Cortical fMRI activation to opponents' body kinematics in sport-related anticipation: expert-novice differences with normal and point-light video.

Badminton players of varying skill levels viewed normal and point-light video clips of opponents striking the shuttle towards the viewer; their task was to predict in which quadrant of the court the shuttle would land. In a whole-brain fMRI analysis we identified bilateral cortical networks sensitive to the anticipation task relative to control stimuli. This network is more extensive and localised than previously reported. Voxel clusters responding more strongly in experts than novices were associated with all task-sensitive areas, whereas voxels responding more strongly in novices were found outside these areas. Task-sensitive areas for normal and point-light video were very similar, whereas early visual areas responded differentially, indicating the primacy of kinematic information for sport-related anticipation.

Using a mammalian cell cycle simulation to interpret differential kinase inhibition in anti-tumour pharmaceutical development.

Systems biology needs to show practical relevance to commercial biological challenges such as those of pharmaceutical development. The aim of this work is to design and validate some applications in anti-cancer therapeutic development. The test system was a group of novel cyclin-dependent kinase (CDK) inhibitors synthesised by Cyclacel Ltd. The measured in vitro IC50s of each compound were used as input data to a proprietary cell cycle model developed by Physiomics plc. The model was able to predict over three orders of magnitude the cytotoxicity of each compound without model adaptation to specific cancer cell types. This pattern matched the experimentally determined data. One class of compounds was predicted to cause an increase of the cell cycle length with a non-linear dose-response curve. Further work will use apoptosis and DNA replication simulations to look at overall cell effects.

The effect of learning condition on perceptual anticipation, awareness, and visual search.

The efficacy of explicit and implicit learning paradigms was examined during the very early stages of learning the perceptual-motor anticipation task of predicting ball direction from temporally occluded footage of soccer penalty kicks. In addition, the effect of instructional condition on point-of-gaze during learning was examined. A significant improvement in horizontal prediction accuracy was observed in the explicit learning group; however, similar improvement was evident in a placebo group who watched footage of soccer matches. Only the explicit learning intervention resulted in changes in eye movement behaviour and increased awareness of relevant postural cues. Results are discussed in terms of methodological and practical issues regarding the employment of implicit perceptual training interventions.

Implicit perceptual training: how, when, and why?

The perceptual skills underlying anticipatory movement in sport have been the focus of much research over the past 20 years. Methods for training such skills have tended to emphasise explicit specification of discriminative cues and the rules linking changes in the perceptual field with required responses. Recently, researchers have begun to examine less prescriptive methods of training. In the present paper, we examine conceptual, methodological, and practical issues associated with whether such skills can or indeed should be trained implicitly. The implications of two ways of conceptualising the explicit-implicit distinction for the methods used to promote implicit learning and the tests used to assess the nature of learning are considered. Finally, potential advantages of implicitly learned skills relating to task complexity and robustness under stress are discussed.

Chromosome-specific desynapsis in the n = 2 race of Haplopappus gracilis (Compositae).

During cytological screening for pollen sterility in a wild population of Haplopappus gracilis (n = 2), several partially sterile plants were found that had good pachytene pairing but varying numbers of univalents. Some plants had chromosome A bivalents or A univalents, while in the same cells chromosome B had only bivalents. In other plants the reverse condition occurred; the B chromosome had B bivalents or B univalents and only A bivalents. This demonstrates a chromosome-specific effect for the desynapsis genes. Hybridization between the two homozygous mutant genotypes produced only normal bivalents; this indicates the two mutants are not alleles and each is recessive. An F2 generation showed independent assortment of the desynaptic mutations. The chromosome A bivalent is the larger of the two and normally has one or two chiasmata; the B bivalent normally has a single chiasma. Chiasmata distribution was tested in the desynaptic mutant A bivalents and showed an acceptable fit to a binomial distribution. This occurs also in heterozygous, asynaptic pairing control gene mutations. Analysis of the NOR bivalent in two hologenomic desynaptic mutations in tomato also showed a good fit to a binomial distribution of chiasmata. This indicates the same methods are applicable to diverse species.

Subchronic toxicity of fish oil concentrates in male and female rats.

