Abundant Cellular Proteins Research Articles

Abstract B41: Proteomic characterization of carboplatin resistance in ovarian cancer cells

Abstract The analysis of quantitative protein expression is necessary for the determination of actual biochemical protein concentrations that can lead to a more accurate description of cellular function. A knowledgebase of cancer function founded on cellular protein levels can more directly implicate potential gene candidates for the detection and treatment of disease. Therefore, we analyzed iTRAQ (Isobaric tag for relative and absolute quantification) data from 11 serous ovarian cancer cell lines and 2 normal ovarian surface epithelial cell lines. We profiled cellular protein abundance in over 2600 proteins with 746 proteins common to all 13 cell lines. Of the 746, 52 proteins exhibited elevated expression and 46 proteins had diminished expression in the cancerous cells when compared to the two normal cell lines. We identified the 300 most variable iTRAQ expressed proteins based on median absolute deviation within 8 carboplatin sensitive and resistant cell lines. Using iTRAQ protein expression profiles from these 300 we successfully segregated the cell lines based on carboplatin sensitivity and resistance measurements. Next, we further examined correlations among iTRAQ protein expression, individual gene RNA expression and DNA methylation data. There was no substantial correlation between protein abundance and RNA expression or epigenetic data. Furthermore RNA expression and DNA methylation data alone did not show any significant discrimination for chemosensitivity or resistance. Our clustering proteomics-based profile used to identify chemotherapy response contained proteins involved in homologous recombination and repair, specifically RPA, MCM2/MCM5 and PALB2. We therefore present the potential of proteomics based discovery for carboplatin response in ovarian cancer and also identify putative gene candidates that may be functionally responsible for chemotherapy response which otherwise would not have been predicted by other singular whole genome data sources. Citation Format: Kazimierz O. Wrzeszczynski, Gaofeng Fan, Cexiong Fu, Darryl J. Pappin, Robert Lucito, Nicholas K. Tonks. Proteomic characterization of carboplatin resistance in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B41.

Proteomics in aortic aneurysm – What have we learnt so far?

Aortic aneurysm is a deceptively indolent disease that can cause severe complications such as aortic rupture and dissection. In the normal aorta, vascular smooth muscle cells within the medial layer produce and sustain the extracellular matrix (ECM) that provides structural support but also retains soluble growth factors and regulates their distribution. Although the ECM is an obvious target to identify molecular processes leading to structural failure within the vessel wall, an in-depth proteomics analysis of this important sub-proteome has not been performed. Most proteomics analyses of the vasculature to date used homogenized tissue devoid of spatial information. In such homogenates, quantitative proteomics comparisons are hampered by the heterogeneity of clinical samples (i.e. cellular composition) and the dynamic range limitations stemming from highly abundant cellular proteins. An unbiased proteomics discovery approach targeting the ECM instead of the cellular proteome may decipher the complex, multivalent signals that are presented to cells during aortic remodelling. A better understanding of the ECM in healthy and diseased vessels will provide important pathogenic insights and has potential to reveal novel biomarkers.

The Unexpected Roles of Eukaryotic Translation Elongation Factors in RNA Virus Replication and Pathogenesis

The prokaryotic translation elongation factors were identified as essential cofactors for RNA-dependent RNA polymerase activity of the bacteriophage Qβ more than 40 years ago. A growing body of evidence now shows that eukaryotic translation elongation factors (eEFs), predominantly eEF1A, acting in partially characterized complexes sometimes involving additional eEFs, facilitate virus replication. The functions of eEF1A as a protein chaperone and an RNA- and actin-binding protein enable its "moonlighting" roles as a virus replication cofactor. A diverse group of viruses, from human immunodeficiency type 1 and West Nile virus to tomato bushy stunt virus, have adapted to use eEFs as cofactors for viral transcription, translation, assembly, and pathogenesis. Here we review the mechanisms used by viral pathogens to usurp these abundant cellular proteins for their replication.

