Murine DNA polymerase alpha fills gaps to completion in a direct assay. Altered kinetics of de novo DNA synthesis at single nucleotide gaps

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.     

Banking brains: a pre-mortem “how to” guide to successful donation

A review of the brain banking literature reveals a primary focus either on the factors that influence the decision to become a future donor or on the brain tissue processing that takes place after the individual has died (i.e., the front-end or back-end processes). What has not been sufficiently detailed, however, is the complex and involved process that takes place after this decision to become a future donor is made yet before post-mortem processing occurs (i.e., the large middle-ground). This generally represents a period of many years during which the brain bank is actively engaged with donors to ensure that valuable clinical information is prospectively collected and that their donation is eventually completed. For the past 15 years, the Essential Tremor Centralized Brain Repository has been actively involved in brain banking, and our experience has provided us valuable insights that may be useful for researchers interested in establishing their own brain banking efforts. In this piece, we fill a gap in the literature by detailing the processes of enrolling participants, creating individualized brain donation plans, collecting clinical information and regularly following-up with donors to update that information, and efficiently coordinating the brain harvest when death finally arrives.     

Synthesis,molecular modelling studies and biological evaluation of new oxoeicosanoid receptor 1 agonists

The oxoeicosanoid receptor 1 (OXER1) is a member of the G-protein coupled receptors (GPCR) family, and is involved in inflammatory processes and oncogenesis. As such it is an attractive target for pharmacological intervention. The present study aimed to shed light on the molecular fundaments of OXER1 modulation using chemical probes structurally related to the natural agonist 5-oxo-ETE. In a first step, 5-oxo-ETE and its closely related derivatives (5-oxo-EPE and 4-oxo-DHA) were obtained by conducting concise and high-yielding syntheses. The biological activity of obtained compounds was assessed in terms of potency (EC₅₀) and efficacy (E_max) for arrestin recruitment. Finally, molecular modelling and simulation were used to explore binding characteristics of 5-oxo-ETE and derivatives with the aim to rationalize biological activity. Our data suggest that the tested 5-oxo-ETE derivatives (i) insert quickly into the membrane, (ii) access the receptor via transmembrane helices (TMs) 5 and 6 from the membrane side and (iii) drive potency and efficacy by differential interaction with TM5 and 7. Most importantly, we found that the methyl ester of 5-oxo-ETE (1a) showed even a higher maximum response than the natural agonist (1). In contrast, shifting the 5-oxo group into position 4 results in inactive compounds (4-oxo DHA compounds (3) and (3a)). All in all, our study provides relevant structural data that help understanding better OXER1 functionality and its modulation. The structural information presented herein will be useful for designing new lead compounds with desired signalling profiles.     

FURTHER SEM STUDY OF MARINE DINOFLAGELLATES: THE GENUS OSTREOPSIS (DINOPHYCEAE)1

This paper presents a comprehensive examination of the taxonomy of the genus Ostreopsis Schmidt. The morphology of six species of marine dinoflagellates, Ostreopsis siamensis Schmidt 1902. Ostreopsis lenticularis Fukuyo 1981, Ostreopsis ovata Fukuyo 1981, Ostreopsis heptagona Norris, Bomber, et Balech 1985, Ostreopsis mascarenensis Quod 1994, and Ostreopsis labens Faust et Morton 1995 from three geographical regions (Japan, Southwest Indian Ocean, and the Caribbean) and three marine habitats (sand, water column, and macroalgal surfaces) are described from scanning electron micrographs. Differences in the following morphological characteristics differentiated the species: cell shape and size, and ornamentation of the epitheca, cingulum, and hypotheca. The thecal plate formula of the six Ostreopsis species is P_o, 3′, 7″, 6C, 6S?, V_p, R_p, 5′″, 1p, 2″″, with differences in thecal plate size and shape. The cingulum in ventral view has two prominent structures: a ventral plate (V_p) with a ventral pore (V_o) and a ridged plate (R_p) that distinguishes Ostreopsis species from any other dinoflagellate taxa. This paper also includes ecological and toxicity information regarding the six Ostreopsis species.     

OBSERVATION OF SAND-DWELLING TOXIC DINOFLAGELLATES (DINOPHYCEAE) FROM WIDELY DIFFERING SITES, INCLUDING TWO NEW SPECIES

Dinoflagellate associations, including toxic and potentially toxic benthic species, were examined in sand from South Water Cay and Carrie Bow Cay, Belize. The inshore sand habitat in localized areas of warm shallow lagoonal waters supported blooms of toxic assemblages of dinoflagellates. In the sand, the dominant microalgae were dinoflagellates; cyanobacteria were a minor component and diatoms were absent. Ciliates and nematodes were present. Assemblages of microorganisms in colored sand were examined for 4 consecutive days after which a storm washed away the patch. The sand-dwelling dinoflagellate assemblage included 16 species where densities ranged from as low as 1.3% to 15% of total cell densities. The dominant species was Scrippsiella subsalsa, having 1.8 × 10⁵ to 2.6 × 10⁵ cells g^-1 sand. Toxic dinoflagellates identified in the sand were Gambierdiscus toxicus, Ostreopsis lenticularis, Prorocentrum lima, Prorocentrum mexicanum, and Amphidinium carteri. The potentially toxic Ostreopsis labens, Gambierdiscus belizeanussp. nov., and Coolia tropicalis sp. nov. were also identified. Toxic and potentially toxic species represented 36% to 60% of total microalgal cell assemblage. The morphology of a new sand-dwelling species, Gambierdiscus belizeanus sp. nov., was examined with the scanning electron microscope. The plate formula was P_o, 3′, 7″, 6c, s?, 5?, 1p, and 2″″.Dimensions of G. belizeanus cells were 53–67 pm long, 54–63 μm wide, and 92–98 μm in dorsoventral depth. Cells were deeply areolated, ellipsoid in apical view, and compressed anteroposteriorly. The cells of G. belizeanus were identified by the cell's long, narrow, pentagonal, posterior intercalary plate (1p) wedged between the wide postcingular plates 2″’and 4″; 1p occupied 20% of the width of the hypotheca. The plate formula for Coolia tropicalis sp. nov. was P_o, 3′, 7″, 7c, 8s?, 5″″, and 2″″, Cell size ranges were 23–40 μm long, 25–39 μm wide, and 35–65 μm in dorsoventral diameter. Cells were spherical, smooth, and covered with scattered round pores. The epitheca was smaller than the hypotheca. Precingular plates 1″ and 7″ were small and narrow, and the first apical plate 1″ and precingular plate 6″ were the largest plates on the epitheca. The apical pore was straight and 7 μm long, and was situated in the apical plate complex. Cells of C. tropicalis were distinguished from C. monotis by the wedge-shaped plate 1′, a four-sided 3’plate, and a short apical pore.     

