Reorganization of actin cytoskeleton by the phosphoinositide metabolite glycerophosphoinositol 4-phosphate

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P.     

CHARACTERIZATION OF OSTREOPSIS AND COOLIA (DINOPHYCEAE) ISOLATES IN THE WESTERN MEDITERRANEAN SEA BASED ON MORPHOLOGY,TOXICITY AND INTERNAL TRANSCRIBED SPACER 5.8S rDNA SEQUENCES1

Several isolates of epiphytic dinoflagellates belonging to the genera Ostreopsis Schmidt and Coolia Meunier from the western Mediterranean Sea were examined by LM and EM, toxicity assays, and internal transcribed spacer (ITS) regions of nuclear rDNA, and 5.8S rDNA were sequenced. Morphological comparisons based on the analyses of cell shape, size, thecal plates, and surface ornamentation revealed two distinct species in the western Mediterranean: O. cf. siamensis Schmidt from the Catalan, Andalusian, and Sicilian coasts and O. ovata Fukuyo from the Ligurian coast, southern Tyrrhenian Sea, and Balearic Islands. Both Ostreopsis species were toxic; however, no differences in toxicity were detected between the two Ostreopsis species. Coolia monotis Meunier was nontoxic. The morphological studies were supported by phylogenetic analyses; all western Mediterranean isolates of O. cf. siamensis showed ITS and 5.8S rDNA sequences identical to each other and so did those of O. ovata, whereas high genetic diversity was detected between the western Mediterranean and Asian isolates of O. ovata. The nucleotide sequence analyses of the C. monotis strains showed that all C. monotis isolates from Europe formed a homogeneous clade. Further, the genetic diversity was high between the European and Asian C. monotis isolates. In this study, genetic markers combined with morphology and toxicity analyses was useful in the taxonomic and phylogenetic studies of the Ostreopsidaceae in a temperate area.     

Enhanced Accumulation of BiP in Transgenic Plants Confers Tolerance to Water Stress

The binding protein (BiP) is an important component of endoplasmic reticulum stress response of cells. Despite extensive studies in cultured cells, a protective function of BiP against stress has not yet been demonstrated in whole multicellular organisms. Here, we have obtained transgenic tobacco (Nicotiana tabacum L. cv Havana) plants constitutively expressing elevated levels of BiP or its antisense cDNA to analyze the protective role of this endoplasmic reticulum lumenal stress protein at the whole plant level. Elevated levels of BiP in transgenic sense lines conferred tolerance to the glycosylation inhibitor tunicamycin during germination and tolerance to water deficit during plant growth. Under progressive drought, the leaf BiP levels correlated with the maintenance of the shoot turgidity and water content. The protective effect of BiP overexpression against water stress was disrupted by expression of an antisense BiP cDNA construct. Although overexpression of BiP prevented cellular dehydration, the stomatal conductance and transpiration rate in droughted sense leaves were higher than in control and antisense leaves. The rate of photosynthesis under water deficit might have caused a degree of greater osmotic adjustment in sense leaves because it remained unaffected during water deprivation, which was in marked contrast with the severe drought-induced decrease in the CO(2) assimilation in control and antisense leaves. In antisense plants, the water stress stimulation of the antioxidative defenses was higher than in control plants, whereas in droughted sense leaves an induction of superoxide dismutase activity was not observed. These results suggest that overexpression of BiP in plants may prevent endogenous oxidative stress.     

Time-resolved methods in Biophysics. 2. Monitoring haem proteins at work with nanosecond laser flash photolysis.