Monitoring epithelial cell responses to an IBD-associated bacterial pathotype using quantitative proteomics (P1352)

Abstract Many lines of evidence suggest that the initiation of inflammatory bowel disease (IBD) involves abnormal host-microbial interactions at the intestinal epithelium. One microorganism that is commonly associated with the intestinal mucosa of IBD patients but not from healthy subjects is called adherent invasive E. coli (AIEC). AIEC are a new pathotype defined by their ability to adhere to and invade epithelial cells in the absence of known virulence factors associated with other pathogenic E. coli. Previous work has demonstrated that AIEC infection of epithelial cells induces cell-intrinsic responses such as the activation of autophagy markers, reduction of barrier function and secretion of IL-8. However, a comprehensive analysis of the cellular response to AIEC has not been performed. Here, we take a global approach to define how protein levels are altered in epithelial cells exposed to AIEC using quantitative mass spectrometry. This experimental strategy allows us to determine how the endogenous abundance of known and novel cellular proteins is altered by AIEC infection. Our preliminary results reveal specific differences between the epithelial cell response to adherent invasive E. coli compared to commensal E. coli not associated with IBD. Ongoing work examines whether any of the changes in protein abundance observed in response to AIEC infection are necessary for AIEC attachment, invasion, or intracellular growth.

Abstract 3272: Differential sensitivities to heat shock protein 90(HSP90) inhibitors in anaplastic lymphoma kinase(ALK)-positive non-small cell ling cancer(NSCLC) cells.

Abstract Objectives: HSP90 is an abundant cellular chaperone protein and its inhibition results in the instability and degradation of client proteins such as HER2, mutant KIT, mutant EGFR, and ALK. Recently, HSP90 inhibitors showed clinical activity against echinoderm microtubule-associated protein-like 4 (EML4)-ALK NSCLC. However, it is unknown whether HSP90 inhibitors remain sensitive against ALK-positive NSCLC cells that show acquired resistance to ALK inhibitors, such as crizotinib. Here, we evaluated the activity of two clinical stage HSP90 inhibitors in ALK-positive NSCLC cells that were sensitive and resistant to crizotinib. Methods: ALK-positive NSCLC cells that were crizotinib-sensitive (SNU-2292 and NCI-H3122) and crizotinib-resistant (SNU-2535 and H3122CR1) were used. In vitro cytotoxicities of HSP90 inhibitors alone (ganetespib and NVP-AUY922), or in combination with crizotinib, were evaluated against ALK-positive NSCLC cells using a modified MTT assay. Cell cycle and Annexin-V binding assays were evaluated by flow cytometry. Expression of ALK and its downstream effectors were detected by Western blot assay after exposure to HSP90 inhibitors ± crizotinib. Results: HSP90 inhibitors were highly potent against crizotinib sensitive SNU-2292 and NCI-H3122 cell lines and displayed enhanced activity when combined with 0.1 μM crizotinib. Consistent with previous reports, crizotinib resistant H3122 cells were ∼5x fold less sensitive to HSP90 inhibitors compared to parental H3122 cells, with IC50 still in the low nanomolar range. In a concentration dependent manner, the HSP90 inhibitors did not suppress ALK downstream signals in H3122CR1 and SNU-2535 cell lines compared to effects on NCI-H3122 and SNU-2292 cell lines. In addition, phospho-ALK signals were significantly diminished in Ba/F3 cells expressing EML4-ALK compared to cell lines expressing L1196M or G1269A after exposure to HSP90 inhibitors. Cell cycle analysis demonstrated HSP90 inhibitors and/or crizotinib significantly increased the sub-G1 populations of NCI-H3122 and SNU-2292 cells compared with those of H3122CR1 and SNU-2535. Similarly, apoptotic cells significantly increased in crizotinib-sensitive cells than in crizotinib-resistant cells treated with HSP90 inhibitors and/or crizotinib. Conclusions: HSP90 inhibitors showed differential sensitivities in ALK-positive NSCLC cells. Our results suggest that HSP90 inhibitors alone or in combination treatment with crizotinib are effective against crizotinib-sensitive, ALK-positive NSCLC cells. Citation Format: SunHwa Lee, Soyeon Kim, Tae Min Kim, Dong-Wan Kim, Dae Seog Heo. Differential sensitivities to heat shock protein 90(HSP90) inhibitors in anaplastic lymphoma kinase(ALK)-positive non-small cell ling cancer(NSCLC) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3272. doi:10.1158/1538-7445.AM2013-3272

Abstract 2502: Proteomic identification of carboplatin resistance in ovarian cancer cells.

