Phylogenetic position of Platanista gangetica: insights from the mitochondrial cytochrome b and nuclear interphotoreceptor retinoid-binding protein gene sequences

The evolutionary relationship of peculiar and poorly known Ganges River dolphin with extinct and extant cetaceans has been in the state of confusion for more than a century. The close resemblance of platanistidae with some of the extinct taxon viz., Dalpiaziniidae and Waipatiidae and their sister group relationship with many of the extant lineages of cetaceans has been reported but none of the alternative hypotheses provide an unambiguous placement for this species. The present study provides insights into the molecular relationships of Platanista with other cetaceans based on comprehensive analyses of the mitochondrial cytochrome b and nuclear interphotoreceptor retinoid-binding protein gene sequences, obtained from 15 specimens of Ganges dolphin from India and Bangladesh. The mean substitution distance analysis of phylogenetically informative characters in the cytochrome b sequences suggested that Platanista gangetica is significantly closer (P<0.001) to Mysticeti than to any other group of toothed whales. However, the conventional methods of phylogenetic reconstruction supported this finding with low to moderate (41-69%) bootstrap values.     

Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen

17beta-Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.     

Modeling the binding sites of anti-hen egg white lysozyme antibodies HyHEL-8 and HyHEL-26: an insight into the molecular basis of antibody cross-reactivity and specificity

Three antibodies, HyHEL-8 (HH8), HyHEL-10 (HH10), and HyHEL-26 (HH26) are specific for the same epitope on hen egg white lysozyme (HEL), and share >90% sequence homology. Their affinities vary by several orders of magnitude, and among the three antibodies, HH8 is the most cross-reactive with kinetics of binding that are relatively invariable compared to HH26, which is highly specific and has quite variable kinetics. To investigate structural correlates of these functional variations, the Fv regions of HH8 and HH26 were homology-modeled using the x-ray structure of the well-characterized HH10-HEL complex as template. The binding site of HH26 is most charged, least hydrophobic, and has the greatest number of intramolecular salt bridges, whereas that of HH8 is the least charged, most hydrophobic and has the fewest intramolecular salt bridges. The modeled HH26-HEL structure predicts the recently determined x-ray structure of HH26, (Li et al., 2003, Nat. Struct. Biol. 10:482-488) with a root-mean-square deviation of 1.03 A. It is likely that the binding site of HH26 is rendered rigid by a network of intramolecular salt bridges whereas that of HH8 is flexible due to their absence. HH26 also has the most intermolecular contacts with the antigen whereas HH8 has the least. HH10 has these properties intermediate to HH8 and HH26. The structurally rigid binding site with numerous specific contacts bestows specificity on HH26 whereas the flexible binding site with correspondingly fewer contacts enables HH8 to be cross-reactive. Results suggest that affinity maturation may select for high affinity antibodies with either "lock-and-key" preconfigured binding sites, or "preconfigured flexibility" by modulating combining site flexibility.     

Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure

Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment.     

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.     

Enhanced protection by melatonin and meloxicam combination in a middle cerebral artery occlusion model of acute ischemic stroke in rat

Mixed efficacy of neuroprotective drugs in clinical trials has led to the emergence of the approach of combination therapy in stroke. The present study was carried out to investigate the effect of the combination of melatonin (potent antioxidant) and meloxicam (preferential inhibitor of cyclooxygenase-2 enzyme) against a middle cerebral artery occlusion model of stroke in rats. Male Wistar rats in the weight range of 250-300 g were used. Rats were anesthetized using chloral hydrate (400 mg/kg i.p) and subjected to 2 h of transient middle cerebral artery occlusion. Melatonin was administered at a dose of 20 mg/kg i.p. four times: at the time of middle cerebral artery occlusion, 1.5 h after middle cerebral artery occlusion, at the time of reperfusion, and 1 h after reperfusion. Meloxicam (2.5 mg/kg) was administered 4 h after middle cerebral artery occlusion. Motor performance tests (grip test, foot fault test, rotarod performance test, spontaneous locomotor activity), markers of oxidative stress, and triphenyltetrazolium chloride staining were carried out 24 h after middle cerebral artery occlusion. A vehicle-treated group was run in parallel. It was observed that melatonin treatment improved the motor performance and significantly attenuated the levels of malondialdehyde (MDA) as compared with the middle cerebral artery occluded group. Meloxicam treatment at the dose used neither showed significant improvement on the motor performance nor decreased the levels of MDA significantly as compared with the middle cerebral artery occluded group. However, when the combination of the two drugs was used, better protection was observed as was evident by the significant decrease in the percent foot fault errors, the increase in the time spent on the rotarod, and the increase in the six-point neurological score and grip test score. There was also a significant decrease in the levels of MDA in the combination group. The results of the present study demonstrate that enhanced protection is observed with the use of a combination of melatonin plus meloxicam in the middle cerebral artery occlusion model of acute ischemic stroke in rats.     

The tRNA-Tyr gene family of Saccharomyces cerevisiae: agents of phenotypic variation and position effects on mutation frequency

Extensive phenotypic diversity or variation exists in clonal populations of microorganisms and is thought to play a role in adaptation to novel environments. This phenotypic variation or instability, which occurs by multiple mechanisms, may be a form of cellular differentiation and a stochastic means for modulating gene expression. This work dissects a case of phenotypic variation in a clinically derived Saccharomyces cerevisiae strain involving a cox15 ochre mutation, which acts as a reporter. The ochre mutation reverts to sense at a low frequency while tRNA-Tyr ochre suppressors (SUP-o) arise at a very high frequency to produce this phenotypic variation. The SUP-o mutations are highly pleiotropic. In addition, although all SUP-o mutations within the eight-member tRNA-Tyr gene family suppress the ochre mutation reporter, there are considerable phenotypic differences among the different SUP-o mutants. Finally, and of particular interest, there is a strong position effect on mutation frequency within the eight-member tRNA-Tyr gene family, with one locus, SUP6, mutating at a much higher than average frequency and two other loci, SUP2 and SUP8, mutating at much lower than average frequencies. Mechanisms for the position effect on mutation frequency are evaluated.