Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation

To attain its native conformation, the cytoskeletal protein tubulin needs the concourse of several molecular chaperones, among others the cytosolic chaperonin CCT. It has been previously described that denatured tubulin interacts with CCT in a quasi-folded conformation using several loops located throughout its sequence. These loops are also involved in microtubule formation and are absent in its prokaryote homologue FtsZ, which in vitro folds by itself and does not interact with CCT. Several FtsZ/tubulin chimeric proteins were generated by inserting consecutively one, two or three of the CCT-binding domains of tubulin into the corresponding sequence of FtsZ from Methanococccus jannaschii. The insertion of any of the CCT-binding loops generates in the FtsZ/tubulin chimeras the ability to interact with CCT. The accumulation of CCT-binding loops induces in the FtsZ/tubulin chimeras unfolding and refolding properties that are more similar to tubulin than to its prokaryote counterpart. Finally, the insertion of some of these loops generates in the FtsZ/tubulin chimeras more complex polymeric structures than those found for FtsZ. These results reinforce the notion that CCT has coevolved with tubulin to deal with the folding problems encountered by the eukaryotic protein with the appearance of the new sequences involved in microtubule formation.     

Lovastatin inhibits the growth and survival pathway of phosphoinositide 3-kinase/protein kinase B in immortalized rat brain neuroblasts

We previously showed that lovastatin, an HMG-CoA reductase inhibitor, suppresses cell growth by inducing apoptosis in rat brain neuroblasts. Our aim was to study intracellular signalling induced by lovastatin in neuroblasts. Lovastatin significantly decreases the phosphoinositide 3-kinase (PI3-K) activity in a concentration-dependent manner. Expression of p85 subunit and its association with phosphotyrosine-containing proteins are unaffected by lovastatin. Lovastatin decreases protein kinase B (PKB)/Akt phosphorylation, and its downstream effectors, p70^S6K and the eukaryotic initiation factor 4E (eIF4E) regulatory protein 1, 4E-BP1, in a concentration-dependent manner, and reduces p70^S6K expression. Lovastatin effects are fully prevented with mevalonate. Only the highest dose of PI3-K inhibitors that significantly reduce PI3-K kinase activity induces apoptosis in neuroblasts but to a lower degree than lovastatin. In summary, this work shows that treatment of brain neuroblasts with lovastatin leads to an inhibition of the main pathway that controls cell growth and survival, PI3-K/PKB and the subsequent blockade of downstream proteins implicated in the regulation of protein synthesis. This work suggests that inactivation of the antiapoptotic PI3-K appears insufficient to induce the degree of neuroblasts apoptosis provoked by lovastatin, which must necessarily involve other intracellular pathways. These findings might contribute to elucidate the molecular mechanisms of some statins effects in the central nervous system.     

Mechanisms of Hedgehog gradient formation and interpretation

Morphogens are molecules that spread from localized sites of production, specifying distinct cell outcomes at different concentrations. Members of the Hedgehog (Hh) family of signaling molecules act as morphogens in different developmental systems. If we are to understand how Hh elicits multiple responses in a temporally and spatially specific manner, the molecular mechanism of Hh gradient formation needs to be established. Moreover, understanding the mechanisms of Hh signaling is a central issue in biology, not only because of the role of Hh in morphogenesis, but also because of its involvement in a wide range of human diseases. Here, we review the mechanisms affecting the dynamics of Hh gradient formation, mostly in the context of Drosophila wing development, although parallel findings in vertebrate systems are also discussed.     

Chloroplast and nuclear microsatellite analysis of Aegilops cylindrica

Aegilops cylindrica Host (2n=4x=28, genome CCDD) is an allotetraploid formed by hybridization between the diploid species Ae. tauschii Coss. (2n=2x=14, genome DD) and Ae. markgrafii (Greuter) Hammer (2n=2x=14, genome CC). Previous research has shown that Ae. tauschii contributed its cytoplasm to Ae. cylindrica. However, our analysis with chloroplast microsatellite markers showed that 1 of the 36 Ae. cylindrica accessions studied, TK 116 (PI 486249), had a plastome derived from Ae. markgrafii rather than Ae. tauschii. Thus, Ae. markgrafii has also contributed its cytoplasm to Ae. cylindrica. Our analysis of chloroplast and nuclear microsatellite markers also suggests that D-type plastome and the D genome in Ae. cylindrica were closely related to, and were probably derived from, the tauschii gene pool of Ae. tauschii. A determination of the likely source of the C genome and the C-type plastome in Ae. cylindrica was not possible.     

Central actions of somatostatin in the generation and control of breathing

The neuropeptide somatostatin is involved in many functions in the central nervous system as well as in the periphery. When it is centrally injected, an irreversible apnea is often developed. In the present review, we discuss the effects of somatostatin as the result of its actions at three levels of the respiratory neural network: a) by modulating the output of cranial or spinal motoneurons; b) by altering the genesis of the respiratory rhythm in the brainstem: and c) by regulating the chemosensory drive input into the respiratory pattern generator.     

