Cytosolic Protein Fractions Research Articles

Spermidine and abscisic acid-mediated phosphorylation of a cytoplasmic protein from rice root in response to salinity stress

Importance of higher polyamines, spermidine, and spermine, in relation to the mechanism and adaptation to combat abiotic stress has been well established in cereals. Owing to their polycationic nature at physiological pH, polyamines bind strongly to negative charges in cellular components such as nucleic acids, various proteins, and phospholipids. To study the physiological role of polyamine during salinity stress, phosphorylation study was carried out in cytosolic soluble protein fraction isolated from the roots of salt tolerant (Nonabokra) and salt sensitive (M-1-48) rice cultivars treated with none (control), NaCl (150 mM, 16 h), spermidine (1 mM, 16 h) or with abscisic acid (100 μM, 16 h). A calcium independent auto regulatory 42 kDa protein kinase was found to phosphorylate myelin basic protein and casein but not histone. Interestingly, this was the only protein to be phosphorylated in root cytosolic fraction during NaCl/abscisic acid/spermidine treatment indicating its importance in salinity mediated signal transduction. This is the first report of polyamine as well as abscisic acid induced protein kinase activity in rice root in response to salinity stress.

Analysis of theArabidopsisCytosolic Proteome Highlights Subcellular Partitioning of Central Plant Metabolism

The plant cell cytosol is a dynamic and complex intracellular matrix that, by definition, contains no compartmentalization. Nonetheless, it maintains a wide variety of biochemical networks and often links metabolic pathways across multiple organelles. There have been numerous detailed proteomic studies of organelles in the model plant Arabidopsis thaliana, although no such analysis has been undertaken on the cytosol. The cytosolic protein fraction from cell suspensions of Arabidopsis thaliana was isolated and analyzed using offline strong cation exchange liquid chromatography and LC-MS/MS. This generated a robust set of 1071 cytosolic proteins. Functional annotation of this set revealed major activities in protein synthesis and degradation, RNA metabolism and basic sugar metabolism. This included an array of important cytosol-related functions, specifically the ribosome, the set of tRNA catabolic enzymes, the ubiquitin-proteasome pathway, glycolysis and associated sugar metabolism pathways, phenylpropanoid biosynthesis, vitamin metabolism, nucleotide metabolism, an array of signaling and stress-responsive molecules, and NDP-sugar biosynthesis. This set of cytosolic proteins provides for the first time an extensive analysis of enzymes responsible for the myriad of reactions in the Arabidopsis cytosol and defines an experimental set of plant protein sequences that are not targeted to subcellular locations following translation and folding in the cytosol.

Manganese Bioconcentration in Aquatic Insects: Mn Oxide Coatings, Molting Loss, and Mn(II) Thiol Scavenging

Streams below mountaintop removal-valley fill coal mining operations often have elevated Mn concentrations, but it remains unclear if Mn plays a role in biodiversity reduction. We examined various aspects of aqueous Mn interactions with aquatic insects exposed to environmentally relevant Mn concentrations, revealing complex behavior. First, Mn accumulation rates varied widely among 9 species. A significant percentage of total Mn accrued (mean 74%, range 24-95%) was associated with the cuticle, predominantly in the form of Mn-oxides, and to a lesser degree Mn(II). Mn II is also absorbed into tissues, possibly through calcium transporters. Increased ambient calcium concentrations decreased both adsorbed and absorbed Mn accumulation from solution. Though species showed similar Mn efflux rate constants (0.032-0.072 d(-1)), the primary mode of Mn loss was through molting. Both adsorbed and absorbed Mn is lost during the molt. Subcellular compartmentalization studies revealed an overwhelming tendency for internalized Mn to associate with the heat stable cytosolic protein fraction. After short dissolved Mn exposures, intracellular glutathione and cysteine levels were markedly reduced relative to controls. These findings suggest that Mn exposure results in transient physiological stress in aquatic insects which is likely relieved, in part, during the molting process.

Identification and characterization of dehydrins in horse chestnut recalcitrant seeds

The fraction of heat-stable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stress-induced dehydrin-like polypeptides. In our experiments, in tissues of dormant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Low-molecular-weight heat-stable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not cross-react with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissue-specificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heat-stable proteins during the entire period of stratification and also radicle emersion. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cycloheximide, or α-amanitin, dehydrins did not disappeared from the fraction of heat-stable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could cross-react with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50-kD heat-stable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heat-sensitive proteins and was named as a dehydrin-like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heat-stable cytosolic proteins. The role and function of major heat-stable proteins in horse chestnut seeds are yet to be studied.

