose-dependent manner in both CCRF-CEM and K562 cells and in lymphoblasts from an adult with newly diagnosed acute myeloid leukaemia treated ex vivo with CPT. In K562 cells, cleavable complexes were found to be maximal between 30 and 90 minutes continuous exposure of CPT, and approximately 78% of cleavable complexes formed in these cells were found to be reversed within 5 minutes of drug removal. It has also been shown that the immunofluorescence detected by the TARDIS assay was specific for topo I-targeting agents. Hence, the TARDIS assay provides a powerful tool to determine the levels of drug-stabilised cleavable complexes in whole cells and thereby aid in the understanding of the mechanism of interaction between topo I-targeting drugs and their target.

Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage.

A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient break in a second duplex. The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DXD. We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium. In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis. It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations. The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s). These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue. A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction.

Murine transgenic cells lacking DNA topoisomerase IIbeta are resistant to acridines and mitoxantrone: analysis of cytotoxicity and cleavable complex formation.

Murine transgenic cell lines lacking DNA topoisomerase II (topo II)beta have been used to assess the importance of topo IIbeta as a drug target. Western blot analysis confirmed that the topo IIbeta -/- cell lines did not contain topo IIbeta protein. In addition, both the topo IIbeta +/+ and topo IIbeta -/- cell lines contained similar levels of topo IIalpha protein. The trapped in agarose DNA immunostaining assay (TARDIS) was used to detect topo IIalpha and beta cleavable complexes in topo IIbeta -/- and topo IIbeta +/+ cells. These results show that both topo IIalpha and beta are in vivo targets for etoposide, mitoxantrone, and amsacrine (mAMSA) in topo IIbeta +/+ cells. As expected, only the alpha-isoform was targeted in topo IIbeta -/- cells. Clonogenic assays comparing the survival of topo IIbeta -/- and topo IIbeta +/+ cells were carried out to establish whether the absence of topo IIbeta caused drug resistance. Increased survival of topo IIbeta -/- cells compared with topo IIbeta +/+ cells was observed after treatment with amsacrine (mAMSA), methyl N-(4'-[9-acridinylamino]-2-methoxyphenyl) carbamate hydrochloride (AMCA), methyl N-(4'-[9-acridinylamino]-2-methoxyphenyl)carbamate hydrochloride (mAMCA), mitoxantrone, and etoposide. These studies showed that topo IIbeta -/- cells were significantly more resistant to mAMSA, AMCA, mAMCA, and mitoxantrone, than topo IIbeta +/+ cells, indicating that topo IIbeta is an important target for the cytotoxic effects of these compounds.

Carbamate analogues of amsacrine active against non-cycling cells: relative activity against topoisomerases IIalpha and beta.

PURPOSE: Methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride (AMCA) and methyl N-(4'-(9-acridinylamino)-2-methoxyphenyl)carbamate hydrochloride (mAMCA) are analogues of the topoisomerase II (topo II) poison amsacrine, and are distinguished from amsacrine by their high cytotoxicity towards non-cycling cells. Since mammalian cells contain two forms (alpha and beta) of topo II and the alpha isoform is down-regulated in non-cycling cells, we have considered whether these carbamate analogues target topo IIbeta selectively. METHODS: A drug permeable yeast strain (JN394 top2-4) was transformed using a shuttle vector containing either human top2alpha, human top2alpha or yeast top2 under the control of a GAL1 promoter. The strain was analysed at a non-permissive temperature, where only the plasmid-borne topo II was active. RESULTS: AMCA and mAMCA produced comparable levels of cell killing with human DNA topo IIalpha, human DNA topo IIbeta and yeast DNA topo II. Two other acridine derivatives N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and its 7-chloro derivative, which like AMCA and mAMCA are able to overcome multidrug resistance mechanisms, were much more active against human DNA topo IIalpha than against human DNA topo IIbeta and yeast DNA topo II. A series of mutant Chinese hamster and human lines with defined topo lesions, including the HL60/MX2 line that lacks topo IIbeta expression, was also used to compare resistance to amsacrine, AMCA and etoposide. Loss of topo IIbeta activity had a greater effect on amsacrine and AMCA than on etoposide. Resistance of murine Lewis lung cultures in exponential and plateau phase was also measured. Loss of topo IIalpha activity, as measured in both mutant cells expressing lower amounts of enzyme and in cells in plateau phase, resulted in concomitant acquisition of resistance that was greatest for etoposide and least for AMCA. CONCLUSION: We conclude that the carbamate analogues of amsacrine recognize both topo IIalpha and beta in cells.

