Nmag_2608, an extracellular ubiquitin-like domain-containing protein from the haloalkaliphilic archaeon Natrialba magadii.

Ubiquitin-like proteins (Ubls) and ubiquitin-like domain-containing proteins (Ulds) found in both eukaryotes and prokaryotes display an ubiquitin fold. We previously characterized a 124-amino acid polypeptide (P400) from the haloalkaliphilic archaeon Natrialba magadii having structural homology with ubiquitin family proteins. The reported N. magadii's genome allowed the identification of the Nmag_2608 gene for the protein containing P400, which belongs to specific orthologs of halophilic organisms. It was found that Nmag_2608 has an N-terminal signal peptide with a lipobox motif characteristic of bacterial lipoproteins. Also, it presents partial identity with the ubiquitin-like domain-containing proteins, soluble ligand binding ß-grasp proteins. Western blots and heterologous expression tests in E. coli evidenced that Nmag_2608 is processed and secreted outside the cell, where it could perform its function. The analysis of Nmag_2608 expression in N. magadii's cells suggests a co-transcription with the adjoining Nmag_2609 gene encoding a protein of the cyclase family. Also, the transcript level decreased in cells grown in low salinity and starved. To conclude, this work reports for the first time an extracellular archaeal protein with an ubiquitin-like domain.

Oxidative stress in mouse liver caused by dietary amino acid deprivation: protective effect of methionine.

The aim of this work was to evaluate the effects of a diet depleted of amino acids (protein-free diet, or PFD), as well as the supplementation with methionine (PFD+Met), on the antioxidant status of the female mouse liver. With this purpose, cytosolic protein spots from two-dimensional non-equilibrium pH gel electrophoresis were identified by several procedures, such as mass spectrometry, Western blot, gel matching and enzymatic activity. PFD decreased the contents of catalase (CAT), peroxiredoxin I (Prx-I), and glutathione peroxidase (GPx) by 67%, 37% and 45%, respectively. Gene expression analyses showed that PFD caused a decrease in CAT (-20%) and GPx (-30%) mRNA levels but did not change that of Prx-I. It was also found that, when compared to a normal diet, PFD increased the liver contents of both reactive oxygen species (+50%) and oxidized protein (+88%) and decreased that of glutathione (-45%). Supplementation of PFD with Met prevented these latter effects to varying degrees, whereas CAT, Prx-I and GPx mRNA levels resulted unmodified. Present results suggest that dietary amino acid deprivation deranges the liver antioxidant defences, and this can be, in part, overcome by supplementation with Met.

Presence of structural homologs of ubiquitin in haloalkaliphilic Archaea.

Ubiquitin, a protein widely conserved in eukaryotes, is involved in many cellular processes, including proteolysis. While sequences encoding ubiquitin-like proteins have not been identified in prokaryotic genomes sequenced so far, they have revealed the presence of structural and functional homologs of ubiquitin in Bacteria and Archaea. This work describes the amplification and proteomic analysis of a 400-bp DNA fragment from the haloalkaliphilic archaeon Natrialba magadii. The encoded polypeptide, P400, displayed structural homology to ubiquitin-like proteins such as those of the ThiS family and Urm1. Expression of the P400 DNA sequence in Escherichia coli cells yielded a recombinant polypeptide that reacted with anti-ubiquitin antibodies. In addition, a putative open reading frame encoding P400 was identified in the recently sequenced genome of N. magadii. Together, these results evidence the presence in Archaea of structural homologs of ubiquitin- related proteins.

[Brain activity associated with a nouns-reading task]. / Activación cerebral asociada a una tarea de lectura de sustantivos.

BACKGROUND: Word reading involves several steps, from the visual perception of each of its constitutent elements to its recognition as an entity with a specific meaning. Various brain structures participate in these processes, depending of the linguistic and cognitive characteristics of the stimulus. Our objective was to characterize brain activity through the use of functional magnetic resonance imaging (FMRI) associated with the process of noun reading. METHODS: Eleven healthy right-handed volunteers participated in a lexical decision task involving 58 written nouns. An equal number of letter sequences were used as control stimuli. Reaction times were also recorded. RESULTS: There was a difference (p < 0.05) in reaction time between nouns and letter sequences in the lexical decision task. FMRI contrasted between conditions revealed significant activations in several areas involved in reading. CONCLUSIONS: The brain activation may reflect the different perceptual demands associated with the initial processing of nouns, as compared to meaningless letter sequences. We attribute the difference between our results and those previously reported to the particular characteristics of the pronunciation rules of written Spanish.

