A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers

A linkage map of cacao based on codominant markers has been constructed by integrating 201 new simple sequence repeats (SSR) developed in this study with a number of isoenzymes, restriction fragment length polymorphisms (RFLP), microsatellite markers and resistance and defence gene analogs (Rgenes-RFLP) previously mapped in cacao. A genomic library enriched for (GA)_n and (CA)_n was constructed, and 201 new microsatellite loci were mapped on 135 individuals from the same mapping population used to establish the first reference maps. This progeny resulted from a cross between two heterozygous cacao clones: an Upper-Amazon Forastero (UPA 402) and a Trinitario (UF 676). The new map contains 465 markers (268 SSRs, 176 RFLPs, five isoenzymes and 16 Rgenes-RFLP) arranged in ten linkage groups corresponding to the haploid chromosome number of cacao. Its length is 782.8 cM, with an average interval distance between markers of 1.7 cM. The new microsatellite markers were distributed throughout all linkage groups of the map, but their distribution was not random. The length of the map established with only SSRs was 769.6 cM, representing 94.8% of the total map. The current level of genome coverage is approximately one microsatellite every 3 cM. This new reference map provides a set of useful markers that is transferable across different mapping populations and will allow the identification and comparison of the most important regions involved in the variation of the traits of interest and the development of marker-assisted selection strategies.Communicated by H. Nybom     

Activity of some platinum(II/IV) complexes with O,O-n-butyl-and O,O-n-pentyl-ethylenediamine-N,N'-di-3-propanoate and halogeno ligands against HeLa and K562 cell lines and human PBMC

This paper reports on syntheses and characterization of chlorotribromo(O,O-n-butyl-ethylenediamine-N,N'-di-3-propanoate)platinum(IV) [II], dichlorodiiodo(O,O-n-butyl-ethylenediamine-N,N'-di-3-propanoate)platinum(IV) [III], and dichloro(O,O-n-butyl-ethylenediamine-N,N'-di-3-propanoate)platinum(II) [V] complexes, with the formulae [Pt(dbeddp)Br(3)Cl], [Pt(dbeddp)Cl(2)I(2)] and [Pt(dbeddp)Cl(2)], respectively. The complexes were characterized by elemental analysis, infrared, (1)H and (13)C NMR spectroscopy and electrospray mass spectrometry. In the aim to assess the selectivity in the antitumor action of these complexes, as well, as tetrachloro(O,O-n-butyl-ethylenediamine-N,N'-di-3-propanoate)platinum(IV) [I] and tetrachloro(O,O-n-pentyl-ethylenediamine-N,N'-di-3-propanoate)platinum(IV) [IV], the antiproliferative action of these compounds was determined to human adenocarcinoma HeLa cells, to human myelogenous leukemia K562 cells and to normal immunocompetent cells, i.e., on human peripheral blood mononuclear PBMC cells.     

Coronary artery reperfusion: The ADP receptor P2Y<Subscript>1</Subscript> mediates early reactive hyperemia <Emphasis Type="BoldItalic">in vivo</Emphasis> in pigs

The physiological mechanisms that regulate reactive hyperemia are not fully understood. We postulated that the endothelial P2Y₁ receptor that release vasodilatory factors in response to ADP might play a vital role in the regulation of coronary flow. Intracoronary flow was measured with a Doppler flow-wire in a porcine model. 2-MeSADP (10^–5 M), ATP (10^–4 M) or UTP (10^–4 M) alone or as co-infusion with a selective P2Y₁ receptor blocker, MRS 2179 (10^–3 M) was locally delivered through the tip of a coronary angioplasty balloon. In separate pigs the coronary artery was occluded with the balloon for 10 min. During the first and tenth minutes of coronary ischemia, 2.5 ml of MRS 2179 (10^–3 M) was delivered distal to the occlusion in 8 pigs, 10 pigs were used as controls. MRS 2179 fully inhibited the 2-MeSADP-mediated coronary flow increase (P < 0.05) with no effect on UTP, indicating selective P2Y₁ inhibition. ATP-mediated flow increase was significantly inhibited by MRS 2179. During reactive hyperemia following coronary occlusion, flow increased by nearly sevenfold. MRS 2179, however, reduced the post-ischemic hyperemia by a mean of 46% during the period 1–2.5 min following balloon deflation (P < 0.05), which corresponds to peak velocity flow during reperfusion. In conclusion, MRS 2179, a selective P2Y₁ receptor blocker, significantly reduces the increased coronary flow caused both by 2-MeSADP and reactive hyperemia in coronary arteries. Thus, ADP acting on the endothelial P2Y₁ receptor may play a major role in coronary flow during post-ischemic hyperemia.     

Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.     

