Increasing Accuracy and Throughput in Large-Scale Microsatellite Fingerprinting of Cacao Field Germplasm Collections

In this study we report on increasing the rate and accuracy of microsatellite fingerprinting of accessions in Theobroma cacao L. field germplasm collections with a medium-throughput capillary sequencing system. We examined the use of a reduced number of microsatellite loci to decrease the time and materials required for fingerprinting and determined the best available microsatellite loci for accurately separating accessions. A subset of nine informative loci that could separate sixty accessions into the same genetic groupings as a complete set of 37 loci was found. Stringent probability of identity values (i.e. chance of unique accession) was highly influenced (r?=??0.996; P?<?0.001) by the number of alleles utilised in the fingerprinting set but the composition of the primer set was more important when choosing discriminatory loci. DNA pooling to reduce the number of samples was also investigated. There was a relatively high level of mixture within plots (59% of 54 plots examined) of the field genebank, which opposed the use of a pooling strategy to fingerprint the multiple trees of an accession plot in the collection.     

Piperazinyl-glutamate-pyrimidines as potent P2Y12 antagonists for inhibition of platelet aggregation

Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the 4-position of the pyrimidine leading to highly potent P2Y₁₂ antagonists. In particular, 4-substituted piperidine-4-pyrimidines provided compounds with exceptional potency. Pharmacokinetic and physicochemical properties were fine-tuned through modifications at the 4-position of the piperidine ring leading to compounds with good human PRP potency, selectivity, clearance and oral bioavailability.     

Piperazinyl-glutamate-pyridines as potent orally bioavailable P2Y12 antagonists for inhibition of platelet aggregation

Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl-glutamate-pyridine as a P2Y₁₂ antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 22J with good human PRP potency, selectivity, in vivo efficacy and oral bioavailability.     

Rotation of Escherichia coli F(1)-ATPase.

By applying the same method used for F(1)-ATPase (TF(1)) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302), we observed ATP-driven rotation of a fluorescent actin filament attached to the gamma subunit in Escherichia coli F(1)-ATPase. The torque value and the direction of the rotation were the same as those observed for TF(1). F(1)-ATPases seem to share common properties of rotation irrespective of the sources.     

Phytoplankton assemblages and steady state in deep and shallow eutrophic lakes – an approach to differentiate the habitat properties of Oscillatoriales

Ecological conditions and phytoplankton succession in two shallow hypertrophic lakes (Langer See and Melangsee) and a dimictic, eutrophic lake (Scharmützelsee) in a lake chain in Eastern Germany were analyzed from 1999 to 2001 in order to find situations of phytoplankton steady state assemblages and variables controlling the phytoplankton composition according to Reynolds et al. (2002). Long term background data from 1993 to 2001 suggest steady state conditions in shallow lakes, whereas the deep lake exhibited irregular fluctuations between various phytoplankton stages. Since the phytoplankton composition in the shallow lakes was similar in all the 3 years, it was highly predictable. Steady state conditions dominated by different species of Oscillatoriales were detected during the summer period 1999 and 2000 in Langer See and in Melangsee (see Mischke & Nixdorf, this volume). This dominant assemblage found in both lakes (group S ₁ acc. to Reynolds et al., 2002): Planktothrix agardhii (Gom.) Anagn. et Kom., Limnothrix redekei (Van Goor) Meffert, Pseudanabaena (Lauterb.) is typical in turbid mixed layers with highly light deficient conditions, but it is also regularly dominant in the dimictic lake Scharmützelsee as observed in 1999 and 2001 (Pseudanabaena limnetica (Lemm.) Kom. The Nostocales Cylindrospermopsis raciborskii (Wolz.) Seenayya et Subba Raju and Aphanizomenon gracile (Lemmerm.) Lemmerm. were important in the shallow lakes as well as in lake Scharmützelsee. Nevertheless, the occurrence of filamentous cyanobacteria in the dimictic lake was not regular and an unpredictable change in phytoplankton development was observed in 2000. It is discussed, whether this phenomenon of regular succession in shallow hypertrophic lakes is caused by adaptation to a resilient and an extreme environment or by the pool of species that can live or survive in that environment. This was checked through comparison of the depth of the mixed layer, the mean daily irradiance within this layer and the nutrient resources. Although the nutrient resources in both types of lake are near threshold levels, indicating growth inhibition by dissolved nutrients (DIP, DIN, TIC, DSi), the under water light supply seems to be the key factor favoring the dominance of filamentous cyanobacteria belonging to the functional group S ₁.     