Control of ribosome turnover during growth of the haloalkaliphilic archaeon Natronococcus occultus.

The metabolism of ribosomes during growth of the haloalkaliphilic archaeon Natronococcus occultus was examined. The ribosome content was higher during exponential growth and diminished to 35% of the maximum in the stationary stage. The incorporation of H3-orotic acid and C14-uracil into rRNA was higher during exponential growth. After that, it decreased to 39% of the maximum in the stationary stage. The labeling of non-ribosomal RNA took place almost exclusively in the exponential stage. From loss of radioactivity, the half-life of rRNA was 11.43, 14.85, 5.28 and 7.14 h during the initial, exponential, late exponential and stationary growth stages, respectively. These results suggested that increased synthesis combined with diminished degradation were responsible for the high ribosome content displayed by Ncc. occultus during exponential growth. In contrast, diminished synthesis together with increased degradation provoked its posterior loss.

Liver damage and caspase-dependent apoptosis is related to protein malnutrition in mice: effect of methionine.

This study aimed to determine whether the effects on the mouse liver caused by three periods of feeding a protein-free diet for 5 days followed by a normal complete diet for 5 days (3PFD-CD) are prevented by a constant methionine supply (3PFD+Met-CD). The expressions of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) were assessed by proteomics and reverse transcriptase-polymerase chain reactions. The liver redox status was examined by measuring the activities of superoxide dismutase (SOD) and catalase (CAT), as well as protein carbonylation. Because oxidative stress can result in apoptosis, the activity and content of caspase-3, as well as the x-linked inhibitor of the apoptosis protein (XIAP) and mitochondrial caspase-independent apoptosis inducing factor (AIF) contents were assessed. In addition, the liver histomorphology was examined. Compared to the controls fed a normal complete diet throughout, feeding with 3PFD-CD increased the FAS content, decreased the CAIII content, decreased both the SOD and CAT activities, and increased protein carbonylation. It also activated caspase-3, decreased the XIAP content, decreased the AIF content, increased the number of GSTP1-positive foci and caspase-3-positive cells, and caused fatty livers. Conversely, the changes were lessened to varying degrees in mice fed 3PFD+Met-CD. The present results indicate that a regular Met supply lessens the biochemical changes, damage, and caspase-dependent apoptosis provoked by recurrent dietary amino acid deprivation in the mouse liver.

The mouse liver content of carbonic anhydrase III and glutathione S-tranferases A3 and P1 depend on dietary supply of methionine and cysteine.

The contents of glutathione S-transferase (GST) subunits, carbonic anhydrase III (CAIII), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a 230 kDa protein are affected by protein deprivation in mouse liver. In order to know if particular amino acids control these contents, the effects of feeding for 5 days with diets containing different amino acids were examined. After an exploration using SDS-PAGE analysis, the action of selected diets was further examined by distinct techniques. The 230 kDa protein was identified as fatty acid synthase (FAS) by both mass spectrometry and amino acid sequence analyses. Dietary tests showed that: (1) a protein-free diet (PFD) increased the content of glutathione S-transferases P1 and M1, and glyceraldehyde-3-phosphate dehydrogenase, while the content of glutathione S-transferase A3, fatty acid synthase and carbonic anhydrase III decreased; (2) a protein-free diet having either methionine or cysteine preserved the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anydrase III; (3) a protein-free diet having threonine preserved partially the normal contents of glutathione S-transferases P1, A3, M1 and carbonic anhydrase III; (4) a protein-free diet having methionine, threonine and cysteine prevented in part the loss of fatty acid synthase; and (5) the glyceraldehyde-3-phosphate dehydrogenase content was controlled by increased carbohydrate level and/or by lower amino acid content of diets, but not by any specific amino acid. These data indicate that methionine and cysteine exert a main role on the control of liver glutathione S-transferases A3 and P1, and carbonic anhydrase III. Thus, they emerge necessary to prevent unsafe alterations of liver metabolism caused by protein deprivation.