sox30: a novel zebrafish sox gene expressed in a restricted manner at the midbrain-hindbrain boundary during neurogenesis.

The Sox family of proteins is thought to act to regulate gene expression in a wide variety of developmental processes. Here we describe the cloning of sox30, a novel sox gene from the zebrafish (Danio rerio). In situ hybridization shows that sox30 is expressed in a restricted manner at the boundary between the midbrain and hindbrain during nervous system development. This expression pattern is in direct contrast to that of most other neuronally expressed Sox genes which are expressed throughout the nervous system.

Molecular cloning and characterization of the human topoisomerase IIalpha and IIbeta genes: evidence for isoform evolution through gene duplication.

Human DNA topoisomerase II is essential for chromosome segregation and is the target for several clinically important anticancer agents. It is expressed as genetically distinct alpha and beta isoforms encoded by the TOP2alpha and TOP2beta genes that map to chromosomes 17q21-22 and 3p24, respectively. The genes display different patterns of cell cycle- and tissue-specific expression, with the alpha isoform markedly upregulated in proliferating cells. In addition to the fundamental role of TOP2alpha and TOP2beta genes in cell growth and development, altered expression and rearrangement of both genes are implicated in anticancer drug resistance. Here, we report the complete structure of the human topoisomerase IIalpha gene, which consists of 35 exons spanning 27.5 kb. Sequence data for the exon-intron boundaries were determined and examined in the context of topoisomerase IIalpha protein structure comprising three functional domains associated with energy transduction, DNA breakage-reunion activity and nuclear localization. The organization of the 3' half of human TOP2beta, including sequence specifying the C-terminal nuclear localization domain, was also elucidated. Of the 15 introns identified in this 20 kb region of TOP2beta, the first nine and the last intron align in identical positions and display the same phases as introns in TOP2alpha. Though their extreme 3' ends differ, the striking conservation suggests the two genes diverged recently in evolutionary terms consistent with a gene duplication event. Access to TOP2alpha and TOP2beta gene structures should aid studies of mutations and gene rearrangements associated with anticancer drug resistance.

Human DNA topoisomerase IIbeta binds and cleaves four-way junction DNA in vitro.

We have used gel retardation analysis to show that human DNA topoisomerase IIbeta can bind a 40 bp linear duplex containing a single DNA topoisomerase IIbeta cleavage site. Furthermore, we demonstrate for the first time that human DNA topoisomerase IIbeta binds to four-way junction DNA. This supports previous suggestions that topoisomerase II may be targeted to supercoiled DNA through the recognition of DNA cruciforms, helix-helix crossovers and hairpins. DNA topoisomerase IIbeta had a 4-fold higher affinity for the four-way junction than for the linear duplex, as demonstrated by protein titration and competition analysis. Furthermore, the DNA topoisomerase IIbeta:four-way junction complex was significantly more salt stable than the complex with linear DNA. The four-way junction contained potential topoisomerase IIbeta cleavage sites straddling the points of strand exchange, and indeed, topoisomerase IIbeta was able to cleave three of these four predicted sites. This indicates that topoiso-merase IIbeta can bind to the centre of the junction. Topoisomerase II has to bind both the transported and the gated DNA helices prior to strand passage, and it is possible that both helices are provided by the four-way junction in this case. The stable complex of DNA topoisomerase IIbeta with four-way junction DNA may provide an ideal substrate for further studies into the mechanism of substrate recognition and binding by DNA topoisomerase II.