Haem proteins have long been the most studied proteins in biophysics, and have become paradigms for the characterization of fundamental biomolecular processes as ligand binding and regulatory conformational transitions. The presence of the haem prosthetic group, the absorbance spectrum of which has a ligation sensitive region conveniently located in the UV-visible range, has offered a powerful and sensitive tool for the investigation of molecular functions. The central Fe atom is capable of reversibly binding diatomic ligands, including O(2), CO, and NO. The Fe-ligand bond is photolabile, and a reactive unligated state can be transiently generated with a pulsed laser. The photodissociated ligands quickly rebind to the haem and the process can be monitored by transient absorbance methods. The ligand rebinding kinetics reflects protein dynamics and ligand migration within the protein inner cavities. The characterization of these processes was done in the past mainly by low temperature experiments. The use of silica gels to trap proteins allows the characterization of internal ligand dynamics at room temperature. In order to show the potential of the laser flash photolysis techniques, combined with modern numerical analysis methods, we report experiments conducted on two non-symbiotic haemoglobins from Arabidopsis thaliana. The comparison between time courses recorded on haemoglobins in solution and encapsulated in silica gels allows for the highlighting of different interplays between protein dynamics and ligand migration.     

Muscular activation patterns during active prone hip extension exercises

BackgroundChanges in activation patterns of hip extensors and pelvic stabilizing muscles are recognized as factors that cause low back disorders and these disturbances could have an impact on the physiological loading and alter the direction and magnitude of joint reaction forces.ObjectiveTo investigate activation patterns of the gluteus maximus, semitendinosus and erector spinae muscles with healthy young individuals during four different modalities of therapeutic exercise.MethodsThirty-one volunteers were selected: (16 men and 15 women), age (24.5 ± 3.47 years), body mass of 66.89 ± 11.89 kg and a height of 1.70 ± 0.09 m). They performed four modalities of therapeutic exercise while the electromyographic activity of the investigated muscles was recorded to determine muscle pattern activation for each exercise.ResultsRepeated measure ANOVA revealed that muscle activation patterns were similar for the four analyzed exercises, starting with the semitendinosus, followed by the erector spinae, and then, the gluteus maximus. The gluteus maximus was the last activated muscle during hip extension associated with knee flexion (p < 0.0001), knee extension (p < 0.0001), and with lateral rotation and knee flexion (p < 0.05).ConclusionFindings of the present study suggested that despite individual variability, the muscle firing order was similar for the four therapeutic exercises.     

Fluorometric quantification of green fluorescent protein in tobacco leaf extracts

The main use of green fluorescent protein (GFP) is as a reporter system, where the existence of the protein is usually determined visually using fluorescent microscopy. Although fluorescence-based quantification of GFP is possible, background fluorescence in plants and in plant extracts was observed by our group. Another phenomenon we observed that makes quantification difficult is the increased level of GFP fluorescence in Nicotiana benthamiana leaf extracts, probably the result of dimerization of GFP molecules promoted by interaction with some component(s) of tobacco extracts. In the current work, the background fluorescence was minimized and the enhancement of GFP fluorescence in tobacco extracts was eliminated with the addition of urea to the measured solution so that a simple quantification assay for the GFP in the tobacco extracts could be established.     

Structure-affinity studies for a novel series of homochiral naphtho and tetrahydronaphtho analogues of alpha 1 antagonist WB-4101

A number of enantiomeric pairs of naphthodioxane, tetrahydronaphthodioxane and naphthoxy analogues of WB-4101 (1) were designed and synthesized in order to improve the selectivity profile of the parent compound, hopefully in favour of the alpha(1a)-AR with respect to the other two alpha(1) subtypes and the 5-HT(1A) receptor. The new compounds 2-8 and, in addition, the two enantiomers of 1 were tested in binding assays on the alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR, and the 5-HT(1A) receptor. Two of them, namely the naphtho- and tetrahydronaphthodioxane derivatives (S)-2 and (S)-3, showed lower, but significantly more specific alpha(1a) affinity than (S)-1, while the two enantiomers of the 2-methoxy-1-naphthoxy analogue 6 maintained most of the very high alpha(1a) affinity of (S)-1 and its alpha(1a) versus alpha(1b) selectivity slightly increasing the alpha(1a)/alpha(1d) and alpha(1a)/5HT(1A) affinity ratios. The SAR data were evaluated in the light of known alpha(1) subtype pharmacophores and of the alpha(1a)-AR binding mode of WB-4101 resultant from literature mutagenesis studies disclosing some interesting consonances with these models.     

