Expression ofruntBIs Modulated during Chondrocyte Differentiation

Theruntlocus inDrosophilaencodes a nuclear protein involved in embryo segmentation, sex determination/X dosage compensation, and neurogenesis.runthomologues have been identified in higher vertebrates. The encoded proteins share a domain of 128 amino acids called the runt domain. It has been reported that this domain mediates DNA binding and heterodimerization. Here, we analyzeruntBexpression during chondrocyte differentiation “in vitro” and “in vivo.” We have first isolated, from a chondrocyte library, a cDNA clone coding for aruntBchicken homologue and containing a complete open reading frame. The predicted protein product is 84% identical to the mouse PEBP2αB2 isoform. By RT-PCR analysis we have also cloned the chicken cDNA fragment coding for ΔαB2, the exon sequence included in the B1 isoform mRNA. On Northern blot analysis of cultured chondrocytes, runtB mRNA levels increase dramatically with the transition from stage 0 (dedifferentiated) to stages I and II (hypertrophic chondrocytes). Moreover, runt polypeptides were demonstrated in chondrocytes bothin vivoandin vitro.These results suggest thatruntplays a role in chondrogenic differentiation.     

An approach for fluorometric determination of glycosyltransferase activities

A new strategy for the fluorometric determination of glycosyltransferase activities is reported. The method involves dansyl chloride derivatization of the reduced form (pNH₂phenyl) of a hydrophobic, aglycon moiety covalently linked to a number of acceptor substrates (pNO₂phenyl). Focusing on the Golgi enzyme core 2N-acetylglucosaminyltransferase, we found that synthesis and fractionation of the dansylated substrate derivative were rapid, easy and inexpensive. Additionally, the corresponding enzyme assay proved reproducible and very sensitive, as 0.4 pmol of reaction product were readily detected. This fluorometric approach appears therefore to be a valid tool for investigating the monitoring differential expression of glycosyltransferases exhibiting low levels of enzyme activity.Abbreviations T transferase - Gal D-N-galactose - GlcNAc D-N-acetylglucosamine - GalNAc D-N-acetylgalactosamine - HPLC high pressure liquid chromatography - UDP uridine diphosphate - TES 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid - pNp para-nitrophenyl - NMR nuclear magnetic resonance - DMSO dimethyl sulphoxide     

Inter-generational competition and selection for helping behaviour

A model is presented which considers the effects of inter-generational competition (IC) for breeding status on selection for helping behaviour. The model shows that for species with both high inter-generational overlap and high frequency of breeding opportunities within the individual's lifespan, helping behaviour is selected if the relatives raised by the helper through helping (i.e. present indirect fitness gains) do not decrease the helper's future direct fitness as a breeder. This effect is dependent on the value of α, the coefficient of IC. The occurrence of high α values can explain those cases in which a non-breeding member of a social group does not help. The effect of α can be reduced by means of establishing an age-based queueing system to attain breeding status. The breeders' acceptance of a non-helping individual within their territory may be explained as extended parental care or parental facilitation.     

A comparative analysis of some life-history traits between cooperatively and non-cooperatively breeding Australian passerines

Summary Comparative analyses were carried out for some life-history traits of cooperatively and non-cooperatively breeding Australian Corvida (i.e. old-endemic passerines). Multivariate statistical analyses at the family and genus levels revealed no significant differences between cooperative and non-cooperative breeders. A matched-pairs analysis between congeneric species showed that cooperatively breeding species lay smaller clutches than non-cooperatively breeding congenerics. Preliminary results also suggest that cooperative breeders have higher probabilities of rearing a second brood in the season and lower probabilities of survival than do non-cooperative breeders. However, the result for survival was significant in only one out of three tests. We conclude that cooperatively and non-cooperatively breeding Australian Corvida cannot be separated into distinct groups showingK- andr-selected life-history traits, respectively. Some life-history traits follow the prediction of ther-K selection model, others show evidence of co-adaptation instead, whereas still others show evidence of trade-offs.     

Electron microscopic observations of young rat liver

Summary Electron microscopic observations on normal liver tissue of four-day-old rats reveal the presence of numerous lamellar structures (lamellar bodies). These can be contained within parenchymal cells or in bile canaliculi, Disse's space, and in the lumen of blood sinusoids. Such bodies can also be found in Kupffer and endothelial cells.The lamellar bodies within hepatic cells are generally seen in very intimate relation to glycogen particles and lipide droplets, but in some to agranular endoplasmic reticulum and Golgi membranes as well.On the basis of this intimate relation to intracellular glycogen granules and lipide droplets, it is presumed that lamellar bodies represent a special intermediate stage in carbohydrate and lipide metabolism.Discontinuities in the endothelial layer of intrahepatic sinusoids are described.This work was supported in part by a N.A.T.O. research fellowship of the Consiglio Nazionale delle Ricerche, Roma.Assistant Professor in the Department of Veterinary Anatomy, Histology and Embryology (Dir.: Prof. A. de Girolamo), University of Naples, Naples, Italy.     

Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium

The isolation of biological clones of Trypanosoma cruzi by microscopically dispensing individual organisms or by serial dilution is laborious and time consuming. The inability to resolve mixed T. cruzi infections, from vectors and hosts, and to isolate clones of slow growing genotypes by efficient plating on solid media, has hindered characterisation studies and downstream applications. We have devised and validated a sensitive, solid medium plating technique for rapid in vitro isolation of clones representative of all the recognised T. cruzi lineages (TCI, TCIIa-e), including the slow growing strain CANIII (TC IIa) and Trypanosoma rangeli, with high plating efficiencies. Furthermore, the method is effective for the isolation of clones directly from silvatic triatomine bugs and from experimentally infected mice harbouring mixed infections, allowing resolution of multiclonal infections from varied sources.     

IQGAP1 stimulates actin assembly through the N-WASP-Arp2/3 pathway

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.     

Detection of S-nitrosothiols in biological fluids: a comparison among the most widely applied methodologies

Many different methodologies have been applied for the detection of S-nitrosothiols (RSNOs) in human biological fluids. One unsatisfactory outcome of the last 14 years of research focused on this issue is that a general consensus on reference values for physiological RSNO concentration in human blood is still missing. Consequently, both RSNO physiological function and their role in disease have not yet been clarified. Here, a summary of the values measured for RSNOs in erythrocytes, plasma, and other biological fluids is provided, together with a critical review of the most widely used analytical methods. Furthermore, some possible methodological drawbacks, responsible for the highlighted discrepancies, are evidenced.     

Release and degradation of amnesic shellfish poison from decaying Pseudo-nitzschia multiseries in presence of bacteria and organic matter

Domoic acid (DA), the neurotoxin produced by diatoms such as Pseudo-nitzschia multiseries is water-soluble and can bioaccumulate, causing mass death of birds and marine mammals worldwide. Humans eating contaminated shellfish most commonly suffer from memory loss but mortalities have been recorded. The fate of particulate and dissolved DA released from the cells or added as standards was studied when incubated with different bacterial abundances, copepod faecal pellets, mussel pseudo-faeces and bottom sediment. Strains of P. multiseries from Canada and Brazil were grown in non-axenic continuous monocultures with different nutrient conditions, or in a follow-up mesocosm experiment. Incubation lasted up to 75 days in the dark under quiescent conditions after the cells had been killed. Release of DA from decaying cells did not depend on bacterial abundance when the bacterial source was cultures of P. multiseries, and the dissolved toxin was stable with bacteria from P. multiseries cultures (at least 20 days with 1× or 4× bacterial concentration), or with a naturally occurring density of bacteria from surface waters of a known P. multiseries bloom area (35 days). However, four-fold concentration of the natural bacterial consortium from the bloom site reduced the onset of DA degradation to 16 days. Thus, this study suggests that when testing toxin degradation by bacteria, it is important to use bacterial consortia from known bloom areas of Pseudo-nitzschia. Copepod faecal pellets did not affect DA degradation, whereas the presence of mussel pseudo-faeces and bottom sediment rapidly removed most of the toxin. We believe that the rapid removal of DA in the two latter treatments was due to higher bacterial abundance and the presence of enzymes from the mussels and/or associated bacteria that are important for the degradation process. The mechanisms underlying the observed effects on DA degradation with mussel pseudo-faeces and sediment require further research, but suggest interesting possibilities as a potential future mitigation technique.     

Molecular modelling studies of four stranded quadruplexes containing a 3'-3' or 5'-5' inversion of polarity site

Recently we reported a preliminary study on the structure of two novel quadruplex structures, Q33 and Q55, formed by the oligodeoxynucleotides (5)'TGG(3)'-(3)'GGT(5)' and (3)'TGG(5)'-(5)'GGT(3)', respectively. Here we report their solution structures at the atomic level. The obtained structures reveal that Q55 and Q33 possess a different stacking among G-quartets and different twist angle (and therefore different helical winding) at the inversion of polarity level.