Enhanced emotional reactivity after selective REM sleep deprivation in humans: an fMRI study.

Converging evidence from animal and human studies suggest that rapid eye movement (REM) sleep modulates emotional processing. The aim of the present study was to explore the effects of selective REM sleep deprivation (REM-D) on emotional responses to threatening visual stimuli and their brain correlates using functional magnetic resonance imaging (fMRI). Twenty healthy subjects were randomly assigned to two groups: selective REM-D, by awakening them at each REM sleep onset, or non-rapid eye movement sleep interruptions (NREM-I) as control for potential non-specific effects of awakenings and lack of sleep. In a within-subject design, a visual emotional reactivity task was performed in the scanner before and 24 h after sleep manipulation. Behaviorally, emotional reactivity was enhanced relative to baseline (BL) in the REM deprived group only. In terms of fMRI signal, there was, as expected, an overall decrease in activity in the NREM-I group when subjects performed the task the second time, particularly in regions involved in emotional processing, such as occipital and temporal areas, as well as in the ventrolateral prefrontal cortex, involved in top-down emotion regulation. In contrast, activity in these areas remained the same level or even increased in the REM-D group, compared to their BL level. Taken together, these results suggest that lack of REM sleep in humans is associated with enhanced emotional reactivity, both at behavioral and neural levels, and thus highlight the specific role of REM sleep in regulating the neural substrates for emotional responsiveness.

Alternation between dietary protein depletion and normal feeding cause liver damage in mouse.

The effect of frequent protein malnutrition on liver function has not been intensively examined. Thus, the effects of alternating 5 days of a protein and amino acid-free diet followed by 5 days of a complete diet repeated three times (3 PFD-CD) on female mouse liver were examined. The expression of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) in liver were assessed by proteomics, reverse transcriptase-polymerase chain reaction and Northern blotting. The activities of liver GSTs, glutathione reductase (GR) and catalase (CAT), as well as serum glutamic-oxaloacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT) were also tested. Additionally, oxidative damage was examined by measuring of protein carbonylation and lipid peroxidation. Liver histology was examined by light and electron microscopy. Compared with control mice, 3 PFD-CD increased the content of FAS protein (+90%) and FAS mRNA (+30%), while the levels of CAIII and CAIII mRNAs were decreased (-48% and -64%, respectively). In addition, 3 PFD-CD did not significantly change the content of GSTP1 but produced an increase in its mRNA level (+20%), while it decreased the activities of both CAT (-66%) and GSTs (-26%). After 3 PFD-CD, liver protein carbonylation and lipid peroxidation were increased by +55% and +95%, respectively. In serum, 3 PFD-CD increased the activities of both SGOT (+30%) and SGPT (+61%). In addition, 3 PFD-CD showed a histological pattern characteristic of hepatic damage. All together, these data suggest that frequent dietary amino acid deprivation causes hepatic metabolic and ultrastructural changes in a fashion similar to precancerous or cancerous conditions.

A germin-like protein of wheat leaf apoplast inhibits serine proteases.

A protein resistant to heat and proteolysis that inhibits serine proteases was isolated from wheat leaf apoplasts. Based on trypsin inhibition, its more active form was a 66-69 kDa oligomer. It was dissociated in an 18-21 kDa monomer having an amino terminal sequence identical to the Box A of germins and germin-like proteins. Like these proteins, it was glycosylated and showed manganese superoxide dismutase activity. The monomer displayed three forms when examined by 2D western blot: two of 19 kDa, pI 5.8 and 6.2; and one of 21 kDa, pI 5.8. It was found that the protein controls serine protease activity in the apoplast of plants challenged with the fungus Septoria tritici.

