60S Acidic Ribosomal Protein Research Articles

Analysis of differentially expressed proteins between the spinetoram-susceptible and -resistant strains of Plutella xylostella (L.)

Plutella xylostella (L.), a worldwide vegetable pest, has developed resistance to spinetoram, which was previously effective for the control of P. xylostella (L.). The insecticidal resistance mechanism is essential to develop effective resistance management strategies. To explore the spinetoram resistance mechanism, a comparative proteomics approach was used to investigate the proteomic differences between the spinetoram-susceptible strain (SS) and spinetoram-resistant strain (RS) of P. xylostella (L.). Approximately 280 protein spots were detected on each SDS-PAGE gel. Of these, 19 proteins were successfully identified by MALDI-TOF-MS.·There were 6 significantly down-regulated spots and 13 up-regulated spots in RS, which showed significantly difference compared to that in SS. Based on the gene ontology(GO) system and KEGG database, the 19 identified proteins were classified into 6 groups including metabolisms, signal transduction, chaperones, transcriptional, protein synthesis, structural protein. Meanwhile, the expression profiles of 5 resistant related protein were further analysed by qRT-PCR. The results showed that 60S acidic ribosomal protein P2, glutathione S-transferase isozyme 3 and glutathione S-transferase delta were significantly up-regulated, while phosphoglycerate mutase and receptor for activated protein kinase C homolog were significantly down-regulated. The expression tendency of mRNA was in accordance with which of protein. This study provided evidences that spinetoram induces proteomic changes in P. xylostella (L.), and it is contributed to help us understand the resistance mechanism of P. xylostella (L.) to spinetoram.

Identification of reference genes for gene expression normalization in safflower (Carthamus tinctorius)

Safflower (Carthamus tinctorius L., Asteraceae) is an important oil crop and medicinal plant. Gene expression analysis is gaining importance in the research of safflower. Quantitative PCR has become a powerful method for gene study. Reference genes are one of the major qualification requirements of qPCR because they can reduce the variability. To identify the reference genes in safflower, nine candidate genes of the housekeeping genes were selected from the EST library of safflower constructed by our lab: CtACT (actin), CtGAPDH (glyceraldehyde 3-phosphate dehydrogenase), CtE1F4A (elongation factor 1 alpha), CtTUA (alpha-tubulin), CtTUB (beta-tubulin), CtPP2A (serine/threonine-protein phosphatase), CtE1F4A (eukaryotic initiation factor 4A), CtUBI (Ubiquitin), and Ct60S (60S acidic ribosomal protein). Expression stability was examined by qPCR across 54 samples, representing tissues at different flowering stages and two chemotype of safflower lines. We assessed the expression stability of these candidate genes by employing four different algorithms (geNorm, NormFinder, ΔCt approach, and BestKeeper) and found that CtUBI and Ct60S were the highly ranked candidate genes. CtUBI and Ct60S were used as reference genes to evaluate the expression of CtFAD2-10 and CtKASII. Our data suggest CtUBI and Ct60S could be used as internal controls to normalize gene expression in safflower.

Rice Diversity Panel Mapping for Identifying Genetics Behind Leaf Vein Density Trait in Rice (<i>Oryza sativa</i> L.)

A screen of rice genotypes was carried out on a total of 327 genotypes of the rice diversity panel for leaf vein density. Identifying locations of genes that impart to leaf vein density in a quantitative way should capable the using of these genes in plant breeding and accelerate producing C4 rice plant. Finding of genotypes of rice that have less distances between veins is still the challenge of the plant breeders. Screen was shown significant variations in leaf vein density. Quantitative trait loci (QTLs) for the leaf vein density trait were identified by using Efficient Mixed Model Analysis (EMMA). QTLs were considered reportable if they had P values (below 0.0001). The most significant Single nucleotide polymorphisms (SNP) associations (EMMA 1.3, EMMA 1.8 and EMMA 10.1) were in each of the rice chromosomes 1 and 10 respectively. All genes positioned 200 kb around associations were selected. The candidacy of the most promising were NADP-dependent malic enzyme, chloroplast precursor (LOC_Os01g09320; ras-related protein, putative, expressed (LOC_Os01g51700); 60S acidic ribosomal protein (LOC_Os01g09510); Auxin-responsive Aux/IAA gene family member (LOC_Os01g09450; myb-related transcription activator, putative, expressed (LOC_Os01g09280); glycine-rich protein (LOC_Os01g09246); phosphofructokinase (LOC_Os01g09570); oxidoreductase, short chain dehydrogenase/reductase family (SDR) LOC_Os10g31780), which have been expressed in leaf tissue and requisite to be investigated further.

Abstract 3306: Aberrant expression of ribosomal protein p1 and its pseudogene in breast tumors