Abstract A115: Mitochondrial analysis and epithelial ovarian carcinoma

Abstract Survival rates of ovarian cancer patients are low, largely due to late diagnosis. Early detection plays a major role towards improving outcomes in ovarian cancer, however few candidate biomarkers have shown efficacy for the detection of preclinical disease. Human mitochondrial gene mutations have increasingly been associated with various cancers. The mitochondrial genome is highly susceptible to oxidative damage and may accumulate damage over a long period of time. Consequently, variations in (mtDNA) may potentially play a role as a modifying risk factor in the development of age-related diseases such as ovarian cancer. The goal of this study is to examine correlations between mitochondrial gene alterations and three subtypes of ovarian tumor progression. Mitochondrial DNA fragments were obtained from 120 epithelial ovarian cancer tissue samples (40 serous; 40 endometrioid and 40 mucinous ovarian tumors respectively). The entire mitochondrial genome was PCR amplified using 9 overlapping primers sets and analyzed using both high resolution restriction digest and PCR-based sequencing techniques. Additionally, the levels of carbonyl proteins in cytosolic fraction of tissues were assessed by standard colorimetric technique, using 2-4-dinitrophenylhydrazine (DNPH) to derivatize protein, and measure the absorbance of the DNP-hydrazones at 370 nm. In this study we revealed the presence thirty-nine polymorphisms of which 28 were unreported. The observed mutations with notably frequencies (41-93%) among these samples were at np C7028T, C7256T, G7520A, T8548G, T8588C, A8860G, C9488G, C9500T, T9540C, C9857T, and T9951C. Furthermore, six unreported point mutations with frequencies of 14-41 % were observed at np G7520A, T8548G, C9488G, C9500T, C9857 and T9951C. A combined mutation of G7520A and C7256T was frequent at 45% in endometrioid stage III only. Interestingly, variants C7020T (56%) and at np A8860G (92%) were evenly distributed in all three studied ovarian tumor subtype and stages. Variants T9540C and C7520T showed significant higher frequency in African American samples compare to Caucasian samples and these may be used for further investigation for cancer disparity study. A number of the observed sequence variants were germ-lines with variants found in these mitochondrial genes/regions; D-Loop, 12S rRNA-tRNAphe, tRNAval, COX I, tRNAser, tRNAasp, COX II, tRNAlys, ATPase 6, ATPase 8, COX III, ND2, and ND3 genes among the three subtypes of ovarian cancer. Our results showed the elevation of the ratio of protein carbonyl/total protein in all three types of serous polyp as compare to their surrounding tissue. The level of the ratio of protein carbonyls/total protein decreased as a result of serous ovarian tumor progression. Our study suggests that certain mitochondrial DNA sequence variants and protein carbonyl (CO) groups, oxidative stress may play a potential role in etiological differences that may exist between the pathogenicity of subtypes and stages of benign and invasive epithelial ovarian tumors. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A115.

Quantitative Proteomics Analysis of Streptomyces coelicolor Development Demonstrates That Onset of Secondary Metabolism Coincides with Hypha Differentiation

Streptomyces species produce many clinically important secondary metabolites, including antibiotics and antitumorals. They have a complex developmental cycle, including programmed cell death phenomena, that makes this bacterium a multicellular prokaryotic model. There are two differentiated mycelial stages: an early compartmentalized vegetative mycelium (first mycelium) and a multinucleated reproductive mycelium (second mycelium) arising after programmed cell death processes. In the present study, we made a detailed proteomics analysis of the distinct developmental stages of solid confluent Streptomyces coelicolor cultures using iTRAQ (isobaric tags for relative and absolute quantitation) labeling and LC-MS/MS. A new experimental approach was developed to obtain homogeneous samples at each developmental stage (temporal protein analysis) and also to obtain membrane and cytosolic protein fractions (spatial protein analysis). A total of 345 proteins were quantified in two biological replicates. Comparative bioinformatics analyses revealed the switch from primary to secondary metabolism between the initial compartmentalized mycelium and the multinucleated hyphae.

Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G(2)/M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G(2) phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.