Abstract The analysis of quantitative protein expression is necessary for the determination of actual biochemical protein concentrations that can lead to a more accurate description of cellular function. A knowledgebase of cancer function based on cellular protein levels can more directly implicate potential gene candidates for the detection and treatment of disease. Therefore, we analyzed iTRAQ (isobaric tag for relative and absolute quantification) data from 11 serous ovarian cancer cell lines and 2 normal ovarian surface epithelial cell lines. We profiled cellular protein abundance in over 3000 proteins with 749 proteins common to all 13 cell lines. Of the 749, 52 proteins exhibited elevated expression and 46 proteins had diminished expression in the cancerous cells when compared to the normal cell lines. We identified the 300 most variable iTRAQ expressed proteins based on median absolute deviation within 8 carboplatin sensitive and resistant cell lines. Using iTRAQ protein expression profiles from these 300 we successfully segregated the cell lines based on carboplatin sensitivity and resistance measurements. We further examined correlations among iTRAQ protein expression, individual gene RNA expression and DNA methylation data. There was no substantial correlation between protein abundance and RNA expression or epigenetic data. Furthermore RNA expression and DNA methylation data alone did not show any significant discrimination for chemosensitivity or resistance. We therefore present the potential of proteomics based discovery for carboplatin response in ovarian cancer and also identify putative gene candidates that may be functionally responsible for chemotherapy response which otherwise would not have been predicted using other singular whole genome data sources. Citation Format: Kazimierz O. Wrzeszczynski, Gaofeng Fan, Cexiong Fu, Darryl J. Pappin, Robert Lucito, Nicholas K. Tonks. Proteomic identification of carboplatin resistance in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2502. doi:10.1158/1538-7445.AM2013-2502

Response of human pancreatic cancer cell xenografts to tetraiodothyroacetic acid nanoparticles.

Tetraiodothyroacetic acid (tetrac) and its nanoparticle formulation (Tetrac NP) act at an integrin cell surface receptor to inhibit tumor cell proliferation and tumor-related angiogenesis. Human pancreatic cancer cell (PANC-1 and MPanc96) xenografts were established in nude mice, and the effects of tetrac versus Tetrac NP on tumor growth and tumor angiogenesis were determined. The in vitro effects of tetrac and Tetrac NP were also determined by reverse transcription polymerase chain reaction or immunoblot on gene expression or gene products relevant to cell cycle arrest, apoptosis, or angiogenesis. Tetrac and Tetrac NP reduced both PANC-1 tumor mass by 45-55 % and PANC-1 tumor hemoglobin content, a marker of angiogenesis, by 50-60 % (*P < 0.05) in treated groups vs. controls by treatment day 15. Comparable results were obtained with tetrac and Tetrac NP in suppressing tumor growth and tumor angiogenesis in MPanc96 xenografts. In vitro studies showed that tetrac and Tetrac NP caused accumulation of pro-apoptotic protein BcLx-s. Tetrac NP was more effective than tetrac in increasing cellular abundance of mRNAs of pro-apoptotic p53 and p21 and anti-angiogenesis thrombospondin 1 protein in PANC-1 and MPanc96 cancer cell lines. Tetrac NP noticeably decreased expression of EGFR and of anti-apoptosis gene XIAP; tetrac did not affect EGFR and increased XIAP mRNA in both MPanc96 and PANC-1. In conclusion, tetrac or Tetrac NP effectively inhibited human pancreatic xenograft growth and tumor angiogenesis via a plasma membrane receptor that downstream modulated cellular abundance of proteins or mRNAs relevant to apoptosis and angiogenesis.

Insights into the Regular Patterns of Protein Abundance Regulation and Distribution

Protein is one of the fundamental building blocks of life, and its amount in one cell is closely related to biological function. Therefore, protein abundance is regulated strictly at multiple levels of gene expression. The determinants of protein abundance include its corresponding mRNA abundance, the rate of protein synthesis and degradation, so the final cellular protein abundance is determined by the balance of these three factors. Benefitting from the rapid advancement of quantitative proteomics and the outputting of large-scale protein abundance data, we can devote to uncovering the relationship between protein abundance and its intrinsic characteristics (such as evolutionary characteristics, structure characteristics and functional categories), which is of great significance for the deep understanding of the basic composition principle of life systems. Here we summarize the recent research advances of protein abundance regulation, and the regular patterns of the relationship between protein abundance and its intrinsic characteristics. In addition, we also discuss the biological significance underlying them.