There are an overwhelming number of reports indicating the beneficial effects of fish oil supplements in human and animal nutrition. The purpose of this study, second in a series, was to evaluate the effects, particularly those that may be harmful, of high-dose, long-term consumption of fish oil concentrates (FOC) using male and female rats. One hundred and twenty male and 120 female rats were gavaged daily with oils and oil mixtures in a volume equal to 0.5% body weight (5 mL/kg/d) for 13 weeks. The administered oils were corn oil, pure menhaden oil (MO), pure MaxEPA fish oil or different mixtures of corn oil with MO. The stability and the homogeneity of the dosing solutions were tested under study conditions. The animals received isocaloric and isonitrogenous diets throughout. Food and pure water were supplied ad libitum. At the end of the in-life phase of the study, the animals were anaesthetized with CO2 and humanely killed by exsanguination. Blood and other tissues were prepared for various clinical, histopathological and laboratory tests. Some beneficial effects of FOC, such as reduction in total serum cholesterol, in rats were confirmed. However, we also observed a significant reduction in absolute amount of serum HDL and a significant increase in relative liver and spleen weights in both sexes with the high dose of FOC. High doses of FOC (5 mL/kg/d) reduced serum iron and vitamin E concentrations. A reduction in osmotic fragility of RBC as well as an increase in RBC deformity were also observed in rats treated with high doses of FOC. These rats showed a significant overall increase in WBC count. We conclude that in rats, subchronic consumption of high levels of FOC can be beneficial but may also be harmful because of induction of clinical abnormalities including increased red cell deformity, increased relative liver and spleen weights, and reduced serum HDL, iron and vitamin E concentrations.

A unique pollen wall mutation in the family Compositae: ultrastructure and genetics.

During a routine screening of pollen fertility in the n = 2 chromosome race of Haplopappus gracilis, a spineless pollen wall mutation was discovered that renders the otherwise functional pollen grains completely unrecognizable as Compositae pollen. Normal Haplopappus pollen is characterized by an outer layer, the ektexine, consisting of large spines supported by a roof (tectum), which in turn is supported by collumellae that are joined basally. A large cavity (cavea) stretches from aperture to aperture and separates columellae bases from the final ektexine unit, the foot layer. The spines, tectum, columellae, and columellae bases are filled with perforations (internal foramina), while the foot layer is without them. Immediately underlying the foot layer is a thickened, lamellate, disrupted, internal foramina-free second exine layer, the endexine. In contrast, the mutant pollen ektexine is a jumble of components with randomly dispersed spines as the only clearly definable unit. The endexine layer is similar to the endexine in normal pollen. The mutation apparently disrupts only the organization of ektexine units, and mutant pollen appears to be without the caveae and foot layer characteristic of normal pollen. In genetic tests, the mutant allele is recessive. There is a simple Mendelian pattern of inheritance of the mutant gene, and its phenotype is under sporophytic control.

Super in vitro synergy between inhibitors of dihydrofolate reductase and inhibitors of other folate-requiring enzymes: the critical role of polyglutamylation.

The combined action among polyglutamylatable and nonpolyglutamylatable antifolates, directed against dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), and thymidylate synthase (TS), in human ileocecal HCT-8 cells was examined in a 96-well plate growth inhibition assay (96-h continuous drug exposure). An interaction parameter, alpha, was estimated for each of 95 experiments by fitting a seven-parameter model to data with weighted nonlinear regression. In a representative experiment, raising the folic acid concentration in the medium dramatically increased the Loewe synergy for the combination of trimetrexate (TMTX) and the GARFT inhibitor AG2034 (from a mean alpha +/- SE of 1.50 +/- 0.25 at 2.3 microM folic acid to 146 +/- 20 at 78 microM folic acid). Enhancements were also found for combinations of TMTX with the GARFT inhibitors AG2032, Lometrexol, and LY309887, the AICARFT inhibitor AG2009, and the TS inhibitors LY231514 and Tomudex but not with the GARFT inhibitor LL95509 or with the TS inhibitors AG337, ZD9331, and BW1843U89. Replacing TMTX with methotrexate in two-drug mixtures decreased the intensity of Loewe synergy. Examination of isobolograms at different effect levels revealed informative reproducible changes in isobol patterns. No two-drug combinations among inhibitors of GARFT, AICARFT, and TS exhibited Loewe synergy at either 2.3 or 78 microM folic acid. Thus, the ideal requirement for the folic acid-enhanced synergy is that a nonpolyglutamylatable DHFR inhibitor be combined with a polyglutamylatable inhibitor of another folate-requiring enzyme. A hypothesis to explain this general phenomenon involves the critical role of folylpoly-gamma-glutamate synthetase and the effect of the DHFR inhibitor in decreasing the protection by folic acid of cells to the other antifolates.