A new regimen for starting warfarin therapy in out-patients.

AIMS: Oral anticoagulation is increasingly used in elderly patients with atrial fibrillation to prevent embolic phenomena. The use of anticoagulants in this population is prophylactic rather than therapeutic and so there is no urgency to establish anticoagulation within the desired therapeutic range. The aim of the study was to develop an out-patient regimen for initiation of oral anticoagulation with warfarin which requires only weekly monitoring of the International Normalized Ratio (INR). METHODS: The study was undertaken in two phases. In the first phase, factors which predict the final maintenance dosage of warfarin were defined and used to build a decision tree and dosage algorithm. In the second study the algorithm was tested. Patients were given 2 mg warfarin daily for 2 weeks and the INR at this time was used to predict the maintenance dose. Patients then attended for weekly measurements of the INR until steady state had been reached. Dosage adjustments were not made unless the INR was >4.0 or <1.5 for 2 consecutive weeks. The accuracy of the prediction was measured by calculating the mean INR of weeks 6-8 and the number of patients in the target range 2.0-3.0 was determined. RESULTS: One hundred and seven consecutive out-patients (mean age 70 years range 64-86) completed the first study. The age, sex, height, weight, alcohol intake, number of cigarettes smoked, concomitant medication, clinical evidence of right heart failure, liver failure, abnormalities in liver enzyme estimations, baseline INR and INR after 2 weeks of 2 mg warfarin daily were used in a polytomous logistic regression analysis with stepwise inclusion of factors to determine which factors influenced the eventual maintenance dosage of warfarin. The INR after 2 weeks of 2 mg warfarin therapy predicted 70% of the variability of the maintenance dose. Of other factors only the sex of the patient had a large enough effect to be included in the prediction algorithm. One hundred and six patients (mean age 71 years range 50-85 years) completed the second study. Only one patient needed a dose adjustment in the first 2 weeks of warfarin 2 mg daily (INR 4.4). Overall, 60% patients were in the narrow target range (INR 2.0-3.0) at steady state. In five patients the INR was >4.0 at any visit after the second week and needed dosage adjustment. In four patients the INR was <1.5 at steady state. CONCLUSIONS: We have developed a method of predicting the maintenance dose of warfarin in an elderly population based on the INR after 2 weeks of warfarin 2 mg daily, and the sex of the patient. This is a safe and convenient way of initiating warfarin therapy as an out-patient which requires only weekly INR checks.

Nuclear distribution of human DNA topoisomerase IIbeta: a nuclear targeting signal resides in the 116-residue C-terminal tail.

We have analyzed the subcellular distribution of the beta isoform of human topoisomerase II using both isoform-specific antisera and an epitope-tagging approach. Previous immunocytochemical studies have yielded differing results with one reporting this isoform to be predominantly nucleolar. Later studies seem to refute this finding, as do our results with isoform-specific antisera reported here. Epitope tagging minimizes potential complications arising from the use of anti-topoisomerase II antisera that may recognize epitopes that are modified or masked in vivo and could lead to misleading results in immunocytochemical studies. A second strength of this approach is that it allowed a comparison with similarly tagged control proteins (derived from the nucleolar transcription factor UBF) that were known to localize unambiguously to the cytoplasmic, nucleoplasmic, or nucleolar compartments. We report that the C-terminal domain of topoisomerase IIbeta fused to a beta-galactosidase tag localizes to the nucleus (but not the nucleolar compartment) and that this is indistinguishable from the localization of native topoisomerase IIbeta detected by isoform-specific antisera. Further analysis revealed that the nuclear localization determinant lies within the 116-residue C-terminal tail of human topoisomerase IIbeta.