Ribosomes take control

The ribosome plays a universally conserved role in catalyzing the translation of all mRNAs in every cell across all kingdoms of life. It is therefore not surprising that the ribosome is considered one of the most complex and elaborately formed “molecular machines” in the cell, whose biogenesis is extraordinarily orchestrated, requiring all three RNA polymerases and more than 150 nonribosomal factors (1). Indeed, ribosomes make up much of the cell’s mass, and the eukaryotic ribosome is comprised of four RNA species and 79 ribosomal proteins (RPs). For decades, the dogma has been that, although the ribosome plays a critical function in translating the genomic code, it is largely a “back-stage” participant in gene regulation. This view has been reinforced by the widespread belief, early in the advent of the molecular era, that transcription rather than translation is the major molecular rheostat of gene expression. On the contrary, recent emerging evidence now reveals tremendous variation between expression of the transcriptome and proteome and that the cellular abundance of proteins may be predominantly controlled at the level of translational control (2). By extension, an interesting emerging question is whether the ribosome exerts a previously unappreciated regulatory function or specificity in translational control (3). A PNAS study by Lee et al. contributes to the growing realization of a surprising and emerging role for the ribosome as a “front-stage” participant in gene regulation (4). Moreover, their studies strongly suggest that much can be learned with respect to specialized mechanisms for cellular mRNA translation through the study of viral gene expression.

Genotoxic Agents Promote the Nuclear Accumulation of Annexin A2: Role of Annexin A2 in Mitigating DNA Damage

Annexin A2 is an abundant cellular protein that is mainly localized in the cytoplasm and plasma membrane, however a small population has been found in the nucleus, suggesting a nuclear function for the protein. Annexin A2 possesses a nuclear export sequence (NES) and inhibition of the NES is sufficient to cause nuclear accumulation. Here we show that annexin A2 accumulates in the nucleus in response to genotoxic agents including gamma-radiation, UV radiation, etoposide and chromium VI and that this event is mediated by the nuclear export sequence of annexin A2. Nuclear accumulation of annexin A2 is blocked by the antioxidant agent N-acetyl cysteine (NAC) and stimulated by hydrogen peroxide (H2O2), suggesting that this is a reactive oxygen species dependent event. In response to genotoxic agents, cells depleted of annexin A2 show enhanced phospho-histone H2AX and p53 levels, increased numbers of p53-binding protein 1 nuclear foci and increased levels of nuclear 8-oxo-2′-deoxyguanine, suggesting that annexin A2 plays a role in protecting DNA from damage. This is the first report showing the nuclear translocation of annexin A2 in response to genotoxic agents and its role in mitigating DNA damage.

An iTRAQ Proteomics Screen Reveals the Effects of the MDM2 Binding Ligand Nutlin-3 on Cellular Proteostasis

Mouse double minute 2 (MDM2) participates in protein synthesis, folding, and ubiquitin-mediated degradation and is therefore a proteostasic hub protein. The MDM2 interactome contains over 100 proteins, yet stratification of dominant MDM2-interacting proteins has not been achieved. 8-plex iTRAQ (nanoLC-MS/MS) of MCF7 cells treated with the MDM2-binding ligand Nutlin-3 identified the most abundant cellular protein changes over early time points; 1,323 unique proteins were identified including 35 with altered steady-state levels within 2 h of Nutlin-3 treatment, identifying a core group of MDM2 related proteins. Six of these proteins were previously identified MDM2 interactors, and the effects of Nutlin-3 on the MDM2-nucleophosmin interaction (NPM) was further validated. This revealed that Nutlin-3 mediates the in vivo conversion of NPM from an oligomer to a monomer as an MDM2-dependent phenomenon, with Nutlin-3 stimulating MDM2 binding to a peptide motif derived from the oligomerization interface of NPM. These data form the first proteomic screen of Nutlin-3 in cells whereby we (i) identify the most abundant MDM2-interacting proteins whose steady-state levels change early after Nutlin-3 treatment; (ii) identify the first protein apart from p53, nucleophosmin (NPM), whose interaction with MDM2 can be stimulated allosterically by Nutlin-3; and (iii) raise the possibility that Nutlin-3 might act as a general agonist of other MDM2 protein-protein interactions.

Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.