THE DINUCLEOTIDE UP4A INDUCE THE PRODUCTION OF REACTIVE OXYGEN SPECIES IN VSMCS VIA NADPH OXIDASE: 1C.02

Objective: There is a growing body of evidence that nucleotides and purinergic signaling play a crucial role in the development of vascular diseases such as atherosclerosis. Uridine adenosine tetraphosphate (Up4A), an endothelium-derived dinucleoside polyphosphate showed inflammatory promoting effects. The inflammatory response is triggered by NADPH oxidase by influencing the production of chemokines. Here, we investigated the effect of Up4A on activation of NADPH oxidase and the production of reactive oxygen species in vascular smooth muscle cells. Methods: Rat vascular smooth muscle cells (VSMCs) were used for experiments. MCP-1 and Nox1/4 expression was measured by real-time PCR. Rac1 phosphorylation was measured by ELISA. Translocation of p47phox was detected by Western Blot technique in membrane fraction and cytosolic protein fraction of the cells. H2O2 production was examined by loading VSMCs with 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein-dieacetate-acetylester (CM-H2DCFDA). Results: Previous work proposed that the MCP-1 expression in VSMCs is controlled by the intracellular redox status. Thus, we investigated the effect of tiron, a vitamin E analog, which is able to significantly diminish the Up4A-induced MCP-1 expression (48 ± 23% decrease, n = 5). Furthermore, incubation of CM-H2DCFDA-labeled VSMCs with Up4A resulted in a significant and dose-dependent increase in DCF fluorescence intensity (n = 6). Thrombin (8 IE/mL) was used as positive control. NADPH oxidase is composed of different subunits. The expression of Nox1 is increased after Up4A stimulation of the cells, whereas Nox4 expression is decreased. The activation of NADPH oxidase requires phosphorylation of Rac1 and translocation of p47phox to the plasma membrane. Up4A induced Rac1 phosphorylation in a significant and time-dependent manner. P47phox amount in membrane fraction increased time-dependently after Up4A stimulation, whereas the amount in the cytosolic protein fraction decreased, respectively. Conclusions: In this study we could show that Up4A is influencing the vascular ROS production in a NADPH oxidase dependent manner. Therefore, the endothelial-derived factor Up4A is not only a potent vasoconstrictor but in addition a potent inductor of pro-inflammatory response in the vascular wall.

Differential regulation of redox proteins and chaperones in HbEβ‐thalassemia erythrocyte proteome

In (hemoglobin, Hb) HbEβ-thalassemia, HbE (β-26 Glu→Lys) interacts with β-thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEβ-thalassemic patients compared to normal. We have used 2-DE and MALDI-MS/MS-based techniques to investigate the differential proteome profiling of membrane and Hb-depleted fraction of cytosolic proteins of erythrocytes isolated from the peripheral blood samples of HbEβ-thalassemia patients and normal volunteers. Our study showed that redox regulators such as peroxiredoxin 2, Cu-Zn superoxide dismutase and thioredoxin and chaperones such as α-hemoglobin stabilizing protein and HSP-70 were upregulated in HbEβ-thalassemia. We have also observed larger amounts of membrane associated globin chains and indications of disruption of spectrin-based junctional complex in the membrane skeleton of HbEβ-thalassemic erythrocytes upon detection of low molecular weight fragments of β-spectrin and decrease in β-actin and dematin content. We have observed interesting changes in the proteomic levels of redox regulators and chaperons in the thalassemic hemolysates and have observed strong correlation or association of the extent of such proteomic changes with HbE levels. This could be important in understanding the role of HbE in disease progression and pathophysiology.

Abstract 5026: βIII-Tubulin regulates expression of proteins involved in tumorigenesis and metastasis in non-small cell lung cancer

