Activation of PMCA by calmodulin or ethanol in plasma membrane vesicles from rat brain involves dissociation of the acetylated tubulin/PMCA complex

We have recently shown that acetylated tubulin interacts with plasma membrane Na(+),K(+)-ATPase and inhibits its enzyme activity in several types of cells. H(+)-ATPase of Saccharomyces cerevisiae is similarly inhibited by interaction with acetylated tubulin. The activities of both these ATPases are restored upon dissociation of the acetylated tubulin/ATPase complex. Here, we report that in plasma membrane vesicles isolated from brain synaptosomes, another P-type ATPase, plasma membrane Ca(2+)-ATPase (PMCA), undergoes enzyme activity regulation by its association/dissociation with acetylated tubulin. The presence of acetylated tubulin/PMCA complex in membrane vesicles was demonstrated by analyzing the behavior of acetylated tubulin in a detergent partition, and by immunoprecipitation experiments. PMCA is known to be stimulated by ethanol and calmodulin at physiological concentrations. We found that treatment of plasma membrane vesicles with these reagents induced dissociation of the complex, with a concomitant restoration of enzyme activity. Conversely, incubation of vesicles with exogenous tubulin induced the association of acetylated tubulin with PMCA, and the inhibition of enzyme activity. These findings indicate that activation of synaptosomal PMCA by ethanol and calmodulin involves dissociation of the acetylated tubulin/PMCA complex. This regulatory mechanism was shown to also operate in living cells.     

A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4

The human CD45 gene encodes a protein–tyrosine phosphatase that exhibits differential isoform expression in resting and activated T cells due to alternative splicing of three variable exons. Previously, we have used biochemical methods to identify two regulatory proteins, hnRNP L and PSF, which contribute to the activation-induced skipping of CD45 via the ESS1 regulatory element in variable exon 4. Here we report the identification of a third CD45 regulatory factor, hnRNP L-like (hnRNP LL), via a cell-based screen for clonal variants that exhibit an activation-like phenotype of CD45 splicing even under resting conditions. Microarray analysis of two splicing-altered clones revealed increased expression of hnRNP LL relative to wild-type cells. We further demonstrate that both the expression of hnRNP LL protein and its binding to ESS1 are up-regulated in wild-type cells upon activation. Forced overexpression of hnRNP LL in wild-type cells results in an increase in exon repression, while knock-down of hnRNP LL eliminates activation-induced exon skipping. Interestingly, analysis of the binding of hnRNP L and hnRNP LL to mutants of ESS1 reveals that these proteins have overlapping, but distinct binding requirements. Together, these data establish that hnRNP LL plays a critical and unique role in the signal-induced regulation of CD45 and demonstrate the utility of cell-based screens for the identification of novel splicing regulatory factors.     

Endogenous morphine in SH-SY5Y cells and the mouse cerebellum

Background

Morphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system.

Methodology and Principal Findings

Microscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca²⁺-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10⁻¹⁰ M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies.

Conclusions/Significance

The presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.     

Genomic diversity within the Enterobacter cloacae complex

Background

Isolates of the Enterobacter cloacae complex have been increasingly isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is unreliable and irreproducible. Identification of species based on currently available genotyping tools is already superior to phenotypic identification, but the taxonomy of isolates belonging to this complex is cumbersome.

Methodology/Principal Findings

This study shows that multilocus sequence analysis and comparative genomic hybridization based on a mixed genome array is a powerful method for studying species assignment within the E. cloacae complex. The E. cloacae complex is shown to be evolutionarily divided into two clades that are genetically distinct from each other. The younger first clade is genetically more homogenous, contains the Enterobacter hormaechei species and is the most frequently cultured Enterobacter species in hospitals. The second and older clade consists of several (sub)species that are genetically more heterogonous. Genetic markers were identified that could discriminate between the two clades and cluster 1.