High-level secretory expression of penicillin amidase from Providencia rettgeri in Saccharomyces cerevisiae: purification and characterization

Heterologous production of the heterodimeric penicillin G amidase (PAC) from Providencia rettgeri was optimized in Saccharomyces cerevisiae. Several factors, including the effect of different growth and induction conditions, were identified to be critical for the enzyme overproduction and secretion. The PAC yield was significantly increased by more than 500-fold compared to that obtained in the native bacterium, and the recombinant enzyme was almost entirely secreted. Electrophoretic characterization of the secreted rPAC(Pr), which was purified over 20-fold by a combination of hydrophobic interaction and ion-exchange chromatography, demonstrated a microheterogeneity of the recombinant enzyme. The recombinant PAC(Pr) was further characterized in terms of specific activity, pH, and temperature profiles and kinetic parameters. The data presented here suggest that by overexpressing rPAC(Pr) in S.cerevisiae and purifying secreted enzyme from culture medium one can readily obtain a large amount of an alternative source of penicillin amidase with properties comparable to that of todays main industrial source of enzyme.     

Provisional stenting in small vessels

Based on the currently available data, the strategy of routine stent placement in unselected lesions located in small coronary arteries provides good immediate results but is still associated with a high incidence of in-stent restenosis. Randomized trials comparing elective stenting with balloon angioplasty have not provided the demonstration that routine stenting is the best strategy for percutaneous intervention in coronary arteries with a reference diameter smaller than 2.75-3.0 mm. This paper describes the rationale for provisional stenting in this clinical setting and reviews the role of quantitative coronary angiography, intracoronary ultrasound and intracoronary Doppler measurements in the identification of lesions that would benefit from adjunctive stent placement after balloon angioplasty and in guiding stent implantation.     

Gustatory responses in primates to the sweetener aspartame and their phylogenetic implications

Two-bottle preference tests have been applied to 70 (sub-) speciesof the order of Primates and, for comparison, to the tree shrew(Tupaia belangeri) to determine their responses to aspartame(APM), the first known sweet-tasting dipeptide which has toman a sweetness potency of about 200 times that of sucrose.It was found that only the Cercopithecidae, the Hylobatidaeand the Pongidae respond like man to this dipeptide and preferit to water. The other primates tested to date, show no responseto this sweetener. From a phylogenetic point of view, we notethat APM shows species specificity similar to thaumatin. Thus,a clear dichotomy exists within the order of Primates with respectto both thaumatin and APM. The results here illustrate oncemore the gustatory diversity among primates and are a compellingargument for the existence of different sweet taste receptorsor recognition sites in primate species.     

Benthic macroinvertebrates associated with four species of macrophytes

Benthic macroinvertebrates associated with four species of macrophytes (Nymphoides peltata, Ceratophyllum demersum, Polygonum amphibium and Carex sp.) were investigated during two growing seasons (2001 and 2002) in the slow-flowing Čonakut Channel in the Kopački rit Nature Park in Croatia. A total of 31 macroinvertebrate taxa were found. C. demersum, a submerged plant with dissected leaves, supported the highest macroinvertebrate abundance, almost seven times more than N. peltata, a floating plant with undissected leaves, which harboured the lowest abundance during the research period. Chironomidae larvae (50–83%) and Oligochaeta (14–46%) were the most abundant groups recorded on all macrophyte species. Water-level fluctuation, because of its influence on the appearance and growth of aquatic vegetation, and the trophic state of water within the macrophyte stands seemed to be the main factors which affected the taxonomic composition and abundance of macroinvertebrates.     

On the Procercoid Protonephridial Systems of Three Diphyllobothrium Species (Cestoda, Pseudophyllidea) and Janicki's Cercomer Theory

Because the types of hooks are so similar in the oncosphacra/procercoid ef cestodes and in several groups of monogenclic trcmatodes and because the exterior of a procercoid with the hooks in a cercomer is so suggestive of a monogenetic nematode, the development of the procercoids of three Diphyllobothrium species was studied. The intention was to determine whether or not the procercoid protonephridial system would have a developmental stage when its type is similar to, or identical with that type which characterizes the monogeneans. Such a conformity would greatly support the theory of a common origin of monogeneans and ceslodes. However, it has emerged that no similar developmental stage exists. The ontogeny revealed a thorough metamorphosis from a very simple primary protonephridial system (identical with that of the miracidium larva in digeneans) to a secondary system, which develops into the system of the adult tapeworm. This fact may be interpreted as an argument against the supposed inter-relalionships between monogeneans and cestodes. However, the type of hooks and the procercoid cercomer still indicate common ancestors. An analysis of the miracidium, the oncosphaera and the oncomiracidium (the monogenean larva) with reference to their different developmental stages when hatching, gave rise to my interpretation of the fundamental structure of both the miracidium and the oncosphaera as primitively simple and not reduced, Especially the identical type of protonephridial system indicates. in my view, that digeneans and ceslodes originally had a common larva type. If the ceslodes and the monogeneans have common ancestors, then the procercoid may be interpreted as the ontogenetic recapitulation of a common hook-armed ancestor, here named hexucanthoid. This rhabdocoelan creature with six hooks in the cercomer and adapted to an ectocommensalic/ectoparasitic mode of life, is thought to have given rise to the monogeneans, the gyrocotylideans, the amphilinideans and the cestodes. The monogeneans were found to have two fundamentally different types of marginal hooks, and on this basis ihe existence of two different lines of evolution in Monogenea is indicated.