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.

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

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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 (AU)

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.

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.

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

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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%) mRNAlevels 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 (AU)

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.

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.

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

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 (AU)