An inhibitor of glycinamide ribonucleotide formyltransferase is selectively cytotoxic to cells that lack a functional G1 checkpoint.

PURPOSE: We studied the effects of purine depletion on the cell cycle using a specific inhibitor of de novo purine biosynthesis, AG2034, an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT). METHODS: Cytotoxicity was determined by clonogenic assays, and cell cycle perturbations by flow cytometry. Ribonucleotide pools were measured by anion exchange high-pressure liquid chromatography, and DNA strand-breaks were determined by alkaline elution and by the TUNEL assay. RESULTS: When cells were maintained in standard tissue culture medium, which contained 2.2 microM folic acid, AG2034 was cytostatic in all the cell lines tested. Under low-folate conditions (50 nM folic acid), AG2034 caused up to 50% cell death in cell lines that possessed a functional G1 checkpoint (A549, MCF-7), but was only cytostatic to the remaining cells, even at very high concentrations (100 microM). In contrast, AG2034 at 10 nM or 100 nM killed all the cells in cultures of HeLa/S3 or SW480 cells, which lack a functional G1 checkpoint. Flow cytometry studies indicated that in G1 checkpoint-competent cells, AG2034 caused a G1 arrest. Those cells (up to 50%) that were already in S phase died, but the cells that were in G1 arrest maintained viability, based upon clonogenic assays, for many days. In G1 checkpoint-deficient cells, no G1 arrest was seen after AG2034 treatment, all cells progressed into S phase, and all cells died. Measurement of DNA strand-breaks, either by alkaline elution or by the dUTP end-labelling technique, indicated no DNA strand-breaks 24 h after AG2034 treatment, indicating that purine nucleotide depletion can trigger the G1 checkpoint in the absence of DNA damage. CONCLUSION: Purine depletion causes slow cell death in cells that have passed the G1 checkpoint, but cytostasis in cells that are arrested at the G1 checkpoint. The GARFT inhibitor, at physiological folate concentrations, thus causes selective cytotoxicity to cells lacking a functional G1 checkpoint.

Contributions of protein structure-based drug design to cancer chemotherapy.

Protein structure-based drug design (SBDD) uses a knowledge of the molecular structure of a target macromolecule, normally obtained by x-ray crystallography, to design potent, selective inhibitors. This technique has played a major role in the design of a number of drugs that have progressed to clinical trials. Most of these compounds have been developed to treat viral diseases and cancer. In the antiviral area, drugs designed by SBDD have been developed for treatment of influenza and acquired immune deficiency syndrome. Three human immunodeficiency virus-I protease inhibitors that were designed at least partially using the x-ray crystal structure of the target enzyme are now approved for sale in the United States. In the anticancer field, no SBDD-designed drugs have yet progressed to market, but several experimental anticancer agents that were designed from a knowledge of the molecular structure of their target enzyme have advanced to clinical trials, of which at least one has shown clinical activity. The present article discusses the strengths and weaknesses of the SBDD approach, and shows its contributions to cancer chemotherapy by discussing rationally designed inhibitors of thymidylate synthase, purine nucleoside phosphorylase, glycinamide ribonucleotide formyltransferase, and matrix metalloproteases.

A pharmacokinetic-pharmacodynamic model of chemotherapy of human immunodeficiency virus infection that relates development of drug resistance to treatment intensity.

RNA viruses, including retroviruses, have mutation rates that are about 100 times higher than those of DNA viruses, bacteria, or eukaryotes, so that resistance to AIDS drugs emerges very rapidly. This has been shown to limit the effectiveness of the treatment of AIDS by reverse transcriptase inhibitors, such as zidovudine (AZT) and resistance to the new class of HIV aspartyl protease inhibitors has already been reported. The technique of pharmacokinetic-pharmacodynamic simulation has now been used to predict ways of delaying the development of resistance to these two classes of antiretroviral agents. A model is described that includes pharmacokinetic, pharmacodynamic, and cytokinetic equations, and expressions describing effects if the HIV on the immune system and destruction of virally infected cells by cellular immunity. The model predicted that the degree of viral drug resistance in relation to the sustainable blood level of drug would be the major determinant of response duration. Early treatment was consistently superior to late treatment, both with a drug that caused cumulative toxicity and with a drug that did not. Making reasonable assumptions about the likely degree of viral resistance, in conjunction with typical blood levels achievable for reverse transcriptase inhibitors or aspartyl protease inhibitors led to predicted response durations of several months to a few years, despite the rapid mutation rate of HIV. Preliminary studies of combination chemotherapy showed that predicted response durations were greater than for monotherapy, though less than the sum of responses to the individual drugs. Strategies for delaying the development of resistance include early treatment, combination chemotherapy, and developing novel agents with a high ratio of plasma level to antiviral efficacy.