Abstract Survival rates for advanced non-small cell lung cancer (NSCLC) remain dismal. Recently, we showed that silencing the microtubule protein βIII-tubulin using siRNA sensitizes NSCLC cells to chemotherapy drugs1. We have also recently shown that βIII-tubulin plays a role in regulating tumor growth both in vitro and in vivo2. Importantly, high levels of βIII-tubulin are correlated with more aggressive and drug refractory tumors in the clinic3. To date, the broader role of βIII-tubulin in NSCLC has not been determined and the aims of this study were to examine the role of βIII-tubulin on the expression of proteins involved in regulating tumorigenesis and metastasis. Methods: Using a functional and differential proteomics approach we examined NSCLC clones stably expressing shRNA against βIII-tubulin (pRS/βIIISH4 and pRS/βIIISH59) or control (pRS/CtrlSH1 and pRS/CtrlSH2). Cytosolic and nuclear protein fractions were prepared and 2-D DIGE was performed over broad (pI 4-7) and narrow (pI 4.5-5.5) pI ranges. Differences in protein expression between pRS/βIIISH4 and pRS/βIIISH59 and pRS/CtrlSH1 and pRS/CtrlSH2 cells were assessed using Decyder software. Protein spots of interest were excised and identified by mass spectrometry. Those identified as being significantly altered were then validated by western blot. Results: Eleven out of a total of 963 proteins (1.1%; pI 4-7) and 53 out of 753 proteins (7%; pI 4.5-5.5) were found to be significantly altered in the cytoplasmic fractions of pRS/βIIISH4 and 59 cells when compared to their controls (pRS/CtrlSH1 and SH2). In addition, 33 out of 1331 proteins (2.5%; pI 4-7) and 42 out of 502 proteins (8.4%; pI 4.5-5.5) were significantly altered in the nuclear fractions of pRS/βIIISH4 and SH59 cells compared to controls. Importantly, a number of proteins which are involved in regulating tumor growth and metastasis were identified as being differentially expressed in the βIII-tubulin knockdown cells. A significant decrease (greater than 2 fold) in heat shock protein 60 expression (promotes metastasis) was observed in the pRS/βIIISH4 and SH59 nuclear fractions when compared to controls. Furthermore, a greater than 6 fold increase in the tumor suppressor protein Tropomyosin 1 and a greater than 2 fold increase in the serpin B5 precursor protein Maspin (inhibitor of metastasis) was identified in the pRS/βIIISH4 and SH59 cytoplasmic fractions when compared to controls. These changes were confirmed by western blotting. Conclusions: This is the first study to show that silencing βIII-tubulin significantly alters the expression of proteins involved in regulating tumorigenesis and metastasis in lung cancer. Targeting βIII-tubulin could be a promising strategy for inhibiting tumor growth and metastasis in lung cancer. 1 Gan et al. Cancer Res, 67:9356-63, 2007 2McCarroll et al. 100th Annual AACR meeting. A3337, 2009 3Seve &Dumontet. Lancet Oncol, 9:168-75, 2008 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5026.

Abstract C32: Proteome analysis of pancreatic cancer tissue for identification of novel biomarkers

Abstract Pancreatic cancer is the 4th largest cancer in US with mortality rate almost equal to incidence. The tumor usually presents at a late stage ruling out any curative surgery. Focus of pancreatic cancer research currently is mainly on early diagnosis and development of novel cancer therapeutics. Identifying sensitive, specific and reliable surrogate markers by genomic and proteomic methods1 in cancer tissue, blood or other bodily secretions can prove promising to reach this goal. We are applying standard proteomic techniques like gel electrophoresis and MS (mass spectrometry) with a goal to identify potential biomarkers and new drug targets for the design and development of effective therapeutics in serum and pancreatic tissue samples. Pancreatic cytosolic and membrane proteins were extracted from the 4 pairs of cancerous and adjacent normal tissue. Cytosolic protein fractions were run on PF-2D, traditional 1D and 2D SDS-PAGE. LC/MS/MS was used to identify differently expressed banding patterns. Multiple proteins were found to be either up (24) or downregulated (11). Compared to cytosolic proteins membrane proteins are priced for its accessibility on tumor tissue making these ideal for diagnosis and therapeutics. Isolating only plasma membrane proteins is very challenging due to low extraction efficiency, extremely low abundancy and poor solubility. Total cellular membrane protein was extracted and analyzed by SDS-PAGE and LC/MS/MS, and significant banding pattern differences were observed with 35 upregulated and 14 downregulated proteins with annexin 1, annexin A2 and annexin 5, tumor rejection antigen 1, prolyl 4-hydroxylase, calreticulin precursor, heat shock 70kDa protein 5, chaperonin and alpha 2 actin being the most significantly overexpressed. Findings were confirmed by probing western blots with anti-annexin 1 and 2 antibodies. Our findings suggest these differently expressed proteins can be explored as potential biomarkers after further validation. In contrast, analysis of serum samples by same techniques was futile due to exceedingly low abundance of protein and peptide fraction containing potential biomarkers due to masking by albumin and globulins as well as enzymatic degradation. At present we are continuing the validation of our preliminary findings on larger sample size by using differential protein profiling technologies, immunohistochemistry and quantitative proteomics. Also we are planning to use affinity bait molecule with nanoparticles2 which can help overcome the limitations faced with serum samples in identification of de novo biomarkers. Citation Information: Cancer Res 2009;69(23 Suppl):C32.

Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when alpha-naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub-cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)-derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional-genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut.

The effects of fluoride on cell migration, cell proliferation, and cell metabolism in GH 4C 1 pituitary tumour cells

The consumption of drinking water rich in fluoride has toxic effects on the central nervous system. In cell biology research, fluoride is currently used as a phosphatase inhibitor. The aim of the present study was to evaluate the effect of fluoride on different physiological processes in GH 4C 1 pituitary tumour cells. We used a range of different fluoride concentrations, from levels below normal human serum concentrations (0.23 and 1.2 μmol/L) to those observed in chronically exposed persons (10.7 μmol/L) and above (107 and 1072 μmol/L). Treatment of 10.7 μmol/L fluoride resulted in a discrete induction of DNA synthesis, without a change in cell number. Cell migration, a behaviour stimulated by growth factors, was increased in cells treated with 2.4 μmol/L. At this fluoride concentration, changes in phosphorylation status of both cytoskeletal and cytosolic protein fractions, as well as in actin cytoskeletal arrangements were observed. The GH 4C 1 fluoride treated cells had significantly less cellular protein than control cells, suggesting an effect of fluoride on hormone secretion and protein synthesis in this endocrine cell. The bioreduction of MTT was significantly increased with a wide range of fluoride concentrations. With the highest fluoride concentration, 1072 μmol/L, all of the analysed parameters were significantly reduced, suggesting that this dose is highly toxic in GH 4C 1 cells. Our results show that biologically relevant concentrations of fluoride are capable of increasing cell migration in tumour cells, suggesting that exposure to fluoride could stimulate tumour invasion.

The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness

The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.

Proteomic analysis of lactose-starvedLactobacillus caseiduring stationary growth phase

Starvation stress is a condition that nonstarter lactic acid bacteria (NSLAB) normally encounter. This study was aimed to investigate starvation-induced proteins in Lactobacillus casei during stationary growth phase. The impact of carbohydrate starvation on L. casei GCRL163 was investigated using two different media (a modified de Man, Rogosa and Sharpe broth and a semi-defined medium). Cells were grown in the presence of excess lactose (1%) or starvation (0%) and differences in the patterns of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of the cytosolic protein fractions were investigated. Differentially regulated proteins were identified by MALDI-TOF/TOF mass spectrometry. Many differentially regulated proteins were enzymes of various metabolic pathways involved in carbohydrate metabolism to yield energy. Differences in protein expression were also observed in the two culture conditions tested in this experiment. Numerous glycolytic enzymes were differentially regulated under lactose starvation. The differential expression of these glycolytic enzymes suggests a potential survival strategy under harsh growth conditions (i.e. lactose starvation). This paper reports improved understanding of stress responses and survival mechanism of NSLAB under lactose-depleted cheese-ripening condition. This knowledge of how NSLAB bacteria adapt to lactose starvation could be applied to predict the performances of bacteria in other industrial applications.

Increased Enzymatic O-GlcNAcylation of Mitochondrial Proteins Impairs Mitochondrial Function in Cardiac Myocytes Exposed to High Glucose

Increased nuclear protein O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function.

Oligomeric β-amyloid(1-42) induces the expression of Alzheimer disease-relevant proteins in cholinergic SN56.B5.G4 cells as revealed by proteomic analysis