Synthesis and alpha4beta2 nicotinic affinity of 2-pyrrolidinylmethoxyimines and prolinal oxime ethers

Homochiral E and Z isomers of N-methylprolinal O-isopropyloxime and (1-methyl-2-pyrrolidinyl)methoxyimines were synthesized as candidate bioisosteres of nicotine and its isoxazolic analogue ABT 418. Two of them, namely (S)-2-isopropylideneaminooxymethyl- and (Z)-(S)-2-ethylideneaminooxymethyl-1-methylpyrrolidine, proved to bind at alpha4beta2 nicotinic acetylcholine receptor with submicromolar affinity and remarkable selectivity over alpha7 and muscarinic receptors thus supporting the hypothesized bioisosteric relationship between their methyloxyimino group and the aromatic heterocycles of the reference ligands.     

Internal dynamics and protein-matrix coupling in trehalose-coated proteins

We review recent studies on the role played by non-liquid, water-containing matrices on the dynamics and structure of embedded proteins. Two proteins were studied, in water-trehalose matrices: a water-soluble protein (carboxy derivative of horse heart myoglobin) and a membrane protein (reaction centre from Rhodobacter sphaeroides). Several experimental techniques were used: Mossbauer spectroscopy, elastic neutron scattering, FTIR spectroscopy, CO recombination after flash photolysis in carboxy-myoglobin, kinetic optical absorption spectroscopy following pulsed and continuous photoexcitation in Q(B) containing or Q(B) deprived reaction centre from R. sphaeroides. Experimental results, together with the outcome of molecular dynamics simulations, concurred to give a picture of how water-containing matrices control the internal dynamics of the embedded proteins. This occurs, in particular, via the formation of hydrogen bond networks that anchor the protein surface to the surrounding matrix, whose stiffness increases by lowering the sample water content. In the conclusion section, we also briefly speculate on how the protein-matrix interactions observed in our samples may shed light on the protein-solvent coupling also in liquid aqueous solutions.     

Immunophenotypic and Ultrastructural Validation of a New Human Glioblastoma Cell Line

1. A human glioma cell line, NG97, was established by Grippo et al. in 2001 from tissue obtained from a grade III astrocytoma (WHO, 2000). In this first study, the cell line grew as two morphologically distinct subpopulations: dendritic/spindle cells and small round cells. The injection of NG97 cells into nude mice induced an aggressive tumor characterized by: severe cytological atypia, vascular proliferation and pseudopalisading necrosis (glioblastoma multiforme features). 2. The purpose of the present study was to characterize the immunophenotype and ultrastructural aspects of this cell line, using the parental tumor, cultured cells and the xenotransplant, in order to assess its glial nature and possible divergent differentiation. 3. NG97 cells and xenotransplant expressed the main neuroglial markers (GFAP, S-100 protein, NSE and Leu-7) and showed no aberrant expression of other histogenetic markers. GFAP was similarly expressed in the parental tumor and in the cells in culture, but decreased in the xenotransplant. NSE expression was reduced in NG97 cells, but substantially recovered in the xenotransplant. This variability in expression of GFAP and NSE was interpreted as either a phenomenon of dedifferentiation or to microenvironmental selection of specific subclones. S-100 was equally expressed in the three contexts. The xenotransplant's ultrastructural features were those of a highly undifferentiated tumor. No significant immunophenotypic or ultrastructural differences between the two morphologically distinct populations were found. 4. Thus, our data demonstrate that NG97 cells constitute a pure glial-committed cell line, which may prove useful as a malignant glioma model in studies addressing pathophysiological, diagnostic and therapeutic issues.