Kinetic effect of a downstream strand and its 5'-terminal moieties on single nucleotide gap-filling synthesis catalyzed by human DNA polymerase lambda

During short-patch base excision repair, the excision of a 5'-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5'-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase beta or lambda is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5'-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase lambda have revealed that the downstream strand and its 5'-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5'-moieties on the polymerase activity of the full-length human polymerase lambda. The downstream strand and its 5'-phosphate were both found to increase nucleotide incorporation efficiency (kp/Kd) by 15 and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant kp rather than the ground state nucleotide binding affinity Kd. With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5'-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase lambda, it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in kp rather than Kd. The implication of the downstream strand and its 5'-moieties on the kinetics of gap-filling synthesis is discussed.     

Chemical contents of Macaranga food bodies: adaptations to their role in ant attraction and nutrition

1. Macaranga (Euphorbiaceae) is a paleotropical tree genus comprising myrmecophytic and non-myrmecophytic species. All species are presumed to possess food bodies (FBs) to maintain or attract ants as anti-herbivore defence.
2. The hypothesis was tested that Macaranga species differing in their mode of association with ants would produce FBs differing in their chemical composition. We investigated contents of carbohydrates, proteins and lipids in FBs of four myrmecophytic and one non-myrmecophytic Macaranga as well as one Parthenocissus (Vitaceae) species.
3. On a dry weight basis, FBs of myrmecophytes contained relatively higher amounts of proteins compared to carbohydrates than those of non-myrmecophytes. Soluble carbohydrates showed species-specific patterns and were found in especially high amounts in both non-myrmecophytes. Furthermore, Parthenocissus FBs contained higher amounts of soluble compared to polymerous substances not only in carbohydrates but also in proteins.
4. FBs seem to be specifically adapted to their respective role in ant attraction and nutrition, with myrmecophytes providing ants with high amounts of lipids and proteins and non-myrmecophytes mainly offering carbohydrates in the form of common soluble sugars.     

Influence of betaine consumption on strenuous running and sprinting in a hot environment

This investigation evaluated the effects of a nutritional supplement (the organic osmolyte betaine) in rehydration solutions, with and without carbohydrate and electrolytes. Ten male runners ((mean +/- SD) age, 20 +/- 2 years; weight, 70.6 +/- 6.8 kg; maximal aerobic power, 63.5 +/- 4.1 mL O2 x kg(-1) x min(-1)) dehydrated to -2.7% of body weight. They next rehydrated to -1.4% of body weight by consuming 1 L fluid during each of four experiments (double-blind, randomized, cross-over design): flavored, non-caloric water (W); W + 5 g x L(-1) betaine (W+B); 6% carbohydrate-electrolyte fluid (C); or C + 5 g x L(-1) betaine (C+B). Subjects then performed prolonged treadmill running (75 minutes at 65%Vo2max) plus a performance sprint to volitional exhaustion (3.1-3.8 minutes at 84%Vo2max) in an environmental chamber (31.1 degrees C, 88.0 degrees F). Only W versus W+B and C versus C+B statistical comparisons were germane to the research questions. Observations indicated that rehydration with fluids containing betaine resulted in significant differences (p < 0.05) of plasma volume, oxygen consumption, plasma lactate concentration, and thermal sensation. The present experiments did not support the use of betaine to improve sprint duration, but nonsignificant trends occurred when betaine trials were compared with non-betaine trials (mean C+B > C by 32 seconds, +16%; mean W+B > W by 38 seconds, +21%). We interpret the increases of both aerobic and anaerobic metabolism (C+B > C) to mean that further investigation of betaine as a nutritional supplement, using other types of exercise, is warranted.     

Semi-Automated Reconstruction of Neural Processes from Large Numbers of Fluorescence Images

We introduce a method for large scale reconstruction of complex bundles of neural processes from fluorescent image stacks. We imaged yellow fluorescent protein labeled axons that innervated a whole muscle, as well as dendrites in cerebral cortex, in transgenic mice, at the diffraction limit with a confocal microscope. Each image stack was digitally re-sampled along an orientation such that the majority of axons appeared in cross-section. A region growing algorithm was implemented in the open-source Reconstruct software and applied to the semi-automatic tracing of individual axons in three dimensions. The progression of region growing is constrained by user-specified criteria based on pixel values and object sizes, and the user has full control over the segmentation process. A full montage of reconstructed axons was assembled from the ∼200 individually reconstructed stacks. Average reconstruction speed is ∼0.5 mm per hour. We found an error rate in the automatic tracing mode of ∼1 error per 250 um of axonal length. We demonstrated the capacity of the program by reconstructing the connectome of motor axons in a small mouse muscle.     

Redescription of Alinema amazonicum (Travassos, 1960) n. comb., a philometrid nematode with unusual morphology

Nematodes (1 male and numerous females) of the Philometridae were collected from the mesentery of 2 species of pimelodid catfishes, Calophysus macropterus and Perrunichthys perruno, from the Amazon River basin (fishmarket in Iquitos, Loreto District) in Peru. A detailed study of their morphology (including scanning electron microscopy) and a reexamination of the type and voucher specimens of Philometra amazonica Travassos, 1960, from Brazilian catfishes confirmed that they belong to this species and that Philometra (Alinema) alii Rasheed, 1963 is its junior synonym. Because of some marked morphological peculiarities of this species (presence of minute peribuccal sclerotized formations, a functional vagina and vulva in gravid female, and structure of the male tail), the validity of an independent genus, Alinema Rasheed, 1963, is confirmed, to which this species is transferred as Alinema amazonicum (Travassos, 1960) n. comb. This is the first record of this parasite from Peru, and P. perruno represents its new host record.     