Cerebral Anatomy of the Spider Monkey Ateles Geoffroyi Studied Using Magnetic Resonance Imaging. First Report: a Comparative Study with the Human Brain Homo Sapiens / Anatomía Cerebral del mono araña Ateles geoffroyi estudiada utilizando imágenes de resonancia magnética. Primer reporte: estudio comparativo con el cerebro humano Homo Sapiens

The objective of the present qualitative study was to analyze the morphological aspects of the inner cerebral anatomy of two species of primates, using magnetic resonance images (MRI): spider monkey (A. geoffroyi) and human (H. sapiens), on the basis of a comparative study of the cerebral structures of the two species, focusing upon the brain of the spider monkey and, primarily, its limbic system. In spite of being an endemic Western hemisphere species, a fact which is by its own right interesting for research due to this animal’s social organization and motor functions, the spider monkey (A. geoffroyi) has hardly been studied in regard to its neuroanatomy. MRI was carried out, in one spider monkey, employing a General Electric Signa 1.5 T scanner. This investigation was carried in accordance to international regulations for the protection of animals in captivity, taking into account all protective means utilized in experimental handling, and not leaving behind any residual effects, either physiological or behavioral. From a qualitative point of view, the brains of the spider monkey and the human were found to have similar structures. In reference to shape, the most similar structures were found in the limbic system; proportionally, however, cervi cal curvature, amygdala, hippocampus, anterior commissure and the colliculi, were larger in the spider monkey than in the human.

El objetivo del presente estudio cualitativo fue analizar los aspectos morfológicos de la anatomía cerebral interna utilizando imágenes de resonancia magnética (IRM) en dos especies de primates, El mono Araña (A. geoffroyi) y el humano (H. sapiens), tomando como base un estudio comparativo de las estructuras cerebrales de las dos especies, concentrándose primordialmente en el sistema límbico del cerebro del mono araña. Aunque es una especie común en el hemisferio occidental, es interesante para estudiar dada su organización social y funciones motoras, el mono araña (A. geoffroyi) ha sido poco estudiado en cuanto a su neuroanatomía. Las IRM fueron hechas a un mono araña utilizando un resonador General Electrics Signa 1.5 T. Esta investigación se llevo a cabo conforme a las leyes internacionales para la protección de animales en cautiverio y teniendo en cuenta todas las medidas de protección para el manejo experimental para evitar cualquier efecto residual de índole comportamental o fisiológico. Desde un punto de vista cualitativo, los cerebros del mono araña y el humano tenían estructuras similares. Con respecto a la forma, las estructuras más parecidas fueron encontradas en el sistema límbico, sin embargo la curvatura cervical, la amígdala, el hipocampo, la comisura anterior y el colículo fueron más grandes proporcionalmente en el mono araña que en el humano.

Proteolytic activities in kidney and liver during refeeding of protein depleted mice

Los contenidos renal y hepático de proteinas disminuyen significativamente en ratones sometidos a una dieta aproteica durante cinco días. La realimentación con una dieta completa induce una rápida recuperación de la masa proteica perdida por ambos tejidos. Esta recuperación es consecuencia de una marcada inhibición de la proteólisis intracelular. El objetivo de este trabajo fue estudiar la contribución de los sistemas proteolíticos lisosomal o ácido y neutro al proceso de recuperación aludido. Para ello se evaluaron las actividades proteolíticas a pH 5.0 y pH 7.4 presentes en los tejidos homogeneizados. La actividad ácida disminuyó en ambos tejidos como consecuencia de la desnutrición proteica y se recuperó luego de 12 horas de realimentación. Sin embargo, las actividades neutras de ambos organos disminuyeron debido a la desnutrición y permanecieron en niveles bajos luego de 12 horas de realimentación. Se estudió, además, el efecto de las dietas sobre la estabilidad osmótica de los lisosomas hepáticos y renales. Esta aumentó durante la realimentación, indicando que se produce una disminución en la actividad autofágica de dichos tejidos. Estos hallazgos indican que tanto la baja actividad del sistema lisosomal vacuolar como la baja actividad del sistema proteolítico neutro serían responsables de la inhibición de la proteólisis in vivo exhibida por los riñones e higados durante la recuperación de su masa proteica