Inflammatory markers in skeletal muscle of older adults

Older adults have an increase in circulating markers of inflammation. The current study examined whether there is an increase in the expression of inflammatory markers within the vastus lateralis, a major locomotive muscle, of older adults, and if so, whether the reduction in muscle strength and aerobic capacity in older adults is related to increased muscle inflammation. Skeletal muscle biopsies were taken from older adults (n=17, 67±1.6years) and young individuals (n=16, 24±0.6years) under resting and fasting conditions. Muscle was analyzed for mRNA levels of intracellular inflammatory molecules (MCP1, TNFα and IL-1β) and total cellular protein abundance of cytokines, chemokines and kinases (IL-6, IL-8, MCP1, TNFα, p65 (NF-κB), JNK1/2 and STAT3). MCP1 expression was significantly higher (p<0.05; 50%, mRNA and 40%, protein) in elderly than younger participants, as was IL-8 (4%). No detectable difference in kinase protein expression was observed for STAT3, JNK or p65 (NF-κB), TNFα or IL-6. Muscle strength was lower in the elderly compared to the young group (1.55±0.17 vs. 2.56±0.13 Nm/kg, p<0.001). The elderly group also had a significantly lower VO(2peak) compared to the young group (24.9±1.9 vs. 39.3±1.9, p<0.001), but muscle strength and VO(2peak) were not correlated with the examined inflammatory markers. Older adults have increased MCP1 (mRNA and protein abundance) and IL-8 (protein abundance) and also reduced muscle strength and VO(2peak). However, the reduction in muscle strength and VO(2peak) was not related to the increase in muscle inflammatory markers in this cohort.

Molecular and Thermodynamic Insights into the Conformational Transitions of Hsp90

Hsp90, the most abundant cellular protein, has been implicated in numerous physiological and pathological processes. It controls protein folding and prevents aggregation, but it also plays a role in cancer and neurological disorders, making it an attractive drug target. Experimental efforts have demonstrated its remarkable structural flexibility and conformational complexity, which enable it to accommodate a variety of clients, but have not been able to provide a detailed molecular description of the conformational transitions. In our molecular dynamics simulations, Hsp90 underwent dramatic structural rearrangements into energetically favorable stretched and compact states. The transitions were guided by key electrostatic interactions between specific residues of opposite subunits. Nucleotide-bound structures showed the same conformational flexibility, although ADP and ATP seemed to potentiate these interactions by stabilizing two different closed conformations. Our observations may explain the difference in dynamic behavior observed among Hsp90 homologs, and the atomic resolution of the conformational transitions helps elucidate the complex chaperone machinery.

Insights into the regulation of protein abundance from proteomic and transcriptomic analyses

Recent advances in next-generation DNA sequencing and proteomics provide an unprecedented ability to survey mRNA and protein abundances. Such proteome-wide surveys are illuminating the extent to which different aspects of gene expression help to regulate cellular protein abundances. Current data demonstrate a substantial role for regulatory processes occurring after mRNA is made - that is, post-transcriptional, translational and protein degradation regulation - in controlling steady-state protein abundances. Intriguing observations are also emerging in relation to cells following perturbation, single-cell studies and the apparent evolutionary conservation of protein and mRNA abundances. Here, we summarize current understanding of the major factors regulating protein expression.

Conformational Transitions of Heat Shock Proteins: The Case of Hsp90

The most abundant cellular protein, Hsp90, partakes in many biological pathways, not only in times of induced stress, but also under normal physiological conditions. Its role in controlling proteostasis, by assisting in protein folding and reducing aggregation, together with its direct involvement in cancer cell survival and neurological disorders, makes this protein an attractive drug target. In solution, Hsp90 is a homodimer, with each subunit composed of three dynamically independent domains, contributing to the great structural flexibility and the ability to accommodate a large number of clients. Our molecular dynamic simulations of the E. coli homologue gave us a unique insight into observing the conformational transitions at atomic resolution. We observed dramatic structural rearrangements, independent of the initial state or the presence of nucleotides. The apo state was free to shift between the compact and fully stretched states, separated by a free energy barrier that was very close to the crystal open conformation. ATP binding stabilized the extended closed state, similar to the closed crystal state of HtpG. In the ADP-bound state the dynamics was limited to local motions in the N-terminal region, although the outward twisting of middle domains indicated the transitioning into an open state. Release of the nucleotides led to the formation of a compact conformation, guided by interactions between Asp287 and Asp292 from the middle domain and Lys103 from the N-terminal domain. The electrostatic interactions between opposite subunits appear to be key in directing the conformational change and making Hsp90 amenable to evolutionary fine-tuning that may regulate the populations of different conformations between homologues.

