Functional response of the small multidrug resistance protein EmrE to mutations in transmembrane helix 2

Escherichia coli EmrE is a small multidrug resistance protein encompassing four transmembrane (TM) sequences that oligomerizes to confer resistance to antimicrobials. Here we examined the effects on in vivo protein accumulation and ethidium resistance activity of single residue substitutions at conserved and variable positions in EmrE transmembrane segment 2 (TM2). We found that activity was reduced when conserved residues localized to one TM2 surface were replaced. Our findings suggest that conserved TM2 positions tolerate greater residue diversity than conserved sites in other EmrE TM sequences, potentially reflecting a source of substrate polyspecificity.     

A new species of Phyllotis (Rodentia,Cricetidae, Sigmodontinae) from Tucumán province,Argentina

A new species of rodent of the genus Phyllotis is described based in cranial and external morphology, as well as morphometric data. Additionally, sequences of the mitochondrial gene cytochrome-b were used to asses the phylogenetic relationships. We have compared our specimens with all the extant species of the genus Phyllotis and also with some species of related genera, particularly with the most similar and with those that occur in the province of Tucumán and northwestern Argentina. The new species is large compared to the average size of the genus, and can be easily distinguished from all other species essentially by coloration and by cranial morphology. It is closely related to the recently described P. anitae, and these two species are, in turn, sister to P. osilae. The only two localities where the new species has been found are in the Upper Montane Forests of the southern portion of the Yungas Ecoregion, in the province of Tucumán, Argentina.     

Catalytic properties of thimet oligopeptidase H600A mutant

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His⁶⁰⁰. In the present work, the role of His⁶⁰⁰ of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S₁ and S₁′ specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His⁶⁰⁰ residue makes important interactions with the substrate, supporting the prediction that His⁶⁰⁰ moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K_m and k_cat, showing the importance of His⁶⁰⁰ for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His⁶⁰⁰ in TOP catalysis, transferring a proton to the newly generated NH₂-terminus or helping Tyr⁶⁰⁵ and/or Tyr⁶¹² in the intermediate oxyanion stabilization.     

Unrevealed structural requirements for auxin-like molecules by theoretical and experimental evidences

An computational-biostatistical approach, supported by ab initio optimizations of auxin-like molecules, was used to find biologically meaningful relationships between quantum chemical variables and fresh bioassay's data. It is proven that the auxin-like recognition requires different molecular assembling states. We suggest that the carboxyl group is not the determining factor in explaining the biological auxin-like conduct. The biological effects depends essentially on the chemical condition of the ring system. The aim to find active molecules (quantum objects) via statistical grouping-analysis of molecular quantum similarity measures was verified by bioactivity assays. Next, this approach led to the discovery of a non-carboxylated active auxin-like molecule (2,6-dibromo-phenol). This is the first publication on structure activity relationship of auxin-like molecules, which relies on highly standardized bioassays of different auxins screened in parallel as well as analysed by multi-dimensional scaling.     

Reorganization of prion protein membrane environment during low potassium-induced apoptosis in primary rat cerebellar neurons

We studied the changes occurring in the membrane environment of prion protein (PrP) during apoptosis induced by low potassium in primary rat cerebellar neurons. Ceramide levels increased during apoptosis-inducing treatment, being doubled with respect to time-matched controls after 24 h. Sphingomyelin levels were parallely decreased, while cholesterol and ganglioside contents were not affected. Changes in ceramide and sphingomyelin composition were exclusively restricted to a detergent-resistant membrane fraction. The pro-apoptotic treatment was accompanied by the down-regulation of PrP and of the non-receptor kinase Fyn. The levels of PrP and Fyn were correspondingly reduced in the detergent-resistant membrane fraction. In control cells, the membrane microenvironment separated by immunoprecipitation with anti-PrP antibody contained 80% of the detergent-resistant PrP and 35% and 38% of the sphingolipids and cholesterol respectively. Upon low potassium treatment, 20% of the PrP originally present in the detergent-resistant fraction was immunoprecipitated, together with 19% of sphingolipids and 22% of cholesterol. Thus, PrP in the immunoprecipitate from apoptotic cells was ninefold less than in control ones, while sphingolipids and cholesterol were about 50% with respect to controls cells. The molar ratio between cholesterol, sphingomyelin and ceramide was 15 : 6 : 1 in the PrP-rich environment from control neurons, and 6 : 2 : 1 in that from apoptotic cells.     

Identification of differentially expressed microRNAs by microarray: a possible role for microRNA genes in pituitary adenomas

MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by targeting mRNA. It has been demonstrated that miRNA expression is altered in many human cancers, suggesting that they may play a role in human neoplasia. To determine whether miRNA expression is altered in pituitary adenomas, we analyzed the entire miRNAome in 32 pituitary adenomas and in 6 normal pituitary samples by microarray and by Real-Time PCR. Here, we show that 30 miRNAs are differentially expressed between normal pituitary and pituitary adenomas. Moreover, 24 miRNAs were identified as a predictive signature of pituitary adenoma and 29 miRNAs were able to predict pituitary adenoma histotype. miRNA expression could differentiate micro- from macro-adenomas and treated from non-treated patient samples. Several of the identified miRNAs are involved in cell proliferation and apoptosis, suggesting that their deregulated expression may be involved in pituitary tumorigenesis. Predictive miRNAs could be potentially useful diagnostic markers, improving the classification of pituitary adenomas.     

Serotonin involvement in the metamorphosis of the hydroid Eudendrium racemosum

Hydroid planulae metamorphose in response to an inducing external stimulus, usually a bacterial cue. There is evidence that neurotransmitters participate in the signal transduction pathway of hydroid metamorphosis. Eudendrium racemosum is a colonial hydroid common in the Mediterranean Sea. It lacks the medusa stage and the planulae develop on female colonies during the fertile season. In this work, serotonin (5-HT) was localized in some planula ectodermal cells. Co-localization of serotonin and beta-tubulin suggested that 5-HT was present in sensory nervous cells and in different ectodermal cells. To investigate the role of neurotransmitters in metamorphosis, E. racemosum planulae were treated with serotonin and dopamine and with agonists and antagonists of the corresponding receptors. Serotonin and a serotonin receptor agonist induced metamorphosis, while a 5-HT receptor antagonist inhibited it. Dopamine and all dopaminergic drugs used did not show any significant effect on the onset of metamorphosis. Results from this work showed that 5-HT could stimulate metamorphosis in E. racemosum planulae in the presence of a natural inducer. A mechanism by which this neurotransmitter could act in this phase is proposed.     

The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.