Update on computer-aided drug design.

Recent advances in computational methods for drug design include developments in quantitative structure-activity relationship approaches as well as novel structure-based strategies. Many new protein structures of pharmaceutical interest have been solved, a number of which contain a bound inhibitor. Continued progress has been reported in algorithms for de novo design, ligand docking, and scoring of protein-ligand binding energy. Meanwhile, several drugs that were designed by intensive use of computational methods are advancing through clinical trials.

Clinical pharmacokinetic and pharmacodynamic studies with the nonclassical antifolate thymidylate synthase inhibitor 3, 4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolone dihydrochloride (AG337) given by 24-hour continuous intravenous infusion.

3,4-Dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolon e dihydrochloride (AG337) is a nonclassical inhibitor of thymidylate synthase (TS) designed to avoid potential resistance mechanisms that can limit the activity of classical antifolate antimetabolites. A clinical pharmacokinetic and pharmacodynamic study of AG337 given as a 24-h i.v. infusion was performed. Thirteen patients received 27 courses over the dose range 75-1350 mg/m2. Plasma AG337 concentrations were achieved which, in preclinical models, were associated with antitumor effects. AG337 clearance was saturable, and the pharmacokinetics of the drug at doses above 300 mg/m2 was best described by a one-compartment model with saturable elimination (median Km = 6.5 microgram/ml; range, 4.1-13 microgram/ml; median Vmax = 2.0 microgram/ml/h/m2; range, 0.96-5.6 microgram/ml/h/m2). Following the end of the infusion, AG337 was cleared rapidly (t1/2, 53-193 min), and levels were less than 0.2 microgram/ml in all patients by 48 h. Plasma protein binding was 96-98%, and the urinary excretion of AG337 as unchanged drug did not exceed 30% of the dose administered. Measurements of plasma deoxyuridine (dUrd) concentrations showed that doses of 600 mg/m2 and above of AG337 produced a consistent elevation in plasma dUrd levels (60-290%), suggesting that TS inhibition was being achieved in patients. However, in all cases dUrd concentrations had returned to pretreatment levels 24 h after the end of the infusion, suggesting that TS inhibition was not maintained. Local toxicity, probably due to the infusate pH, was the only significant adverse effect observed. These studies have shown that cytotoxic AG337 plasma concentrations can be readily achieved without acute toxicity and that these concentrations are associated with elevations in plasma dUrd levels. The lack of prolonged dUrd elevations indicates that extended administration should be explored using central line or p.o. administration to avoid local toxicity.

Toxicity prediction from metabolic pathway modelling.

The technique of kinetic simulation, in which a metabolic pathway is modelled by a system of differential equations, has been used to study several questions concerning the teratogenic effects of antifolate drugs. These studies made use of BioCHIMICA, a new biochemical modelling software package. This program is a general-purpose system for simulation of reaction networks. It can simulate systems of over 100 reactions, and can make use of a wide variety of rate equations. Several applications of this program in predicting the teratogenicity of antifolates are described: (a) prediction of relative toxic potency of related analogues, (b) prediction of shapes of dose-response curves in an attempt to determine whether safe exposure levels may exist, (c) prediction of reversibility, i.e. can we distinguish between reversible damage and effects likely to be irreversible, (d) prediction of combined inhibitor effects, i.e. can we identify which teratogenic effects are likely to be synergistic and which antagonistic? These questions were asked in an attempt to predict the toxic effects of a group of experimental inhibitors of thymidylate synthase and dihydrofolate reductase.

[202 toe transfers to the hand]. / 202 Zehentransplantate an die Hand.

This retrospective study analyzed 202 toe-to-hand transplants performed over the last 20 years at the Davies Medical Center, San Francisco (USA). The overall success rate was 97%. Toe transplants for finger reconstruction yielded optimal functional and cosmetic results due to their anatomical similarity to fingers. The great toe was preferably used for thumb reconstruction, whereas the other toes were used for reconstruction of the long fingers. Early reconstructions, multiple simultaneous toe transplants, and interventions combining toe transplantation with free flaps seemed to be advantageous because of shorter rehabilitation and comparable results.