Latexin regulates the abundance of multiple cellular proteins in hematopoietic stem cells

Latexin is the only known carboxypeptidase A inhibitor in mammals and shares structural similarity with cystatin C, suggesting that latexin regulates the abundance of as yet unidentified target proteins. A forward genetic approach revealed that latexin is involved in homeostasis of hematopoietic stem cells (HSCs) in mice; however, little is known about the mechanisms by which latexin negatively affects the numbers of HSCs. In this study, we found that latexin is preferentially expressed in hematopoietic stem/progenitor cells, and is co-localized with the molecules responsible for the interaction of HSCs with a bone marrow niche, such as N-cadherin, Tie2, and Roundabout 4. Latexin-knockout young female mice showed an increase in the numbers of KSL (c-Kit(+)/Sca-1(+)/linegae marker-negative) cells, which may be attributable to enhanced self-renewal because latexin-deficient KSL cells formed more colonies than their wild-type counterparts in methylcellulose culture. Proteomic analysis of Sca-1(+) bone marrow cells demonstrated that latexin ablation reduced the abundance of multiple cellular proteins, including N-cadherin, Tie2, and Roundabout 4. Finally, we found that latexin expression was lost or greatly reduced in approximately 50% of human leukemia/lymphoma cell lines. These results imply that latexin inhibits the self-renewal of HSCs by facilitating the lodgment of HSCs within a bone marrow niche to maintain HSC homeostasis.

Acid increases NHE8 surface expression and activity in NRK cells

We previously demonstrated that there is a paucity of brush-border membrane NHE3 in neonates, the predominant Na(+)/H(+) exchanger in the adult proximal tubule, while NHE8 is relatively highly expressed in neonates compared with adults. We recently showed that metabolic acidosis in neonatal rodents can increase brush-border membrane NHE8 protein expression and Na(+)/H(+) exchange activity. To further examine the regulation of NHE8 by acid, we incubated NRK cells, which express NHE8 but not NHE3, with either acid or control media (6.6 vs. 7.4). There was an increase in Na(+)/H(+) exchanger activity within 6 h of incubation with acid media assessed as the rate of sodium-dependent recovery of pH from an acid load (dpH(i)/dt). The acid stimulation persisted for at least 24 h. The increase in Na(+)/H(+) exchange activity was paralleled by an increase in surface expression of NHE8, assessed by surface biotinylation and streptavidin precipitation. The increase in both apical membrane NHE8 protein expression and Na(+)/H(+) exchange activity with pH 6.6 media compared with 7.4 media was not affected by actinomycin D or cycloheximide consistent with an increase in surface expression independent of mRNA or protein synthesis. Furthermore, there was no increase in total cellular NHE8 protein abundance or mRNA abundance with acid media. Finally, we demonstrate that the increase in surface expression of NHE8 with acid media was blocked by colchicine and cytochalasin D and mediated by acid increasing the rate of exocytosis. In conclusion, NHE8 surface expression and activity are regulated by acid media by increasing the rate of trafficking to the apical membrane.

Survey role of nano structure surface layer in Bacillus cereus strains resistant to antibiotics (Iran-Azzahra Hospital and Esfahan University)

The most common surface structures bacteria are monomolecular crystalline arrays of proteinaceous subunits termed surface layers or S-layers. Since S-layer-carrying organisms are ubiquitous in the biosphere and because S-layers represent one of the most abundant cellular proteins, it is now obvious that these metabolically expensive products must provide the organisms with an advantage of selection in very different habitats. S-layers have been associated with a number of possible functions that relate to pathogenicity. S-layers can function as adhesins, enabling the bacterium to adhere to host cell membranes and tissue surfaces in order to colonize and protect bacteria from harmful enzymes and antimicrobial agents or changes in pH. Bacillus cereus is one of nosocomial infections bacteria. B. cereus produces several potential virulence factors such as S-layer. A total of 274 strains were isolated from staff hand and hospital surfaces of Azzahra-hospital during 2005 to 2007 years. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from B. cereus strains by using Tris-HCl (pH 8) showed that the S-layer proteins of different strains isolate of staff hands and hospital surfaces had subunit molecular weight of 97-kDa. Antibiotic susceptibility was performed according to antibiotic susceptibility standard disc diffusion agar. All the statistical analyses carried out using SPSS version 14. Chi-square and fisher test used for determination of significance of association. The p≤ 0.05 was considered significant. From 247 bacteria, frequency of B. cereus strains was 9.49%. From 13 isolated B. cereusof, staff hand 11 strain (84/6%) and from 13 isolated B. cereus from hospital surfaces, 1 strain (7/7%) production S-layer. According to the antibiogram result, S-layer non producer strains, in comparison with S-layer producer strains, were more sensitive to antibiotics. The result showed high prevalence of S-layer producer of B. cereus strains in hospital; and this point is due to the increased antibiotic resistance of nosocomial infections. Key words: S-layer, Bacillus cereus, antibiotic resistant, nosocomial infections.

Global quantification of mammalian gene expression control.

Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.