Equilibrium phase conditions in shallow German lakes: How Cyanoprokaryota species establish a steady state phase in late summer

In 2000, a field study in two shallow, polytrophic lakes (Langer See and Melangsee) in eastern Germany revealed an equilibrium state assemblage of Cyanoprokaryota in late summer. During 4 successive weeks in Langer See Planktothrix agardhii (Gom.) Anagn. et Kom., Aphanizomenon gracile (Lemmerm.) Lemmerm. and Pseudanabaena limnetica Lemmerm. were more than 80% of the standing biomass of phytoplankton, and their cumulative biovolume was around 33 mm³ l^?1 ((±3.2 SD). In Melangsee, the very small Limnothrix species L. amphigranulata (Van Goor) Meffert was the most common species, accompanied by Pseudanabaena limnetica and Planktothrix agardhii. For 3 weeks, their cumulative biovolume was about 23 mm³ l^?1 ((±3.4 SD), which represented 75 – 82% of total biovolume. The dominant species all belong to the functional group S1 defined by Reynolds (1997), except for A. gracile, which we suggest to be included in group S _N. In both lakes mean light intensities ranged between 2.2 and 8.3 E m^?2 d^?1. Overall species spectra were very similar in both lakes, but dominance by Limnothrix and by Planktothrix in the respective lakes is observed repeatedly. The success of these species is discussed in the context of the habitat properties in August/September. Summer mixing events represented no disturbances in the sense of Connell (1978), since they do not interrupt the species dominance. More frequent mixing events and higher concentrations of dissolved nitrogen occurred in Langer See than in the more shallow, but wind protected Melangsee. In Langer See light deficient conditions were intensified by an increasing biomass of P. agardhii, and this species probably benefited from nutrient input by more frequent resuspension. The light deficiency also affected the diversity, expressed as Shannon–Wiener Index (H), which was reduced more in lake Langer See (H = 0.51) than in Melangsee (0.74) during steady state periods. Recognizing the important effects of mixing, we suggest an additional variable to describe habitat properties: the number of full mixing days as a proportion of total days of observation should help to discriminate between shallow habitats with intermittent mixing events, and those with more regularly mixing in summer period.     

The regulation of DNA replication during senescence and rejuvenation of Dunaliella tertiolecta by factors other than available metabolic energy

The unicellular green alga, Dunaliella tertiolecta undergoes a 4-fold reduction in DNA while progressing from early to late log phase of culture. During the period when the reduction in DNA occurs, the cells continue to divide at the maximal rate. Pulse labelling indicates little incorporation of [³H]thymidine into DNA during late log phase. Stationary phase cultures diluted with fresh medium undergo a lag period during which there is a 4-fold increase in DNA and a rapid incorporation of [³H]thymidine into DNA into DNA before division. The evidence indicates that the differences in the levels of DNA are not attributable to tetraploidy, multinucleated cells or a high level of redundancy of G-C-rich satellite DNA in early log phase cells. During stationary phase there is an increase in cellular starch and a decrease in free nucleotides. Electron microscopy reveals that stationary phase cells are distorted by many granules proven to be starch by their susceptibility to amylase treatment. The production of starch by stationary phase cells indicates that DNA replication does not cease because of a deficit of metabolic energy but because of some direct function of culture density.     

Subunit delta of H(+)-ATPases: at the interface between proton flow and ATP synthesis

The ATP synthases in photophosphorylation and respiration are of the F-type with a membrane-bound proton channel, F0, and an extrinsic catalytic portion, F1. The properties of one particular subunit, delta (in chloroplasts and Escherichia coli) and OSCP (in mitochondria), are reviewed and the role of this subunit at the interface between F0 and F1 is discussed. Delta and OSCP from the three sources have in common the molecular mass (approximately 20 kDa), an elongated shape (axial ratio in solution about 3:1), one high-affinity binding site to F1 (Kd approximately 100 nM) plus probably one or two further low-affinity sites. When isolated delta is added to CF1-depleted thylakoid membranes, it can block proton flow through exposed CF0 channels, as do CF1 or CF1(-delta)+ delta. This identifies delta as part of the proton conductor or, alternatively, conformational energy transducer between F0 (proton flow) and F1 (ATP). Hybrid constructs as CF1(-delta)+ E. coli delta and EF1(-delta)+ chloroplast delta diminish proton flow through CF0.CF1(-delta) + E. coli delta does the same on EF0. Impairment of proton leaks either through CF0 or through EF0 causes "structural reconstitution' of ATP synthesis by remaining intact F0F1. Functional reconstitution (ATP synthesis by fully reconstructed F0F1), however, is absolutely dependent on the presence of subunit delta and is therefore observed only with CF1 or CF1(-delta) + chloroplast delta on CF0 and EF1 or EF1(-delta) + E. coli delta on EF0. The effect of hybrid constructs on F0 channels is surprising in view of the limited sequence homology between chloroplast and E. coli delta (36% conserved residues including conservative replacements). An analysis of the distribution of the conserved residues at present does not allow us to discriminate between the postulated conformational or proton-conductive roles of subunit delta.     

Protein structure and the sequential structure of mRNA: α-Helix and β-sheet signals at the nucleotide level

A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed. We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting protein. The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain. A complete search for GenBank nucleotide sequences coding for structural entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment. By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets. These signals do not originate from the clustering of rare codons, but from the similarity of codons coding for very abundant amino acid residues at the N- and C-termini of helices and sheets. No correlation between the positioning of rare codons and the location of structural units was found. The mRNA signals were also compared with conserved nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome. © 1996 Wiley-Liss, Inc.