The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3⁺ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.     

Cardiospheres and tissue engineering for myocardial regeneration: potential for clinical application

Tissue engineering is an increasingly expanding area of research in the cardiovascular field that involves engineering, chemistry, biology and medicine. Cardiac tissue engineering (CTE) aims to regenerate myocardial damage by combining cells, matrix, biological active molecules and physiological stimuli. The rationale behind CTE applications is that in order to regenerate the ventricular wall after a myocardial infarction it is necessary to combine procedures that regenerate both cardiomyocytes and the extracellular matrix. The application of (stem) cells together with a matrix could represent an environment protected from the inflammatory and pro-apoptotic signals, a stemness/survival reservoir slowly releasing cells and factors promoting tissue regeneration and angiogenesis. This review will focus on the applications and advantages that CTE application could offer compared to conventional cell therapy.     

Characterization of an RNA granule from developing brain

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.     

Evolution of hypervariable microsatellites in apomictic polyploid lineages of Ranunculus carpaticola: directional bias at dinucleotide loci

Microsatellites are widely used in genetic and evolutionary analyses, but their own evolution is far from simple. The mechanisms maintaining the mutational patterns of simple repeats and the typical stable allele-frequency distributions are still poorly understood. Asexual lineages may provide particularly informative models for the indirect study of microsatellite evolution, because their genomes act as complete linkage groups, with mutations being the only source of genetic variation. Here, we study the direction of accumulated dinucleotide microsatellite mutations in wild asexual lineages of hexaploid Ranunculus carpaticola. Whereas the overall number of contractions is not significantly different from that of expansions, the within-locus frequency of contractions, but not of expansions, significantly increases with allele length. Moreover, within-locus polymorphism is positively correlated with allele length, but this relationship is due solely to the influence of contraction mutations. Such asymmetries may explain length constraints generally observed with microsatellites and are consistent with stable, bell-shaped allele-frequency distributions. Although apomictic and allohexaploid, the R. carpaticola lineages show mutational patterns resembling the trends observed in a broad range of organisms, including sexuals and diploids, suggesting that, even if not of germline origin, the mutations in these apomicts may be the consequence of similar mechanisms.     

Solution structure of a chemosensory protein from the desert locust Schistocerca gregaria

Chemical stimuli, generally constituted by small volatile organic molecules, are extremely important for the survival of different insect species. In the course of evolution, insects have developed very sophisticated biochemical systems for the binding and the delivery of specific semiochemicals to their cognate membrane-bound receptors. Chemosensory proteins (CSPs) are a class of small soluble proteins present at high concentration in insect chemosensory organs; they are supposed to be involved in carrying the chemical messages from the environment to the chemosensory receptors. In this paper, we report on the solution structure of CSPsg4, a chemosensory protein from the desert locust Schistocerca gregaria, which is expressed in the antennae and other chemosensory organs. The 3D NMR structure revealed an overall fold consisting of six alpha-helices, spanning residues 13-18, 20-31, 40-54, 62-78, 80-90, and 97-103, connected by loops which in some cases show dihedral angles typical of beta-turns. As in the only other chemosensory protein whose structure has been solved so far, namely, CSP from the moth Mamestra brassicae, four helices are arranged to form a V-shaped motif; another helix runs across the two V's, and the last one is packed against the external face. Analysis of the tertiary structure evidenced multiple hydrophobic cavities which could be involved in ligand binding. In fact, incubation of the protein with a natural ligand, namely, oleamide, produced substantial changes to the NMR spectra, suggesting extensive conformational transitions upon ligand binding.     

A new method for determination of plasma homocystine by isotope dilution and electrospray tandem mass spectrometry

A new analytical determination method of homocystine in human plasma has been developed. The method utilises liquid chromatography coupled to ionspray tandem mass spectrometry. Quantitative analysis was achieved using as an internal standard homocystine-d8. Mass spectrometer operated in the multiple reaction mode: homocystine and homocystine-d8 were detected through the transition from the precursor to the product ion (from m/z 269.3 to 90.0, and m/z 277.3 to 94.0, respectively). The method is extremely sensitive, with limit of detection in the range of 6 fmol/L. The interassay and intraassay coefficients of variation for homocystine were 6.22% and 3.4%, respectively. The accuracy for the added homocystine ranged from 85% to 110%. High specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for a rapid and reliable assay of homocystine.     

Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.     

Factors controlling hydrochemical and trophic state variables in 86 shallow lakes in Europe

The total nitrogen mass balance was studied over a two-year period in two reservoirs located in south-eastern Poland. Theobserved nitrogen retention (N_ret) inthe reservoirs ranged from 0.18 to 2.12g m^-2d^-1 (mean 0.83g m^-2d^-1) in case of theRzeszów Reservoir and from -0.02 to0.41 g m^-2d^-1 (mean 0.13g m^-2d^-1) in case of the SolinaReservoir, while the percentage retention(N_ret) ranged from 7 to 42% (mean22%) and -15 to 73% (mean 35%),respectively. A good relationship betweenN_ret and nitrogen loads was observedfor the Solina Reservoir. The data obtainedby the author and collected from theresearches of 6 other reservoirs located insouthern Poland were used to develop anempirical model suitable to predict thenitrogen seasonal dynamics in a reservoir.The model allows for the determination ofboth the annual mean N_ret value andits seasonal variation in a reservoir.     

Gross mineralization and plant N uptake from animal manures under non-N limiting conditions, measured using 15N isotope dilution techniques

To utilise wisely the manure resource, a better understanding of the processes that control the breakdown of organic N to inorganic N (mineralization) is required. ¹⁵N isotope dilution techniques should allow estimates of plant N uptake and gross mineralization from organic manures under non-N limiting conditions to be made. In natural systems the study of organic nitrogen breakdown to inorganic nitrogen, mineralization, is confounded by the processes of nitrification, nitrate leaching, gaseous N losses and plant N uptake. The ¹⁵N isotope dilution approach allows measurement of gross mineralization independently of these processes. Greenhouse experiments were conducted to determine plant N uptake from organic manures under non-N limiting conditions using the soil pre-labelling isotope dilution approach. The soil was pre-labelled with ¹⁵N and maize plants were then grown on the control treatments (no organic amendment) or on the manure treatments. The principle is thus that the control crop has a ¹⁵N abundance which reflects the ¹⁵N status of the soil and the treatment crop has a ¹⁵N enrichment diluted by the contribution of mineralized unlabelled manure N. Using this technique, it was estimated that maize plants derived 17 and 34% of their N from sewage sludge and turkey manure, respectively. The soil pre-labelling isotope dilution approach allowed yield-independent estimation of nitrogen derived from manures under non-N limiting conditions. Estimates of gross N mineralization were made to determine the breakdown of manure under field conditions. Results suggested that there was a rapid mineralization of turkey manure N in the initial weeks after application, in the order of 50 kg N ha^?1, which tailed off in the following weeks. The technique suggested that the soil used in the study had an extremely low basal mineralization rate, and a high nitrification rate.     

Tree community structure and stem mortality along a water availability gradient in a Mexican tropical dry forest

We document spatial changes in species diversity, composition, community structure, and mortality of trees across a gradient of water availability in a tropical dry forest in western Mexico. This gradient occurs along the main stream of a small watershed of less than 1 km in length. Four 30 × 80 m plots were established systematically to include the driest (ridge top of the watershed) to the wettest sites (watershed bottom) within this watershed. All stems larger than 5 cm were identified, and measured for diameter and height. Dead stems larger than 5 cm were measured and classified as: a) found on live or dead trees, and b) standing (“snags”) or lying (“downlogs”) on the ground. The number of recorded species per plot declined from 73 to 44 species as water availability decreased. A decline in estimated total richness, and in Shannon-Wiener and Simpson diversity indices was also observed in the drier plots. Species composition strongly changed along the gradient, with the two ends of the gradient sharing only 11% of the species. Stem density and percentage of dead stems and trees increased in abundance and basal area from the wetter to the drier sites. Tree and stem size (basal area, height and stem diameter) showed the opposite trend. Nonetheless, total basal area of live trees was largest at the two end gradient locations and oscillated between 12.22 m² ha⁻¹ and 7.93 m² ha⁻¹. Proportion of snags increased towards the driest site (from 46 to 72%), while that of down logs decreased. Overall, our results suggest that small-scale gradients of water availability play a paramount role in the spatial organization of tree communities in seasonal tropical environments. This revised version was published online in June 2006 with corrections to the Cover Date.