Seasonal changes of pCO2 over a subantarctic Macrocystis kelp bed

The partial pressure of carbon dioxide (pCO₂), calculated from pH and total alkalinity measurements, was monitored together with chlorophyll a and bacterioplankton biomass in shallow coastal water located inside and outside a giant kelp bed (Macrocystis pyrifera) situated in the Kerguelen Archipelago, Southern Ocean. In spite of large changes over a short time-scale, pCO₂ variations over the year are large and exhibit a seasonal pattern in which the different stages of the annual biological turnover are well marked. The overall pattern of pCO₂ variations is related to biological activity (development of both photosynthesis and respiration) during almost the whole year. However, physical and thermodynamical constraints exert a strong influence on pCO₂ at meso time-scale (10 days) and/or when biological activity is weak. Macrocystis acts to maintain pCO₂ below saturation almost the whole year and large undersaturations (pCO₂ as low as 20 μatm) were observed within the kelp bed. Furthermore, primary production of Macrocystis covers a period of 8 ∼ 9 months a year from winter to late summer and the kelp bed seems to favour the spring phytoplanktonic bloom. The buffer factor β indicates that, outside the kelp bed, inorganic carbon dynamics are mainly influenced by air-sea exchange and photosynthesis without calcification. Inside the kelp bed, β suggests calcification by the epiphytic community. Accepted: 1 April 2000     

FACTORS DETERMINING THE ACCURACY OF CLADOGRAM ESTIMATION: EVALUATION USING COMPUTER SIMULATION

We developed a simulation model of phylogenesis with which we generated a large number of phylogenies and associated data matrices. We examined the characteristics of these and evaluated the success of three taxonomic methods (Wagner parsimony, character compatibility, and UPGMA clustering) as estimators of phylogeny, paying particular attention to the consequences of changes in certain evolutionary assumptions: relative rate of evolution in three different evolutionary contexts (phyletic, parent lineage, and daughter lineage); relative rate of evolution in different directions (novel forward, convergent forward, or reverse); variation of evolutionary rates; and topology of the phylogenetic tree. Except for variation of evolutionary rates, all the evolutionary parameters that we controlled had significant effects on accuracy of phylogenetic reconstructions. Unexpectedly, the topology of the phylogeny was the most important single factor affecting accuracy; some phylogenies are more readily estimated than others for simply historical reasons. We conclude that none of the three estimation methods is very accurate, that the differences in accuracy among them are rather small, and that historical effects (the branching pattern of a phylogeny) may outweigh biological effects in determining the accuracy with which a phylogeny can be reconstructed.     

Multivariate Analysis of Correspondence between Left Atrial Volumes Assessed by Echocardiography and 3-Dimensional Electroanatomic Mapping in Patients with Atrial Fibrillation

Background

Left atrial (LA) enlargement is a predictor of worse outcome after catheter ablation for atrial fibrillation (AF). Widely used two-dimensional (2D)-echocardiography is inaccurate and underestimates real LA volume (LAV). We hypothesized that baseline clinical characteristics of patients can be used to adjust 2D-ECHO indices of LAV in order to minimize this disagreement.

Methods

The study enrolled 535 patients (59 ± 9 years; 67% males; 43% paroxysmal AF) who underwent catheter ablation for AF in three specialized centers. We investigated multivariately the relationship between 2D-echocardiographic indices of LA size, specifically LA diameter in M-mode in the parasternal long-axis view (LAD), LAV assessed by the prolate-ellipsoid method (LAV_Ellipsoid), LAV by the planimetric method (LAV_Planimetry), and LAV derived from 3D-electroanatomic mapping (LAV_CARTO).

Results

Cubed LAD of 106 ± 45 ml, LAV_Ellipsoid of 72 ± 24 ml and LAV_Planimetry of 88 ± 30 ml correlated only modestly (r = 0.60, 0.69, and 0.53, respectively) with LAV_CARTO of 137 ± 46 ml, which was significantly underestimated with a bias (±1.96 standard deviation) of -31 (-111; +49) ml, -64 (-132; +2) ml, and -49 (-125; +27) ml, respectively; p < 0.0001 for their mutual difference. LA enlargement itself, age, gender, type of AF, and the presence of structural heart disease were independent confounders of measurement error of 2D-echocardiographic LAV.

Conclusion

Accuracy and precision of all 2D-echocardiographic LAV indices are poor. Their agreement with true LAV can be significantly improved by multivariate adjustment to clinical characteristics of patients.     

THE ACTION OF CORTICOTROPIN (ACTH) ON NUCLEIC ACIDS AND SUBCELLULAR ELEMENTS OF THE ADRENAL CORTEX

It was shown experimentally that ACTH causes an increase of cytoplasmic RNA and subcellular granules of the adrenal cortex shortly after administration. This effect was found to be concentrated primarily on the basophilic cytoplasmic granules and to be largely independent of cell division. The increase of nuclear RNA, DNA, and nuclear mass followed that of the cytoplasm. The observations indicate also that the mass increase of mitochondrial and chromidial systems is closely related to the increase in cytoplasmic, largely chromidial RNA.