Biological activity of a novel rationally designed lipophilic thymidylate synthase inhibitor.

AG-331 (N6[4-(N-morpholinosulfonyl)benzyl]-N6-methyl-2,6-diamino- benz[cd]indole glucuronate) is a novel lipophilic thymidylate synthase (TS) inhibitor. The properties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mechanisms. There was no evidence for any intracellular effect of AG-331 on dihydrofolate reductase (DHFR); however, the low degree of cross-resistance found for the H35FF line, which has elevated TS levels, suggested that TS may not be the sole locus of action of AG-331 in hepatoma cells. TS-directed effects of AG-331 were suggested by the pattern of its inhibition of deoxyuridine incorporation into DNA and the lesser effects of purine incorporation. In addition, H35 cells treated with 10 microM AG-331 were shown to accumulate in the S phase of the cell cycle, and this effect could be reversed by coadministration of thymidine. However, when treatments were conducted at a 5-fold higher concentration of AG-331, no S-phase block was apparent, suggesting the loss of a TS-directed effect at high inhibitor concentrations. Thymidine and folinic acid also failed to protect cells against AG-331 cytotoxicity, suggesting an alternate mode of action. Similar results were also obtained in protection experiments with a human hepatoma cell line, HEPG2, although previous results obtained in colon- and breast-cancer cell lines have suggested TS specific effects for AG-331. The possibility that biotransformation of AG-331 to other toxic species may occur in liver-derived cell lines has yet to be investigated.

Amphibolic drug combinations: the design of selective antimetabolite protocols based upon the kinetic properties of multienzyme systems.

Computer simulation of a simple biochemical pathway containing a divergent branch has been used to study the interaction of two inhibitors that straddle the branch point. The combined effects of the two sequentially acting inhibitors could be synergistic, additive, or antagonistic, depending upon the regulation of the pathway. Factors that influenced the interaction included relative Vmax and Km values for the competing enzymes, the sink capacity of the system (i.e., the capacity of the system for eliminating the shared intermediate in relation to its capacity for producing it), the competitive or noncompetitive nature of the second inhibitor, and the presence or absence of feedback in the system. In certain cases, a two-fold change in the Vmax value of one of the competing enzymes was sufficient to change the interaction from synergistic to antagonistic. The existence of such drug combinations (which this article terms amphibolic combinations) means that it is possible in principle to identify binary drug combinations that will be synergistic against a tumor cell but antagonistic against normal cells. Identification of amphibolic drug combinations should be a means of designing more selective chemotherapy.

Inhibition of 5-phosphoribosyl-1-pyrophosphate synthetase by the monophosphate metabolite of 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine: a novel mechanism for antitumor activity.

The aminopyrimidopyrimidine nucleoside 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (APP), which was previously shown to possess experimental antitumor and antiviral activity, was metabolized within WI-L2 human lymphoblastoid cells to a derivative identified as the beta-D-ribonucleotide (APP-MP). In a subline of WI-L2 cells deficient in adenosine kinase, this metabolite was not formed and APP was not cytotoxic, suggesting that APP is converted by adenosine kinase to its 5'-monophosphate. Because no evidence of di- or triphosphates was seen, the monophosphate appeared to be the active species. Treatment of WI-L2 or L1210 cells with APP (10 microM) for 30 min caused extensive depletion of both purine and pyrimidine ribonucleotides. Purine and pyrimidine deoxyribonucleotides were also depleted. Cells were not protected from the cytotoxicity of APP by hypoxanthine plus uridine, but uridine plus adenosine plus 2-deoxycoformycin gave considerable protection. This result was consistent with APP-MP acting as an inhibitor of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase, a hypothesis that was confirmed by preparing PRPP synthetase from Novikoff hepatoma cells; APP-MP was a noncompetitive inhibitor, with a Ki of 0.43 mM. APP-MP was found to accumulate in APP-treated cells to a concentration of almost 3 mM. The relevance of PRPP synthetase inhibition to the cytotoxic mechanism of APP is indicated by the fact that depletion of the PRPP pool was seen as early as 15 min after treatment, before any change was apparent in cellular levels of ATP or UTP. DNA synthesis was markedly suppressed within 30 min of APP treatment of WI-L2 cells, and a lesser degree of inhibition of RNA synthesis was apparent after 45 min.