Alzheimer's disease (AD) is characterized by cholinergic dysfunction and progressive basal forebrain cell loss which has been hypothesized to be associated with extensive accumulation of β-amyloid (Aβ). To reveal whether oligomeric Aβ displays a particular toxicity for cholinergic neurons, the cholinergic cell line SN56.B5.G4 (SN56) was used as a model. Recently performed microarray analyses demonstrated that genes affected by exposure of SN56 cells with 50μM oligomeric Aβ(1-42) for 24h were involved in protein modification and degradation [Heinitz, K., Beck, M., Schliebs, R., Perez-Polo, J.R., 2006. Toxicity mediated by soluble oligomers of beta-amyloid(1-42) on cholinergic SN56.B5.G4 cells. J. Neurochem. 98, 1930–1945]. Using a proteomic approach, we compared the levels of proteins and specially of phosphorylated proteins in cytosolic fractions of cell lysates from cholinergic SN56 cells exposed to 50μM Aβ(1-42) for 24h to those in control incubations. We show here that the levels of calreticulin, and mitogen-activated protein kinase (MAPK) kinase 6c were up-regulated in cholinergic SN56 cells exposed to Aβ(1-42), while γ-actin appeared down-regulated. Aβ(1-42) exposure of cholinergic SN56 cells led to decreased phosphorylation of phosphoproteins, such as the Rho GDP dissociation inhibitor, the ubiquitin carboxyl terminal hydrolase-1, and the tubulin α-chain isotype Mα6, as compared to untreated control lysates. The proteins identified have also been reported to be affected in brains of AD patients, suggesting a potential role of Aβ in influencing the integrity and functioning of the proteome in AD.

Divergent Regulation of Protein Synthesis in the Cytosol and Endoplasmic Reticulum Compartments of Mammalian Cells

In eukaryotic cells, mRNAs encoding signal sequence-bearing proteins undergo translation-dependent trafficking to the endoplasmic reticulum (ER), thereby restricting secretory and integral membrane protein synthesis to the ER compartment. However, recent studies demonstrating that mRNAs encoding cytosolic/nucleoplasmic proteins are represented on ER-bound polyribosomes suggest a global role for the ER in cellular protein synthesis. Here, we examined the steady-state protein synthesis rates and compartmental distribution of newly synthesized proteins in the cytosol and ER compartments. We report that ER protein synthesis rates exceed cytosolic protein synthesis rates by 2.5- to 4-fold; yet, completed proteins accumulate to similar levels in the two compartments. These data suggest that a significant fraction of cytosolic proteins undergo synthesis on ER-bound ribosomes. The compartmental differences in steady-state protein synthesis rates correlated with a divergent regulation of the tRNA aminoacylation/deacylation cycle. In the cytosol, two pathways were observed to compete for aminoacyl-tRNAs-protein synthesis and aminoacyl-tRNA hydrolysis-whereas on the ER tRNA deacylation is tightly coupled to protein synthesis. These findings identify a role for the ER in global protein synthesis, and they suggest models where compartmentalization of the tRNA acylation/deacylation cycle contributes to the regulation of global protein synthesis rates.

Cadmium biodynamics in the oligochaete Lumbriculus variegatus and its implications for trophic transfer

It has become increasingly apparent that diet can be a major source of trace metal bioaccumulation in aquatic organisms. In this study, we examined cadmium uptake, efflux, and subcellular compartmentalization dynamics in the freshwater oligochaete Lumbriculus variegatus. L. variegatus is an important component of freshwater food webs in Europe and North America and is potentially useful as a standard food source for laboratory-based trophic transfer studies. Cadmium accumulation and depuration were each followed for 10 days. Rate constants of uptake ( k u) and efflux ( k e) were estimated and subcellular Cd compartmentalization was followed over the course of uptake and efflux. The partitioning of Cd into operationally-defined subcellular compartments was relatively consistent throughout the 20-day experiment, with the majority of Cd accumulating in the cytosol. No major changes in Cd compartmentalization were observed over uptake or depuration, but there appeared to be some exchange between heat-stable and heat-labile cytosolic protein fractions. Cadmium accumulation from solution was strongly affected by ambient calcium concentrations, suggesting competition between Cd and Ca for uptake sites. Finally, we demonstrate the ability to manipulate the whole body calcium content of L. variegatus as a potential tool for examining calcium influences on dietary Cd dynamics. The potential for this species to be an important conduit of Cd to higher trophic levels is discussed, along with its potential as a standardized food source in metal trophic transfer studies.

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (MΦ) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDMΦ) as target cells. Various B. abortus antigen preparations were tested including whole γ-irradiated B. abortus bacteria (γBA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDMΦ targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with γBA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4 +, CD8 − cells indicating that the observed cytotoxic responses were most likely due to an inflammatory T h1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.