Endometritis treatment with a PGF2alpha analog does not improve reproductive performance in a large dairy herd in Argentina

In Argentina, most dairy cows with endometritis are treated with prostaglandin (PGF(2alpha) or its analogs) and insemination is withheld until there are no signs of endometritis. The objective of the present study was to evaluate if this method of managing endometritis enhances reproductive performance. Three experiments were conducted over 4 years in a large farm in the west of Buenos Aires province. In Experiment 1, half of the cows diagnosed with endometritis (>1.5-fold difference in diameter of uterine horns, as determined by rectal palpation) received standard endometritis management (treatment with tiaprost, a PGF(2alpha) analog, rectal palpation every 20 days, and withholding of AI until endometritis apparently resolved) and the other half was untreated, with AI at the first estrus after the voluntary waiting period. Untreated cows were inseminated and conceived 20 days earlier than treated cows, and the pregnancy rate by Day 90 postpartum was higher in the untreated group. In Experiment 2, cows with endometritis were divided into four groups according to the severity of symptoms; within each group, cows were allocated to treatment or control, as in Experiment 1. Although first service conception rate decreased as endometritis severity increased, reproductive performance in treated versus control cows was similar to that of Experiment 1 (with no interaction due to degree of endometritis). Re-evaluation of the treated cow (to confirm uterine "normality") may have been responsible for the delay in conception in both experiments. The objective of Experiment 3 was to determine the effects of tiaprost treatment on clinically normal postpartum cows (no evidence of endometritis). Tiaprost treatment reduced the interval from calving to conception in multiparous cows, but it delayed conception and reduced the conception rate in primiparous cows. In conclusion, treatment with tiaprost impaired reproductive performance in primiparous cows (in the absence of endometritis). Furthermore, the standard treatment for endometritis (treatment with a prostaglandin analog and withholding insemination until clinical signs abated) impaired reproductive performance and increased costs.     

Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein alpha subunit that belongs to subgroup III of fungal Galpha proteins. Deltatga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.     

Metagenomic analysis of the microbial community at Zodletone Spring (Oklahoma): insights into the genome of a member of the novel candidate division OD1

A metagenomic library was constructed from the anaerobic sediments of a mesophilic sulfur spring. Thirty-five bacterial 16S rRNA gene-containing clones were identified in this library. Analysis of a genomic fragment belonging to candidate division OD1 provided useful insights into the physiology and biochemistry of this novel, yet-uncultured candidate division.     

Studies on the interaction mechanism between hexakis(imidazole) manganese(II) terephthalate and DNA and preparation of DNA electrochemical sensor

The interaction between hexakis(imidazole) manganese(II) terephthalate ([Mn(Im)(6)](teph).4H(2)O) and salmon sperm DNA in 0.2M pH 2.30 Britton-Robinson buffer solution was studied by fluorescence spectroscopy and cyclic voltammetry. Increasing fluorescence was observed for [Mn(Im)(6)](2+) with DNA addition, while quenching fluorescence phenomenon appeared for EB-DNA system when [Mn(Im)(6)](2+) was added. There were a couple quasi-reversible redox peaks of [Mn(Im)(6)](2+) from the cyclic voltammogram on the glassy carbon electrode. The peak current of [Mn(Im)(6)](2+) decreased with positive shift of the formal potential in the presence of DNA compared with that in the absence of DNA. All the experimental results indicate that [Mn(Im)(6)](2+) can bind to DNA mainly by intercalative binding mode. The binding ratio of the DNA-[Mn(Im)(6)](2+) association complex is calculated to be 1:1 and the binding constant is 4.44x10(3) M(-1). By using [Mn(Im)(6)](teph).4H(2)O as the electrochemical hybridization indicator, the DNA electrochemical sensor was prepared by covalent interaction and the selectivity of ssDNA modified electrode were described. The results demonstrate the use of electrochemical DNA biosensor in the determination of complementary ssDNA.     

The first enantioselective synthesis of the amavadin ligand and its complexation to vanadium

The ligand of the naturally occurring vanadium compound amavadin found in Amanita muscaria, (2S, 2'S)-N-hydroxyimino-2,2'-dipropionic acid (1), was synthesized stereoselectively in two steps with 43% overall yield. After complexation of this ligand to vanadyl acetate, amavadin was isolated in quantitative yield. Due to the chirality at vanadium amavadin consists of a mixture of delta and lambda diastereoisomers. Directly after its synthesis, the delta to lambda ratio of amavadin is 2.27 and it decreases to 0.80 after equilibrium has been reached. During this epimerization the optical rotation for V[(2S,2'S)-N-hydroxyimino-(2,2')-dipropionate]2 (=amavadin) changes from [alpha](D)25 = +36 degrees to +114.0 degrees (c = 0.5, H2O). For V[(2R,2'R)-N-hydroxyimino-(2,2')-dipropionate] the optical rotation changes from [alpha](D)25 = -36 degrees to -113.2 degrees (c = 0.5, H2O).