Conclusions/Significance

Based on genomic differences it is concluded that some previously defined (clonal and heterogenic) (sub)species of the E. cloacae complex have to be redefined because of disagreements with known or proposed nomenclature. However, further improved identification of the redefined species will be possible based on novel markers presented here.     

Outer membrane modifications of Pseudomonas fluorescens MF37 in response to hyperosmolarity

The effect of hyperosmotic condition on the outer membrane protein (Omp) composition of Pseudomonas fluorescens was investigated by proteomic analyses. The abundances of 12 proteins, including porins, lipoproteins, and the flagella subunit FliC, were modified. This was at least partly explained by altered gene expression, as shown by mRNA level study. In agreement with Omp changes, hyperosmotic condition resulted in vesicle formation and modifications of mobility and antibiotic susceptibility.     

Quantitative analysis of snake venoms using soluble polymer-based isotope labeling

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-containing peptides, and the subsequent tagged peptides are released and analyzed using nanoflow liquid chromatography-tandem mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The hemorrhagic, hemolytic, clotting ability, and fibrinogenolytic activities of crude venoms were measured and correlated with difference in protein abundance determined by the SoPIL analysis. The SoPIL approach could provide an efficient and widely applicable tool for quantitative proteomics.     

Seeing tissue as a 'phase of matter': exploring statistical mechanics for the cell

The field of tissue engineering aims to produce living, biological constructs which possess the appropriate spatial ordering of cells and their extra cellular matrix products. The complexity of a single cell and its interactions in a large collective have made development of useful models to assist in tissue culture difficult, and consequentially most tissue culture endeavors are limited to trial and error approaches. Some cell types display a natural tendency to spontaneously self-assemble into large domains of parallel-oriented cells. In this work, we show that these cell culture systems can be studied in the context of continuous disorder-order phase transformations. We suggest that collective ordering of the cells is controlled by the amount of noise in the walk of the individual cells (directional persistence) because undifferentiated mesenchymal stem cells display a seven-times higher directional persistence than mature fibroblasts and have a 24-times larger final-oriented domain size, an observation that corresponds with collective ordering in self-propelled particle systems. The study of cell culture systems using analogies derived from statistical mechanics yields simple, practical models offering insight into how a long-range order can be obtained in tissue-engineered constructs, providing a new paradigm for managing operations with large collectives of living cells.     

Diversity,origins and virulence of <Emphasis Type="Italic">Avipoxviruses</Emphasis> in Hawaiian Forest Birds

We cultured avian pox (Avipoxvirus spp.) from lesions collected on Hawai‘i, Maui, Moloka‘i, and ‘Oahu in the Hawaiian Islands from 15 native or non-native birds representing three avian orders. Phylogenetic analysis of a 538 bp fragment of the gene encoding the virus 4b core polypeptide revealed two distinct variant clusters, with sequences from chickens (fowlpox) forming a third distinct basal cluster. Pox isolates from one of these two clusters appear closely related to canarypox and other passerine pox viruses, while the second appears more specific to Hawai‘i. There was no evidence that birds were infected simultaneously with multiple pox virus variants based on evaluation of multiples clones from four individuals. No obvious temporal or geographic associations were observed and strict host specificity was not apparent among the 4b-defined field isolates. We amplified a 116 bp 4b core protein gene fragment from an ‘Elepaio (Chasiempis sandwichensis) collected in 1900 on Hawai‘i Island that clustered closely with the second of the two variants, suggesting that this variant has been in Hawai‘i for at least 100 years. The high variation detected between the three 4b clusters provides evidence for multiple, likely independent introductions, and does not support the hypothesis of infection of native species through introduction of infected fowl. Preliminary experimental infections in native Hawai‘i ‘Amakihi (Hemignathus virens) suggest that the 4b-defined variants may be biologically distinct, with one variant appearing more virulent. These pox viruses may interact with avian malaria (Plasmodium relictum), another introduced pathogen in Hawaiian forest bird populations, through modulation of host immune responses.