MORPHOLOGY AND ECOLOGY OF THE MARINE DINOFLAGELLATE OSTREOPSIS LABENS SP. NOV. (DINOPHYCEAE)1

A new species, Ostreopsis labens Faust et Morton sp. nov., is described from three marine habitats: lagoonal water and lagoonal sand from the barrier reef of Belize, and associated with macroalgae from coral reef habitats of Oshigaki and Iriomote Islands, Japan. Dimensions of Ostreopsis labens cells are 60–86 μm long, 70–80 μm wide, and 81–110 μm in dorsoventral depth. Cells are broadly ovoid, anterioposteriorly compressed bearing a spherical nucleus and many chloroplasts. The epitheca is convex and composed of three apical plates, seven precingular plates, and an apical pore plate. The cingulum is composed of six plates. The hypotheca is constructed of five postcingular plates, one posterior intercalary, and two antapical plates. The sulcus is small, recessed, and hidden and exhibits a ventral pore and a ridged, curved plate. The thecal arrangement of O. labens is P_o, 3′, 7″ 6C, 6S(?), V_p, R_p, 5″, 1p, 2″. Only one sulcal list is present. The thecal plates have a smooth surface with distinct round pores. The intercalary band between the thecal plates is smooth. A row of marginal pores line the lipped cingulum. Ostreopsis species are anteroposteriorly flattened, photosynthetic, benthic dinoflagellates that are more diverse in ecology than previously known. Ostreopsis labens is capable of living in three marine habitats: in the water column, in sand, and on macroalgal surfaces. It was most numerous in sand and less in lagoonal waters, and only a few cells were associated with macroalgae. Light and scanning electron microscopy studies revealed engulfed cells within O. labens, which indicates mixotrophic/phagotrophic behavior. A ventral opening situated in the cingulum of O. labens exhibits size variability; it may serve as an opening for engulfiing food particles because it varies in size. We propose that ingestion of prey by O. labens occurs through the ventral opening, the proposed feeding apparatus of this species, which is similar to the function of the peduncle-like structure of mixotrophic dinoflagellates. The behavior of O. labens appears similar to that previously described for Dinophysis species.     

Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewisx bound to E-selectin

The conformation of the sialyl Lewis ^x tetrasaccharidebound to E-selectin was previously determined from transfer NOE (trNOE)experiments in conjunction with a distance-geometry analysis. However, theorientation of the tetrasaccharide ligand in the binding site of E-selectinis still unknown. It can be predicted that the accurate quantitativeanalysis of all trNOEs, including those originating from spin diffusion, isone key to analyze the orientation of sialyl Lewis^x in thebinding pocket of E-selectin. Therefore, we applied homonuclear 3D NMRexperiments and 1D analogs to obtain trNOEs that could not unambiguously beassigned from previous 2D trNOESY spectra, due to severe resonance-signaloverlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a1D trNOESY-TOCSY experiment of the sialyl Lewis^x/E-selectincomplex furnished new interglycosidic trNOEs and provided additionalinformation for the interpretation of trNOEs that have been describedbefore. A 2D trROESY spectrum of the sialyl Lewis^x/E-selectincomplex allowed one to identify the amount of spin-diffusion contributionsto trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysisof spin-diffusion pathways, was obtained, creating a basis for aquantitative analysis of trNOEs in the sialylLewis^x/E-selectin complex.     

Interspecific relationship of ten European orchid species as revealed by enzyme electrophoresis

Ten species of orchid plants belonging to the generaOrchis (7),Dactylorhiza (2), andGymnadenia (1) were analyzed by enzyme electrophoresis. Each species can be identified by a combination of enzyme bands different from those of all other species examined. The electrophoretic data were used for the construction of phenetic and phylogenetic trees with the help of computer programs. The trees were almost identical regardless which method was used. Our results differ considerably from a classification based on morphological evidence. The electrophoretic data indicate that the genusOrchis is not a monophyletic group.     

Calcium transport in apple trees

⁴⁵Ca, applied to roots of apple seedlings, moved readily to the developing leaves. Kinetin, benzyladenine, and B sprays increased movement. NO₃⁻ as the source of N increased movement and accumulation of Ca into mature leaves; NH₄⁺ increased movement into new leaves. Translocation in the stem is effected by a nonspecific ion exchange. Any divalent cation can free Ca for ascent. The exchange may be a property of lignin. Genetic differences in the uptake and translocation of Ca exist among apple seedlings. Those which show juvenile leaf characteristics translocate Ca into mature leaves more readily. Translocation of Ca in woody species appears to be similar to that reported for herbaceous plants.