Abstract Introduction: Ribosome biogenesis and translation control are essential cellular processes in growing cancer cells. Defects in ribosomal components can alter the pattern of gene expression, cell cycle progression and cell growth. Human ribosomes consist of four RNA species and 79 ribosomal proteins with 2090 processed and 16 duplicated pseudogenes. Specifically, 60S Acidic Ribosomal Protein P1 (RPLP1) is expressed in a tissue specific manner with multiple processed pseudogenes. We accidentally identified the expressed pseudogene 4 of RPLP1 (RPLP1P4) and determined genetic and molecular mechanism of RPLP1 and RPLP1P4 expression in breast cancer cells. Methods: Expression levels of RPLP1 and RPLP1P4 were quantified using qRT-PCR with total RNAs isolated from 7 normal tissues and 20 breast tumor tissues of 5 different subtypes. Cell cycle progression was analyzed cytofluorimetrically with the Beckman Coulter Gallios Flow Cytometer. Gene expression profiles were performed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Array and analyzed using Partek Genomic Suite. Somatic copy number alterations (SCNA) of RPLP1/RPLP1P4 were detected with the custom designed TaqMan assays using ABI 7900HT real time PCR system. CopyCaller™ Software was used for the SCNA analysis with RNase P as a reference. Results: Because breast cancer subtypes have distinct origins and molecular characteristics, we compared expression of RPLP1 and RPLP1P4 in 5 subtypes of breast tumors. Although RPLP1/RPLP1P4 is ubiquitously expressed in normal and cancer tissues, their expression levels are significantly increased in the basal-like subtype of breast cancer compared to other subtypes with a 2.23 ± 1.06 fold increase in basal-like subtype compared to non-basal subtypes. Interfering RPLP1 expression with siRNA dramatically decreased cell proliferation up to the level of complete inhibition of cell proliferation. FACS data revealed reduced S phase cells in RPLP1 silencing-cells compared to control, suggesting that RPLP1-mediated regulation of cell proliferation is partly mediated through regulation of cell cycle progression. Analysis of gene expression profiles displayed down-regulation of genes involved in cellular metabolism and DNA replication. When we assessed 44 formalin fixed paraffin embedded breast tissues for SCNA of RPLP1/RPLP1P4, we observed RPLP1 amplifications in 44% of tumor DNAs with no amplification observed in normal tissues. The clear difference between tumors and normal tissues demonstrates frequent RPLP1 amplification events during tumorigenesis, which possibly drives an increase in RPLP1 expression and cell proliferation in cancer. Conclusion: Our data suggest a subtype-specific increased expression of a ribosomal protein and its pseudogene, potentially driven by copy number alterations in breast tumors. Future studies to understand the contributions of ribosomal protein genes and their pseudogenes to human neoplasia are warranted. Citation Format: Yoo J. Han, Olufunmilayo I. Olopade. Aberrant expression of ribosomal protein p1 and its pseudogene in breast tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3306. doi:10.1158/1538-7445.AM2014-3306

Comparative proteomic profiling reveals aberrant cell proliferation in the brain of embryonic Ts1Cje, a mouse model of Down syndrome

To identify molecular candidates involved in brain disabilities of Ts1Cje, a mouse model of Down syndrome (DS), we performed comparative proteomic analyses. Proteins extracted from the brains of postnatal wild-type (WT) and Ts1Cje mice were analyzed by two-dimensional gel electrophoresis (2-DE). No differences were detected in the proteins expressed in the whole brain between WT and Ts1Cje mice at postnatal day 0 and 3months of age. Five spots with differential expression in the brains of Ts1Cje mice were detected by 2-DE of brain proteins from WT and Ts1Cje embryos at embryonic day 14.5 (E14.5). These differentially expressed proteins in Ts1Cje embryos were identified as calcyclin-binding protein (CACYBP), nucleoside diphosphate kinase-B (NDPK-B), transketolase (TK), pyruvate kinase (PK), and 60S acidic ribosomal protein P0 (RPLP0) by peptide mass fingerprinting. CACYBP and NDPK-B were involved in cell proliferation, whereas TK and PK were associated with energy metabolism. Experiments on cell proliferation, an in vivo bromodeoxyuridine (BrdU)-labeling experiment, and immunohistochemical analysis for phospho-histone H3 (an M-phase marker) demonstrated increased numbers of BrdU-positive and M-phase cells in the ganglionic eminence. Our findings suggest that the dysregulated expression of proteins demonstrated by comparative proteomic analysis could be a factor in increased cell proliferation, which may be associated with abnormalities in DS brain during embryonic life.

Identification of the Novel Interacting Partners of the Mammalian Target of Rapamycin Complex 1 in Human CCRF-CEM and HEK293 Cells

The present study was undertaken to identify proteins that interact with the mammalian target of rapamycin complex 1 (mTORC1) to enable it to carry out its crucial cell signaling functions. Endogenous and myc-tag mTORC1 was purified, in-gel tryptic digested and then identified by nano-LC ESI Q-TOF MS/MS analysis. A total of nine novel interacting proteins were identified in both endogenous and myc-tag mTORC1 purifications. These new mTORC1 interacting partners include heterogeneous nuclear ribonucleoproteins A2/B1, enhancer of mRNA decapping protein 4, 60S acidic ribosomal protein, P0, nucleolin, dynamin 2, glyceraldehyde 3 phosphate dehydrogenase, 2-oxoglutarate dehydrogenase, glycosyl transferase 25 family member 1 and prohibitin 2. Furthermore hnRNP A2/B1 and dynamin 2 interaction with mTORC1 was confirmed on immunoblotting. The present study has for the first time identified novel interacting partners of mTORC1 in human T lymphoblasts (CCRF-CEM) and human embryonic kidney (HEK293) cells. These new interacting proteins may offer new targets for therapeutic interventions in human diseases caused by perturbed mTORC1 signaling.

Выбор эндогенных контрольных генов для изучения экспрессии генов в периферической крови больных ишемической болезнью сердца

The selection of stable endogenous control genes is critical for normalization of quantitative real-time PCR (qPCR) data. In this study, we aimed to identify a suitable set of control genes to be used as endogenous references for gene expression evaluation in human peripheral blood samples among coronary artery disease patients. The expression levels of 12 endogenous control genes procured from TATAA Biocenter (Goteborg, Sweden) were measured in five acute coronary syndrome patients and five chronic stable angina patients. Gene expression stability was analyzed using two different software applications i.e geNorm and NormFinder. Results suggested that beta-glucuronidase is the most stable endogenous control, followed by hypoxanthine-guanine phosphoribosyltransferase. The NormFinder analysis further confirmed that beta-glucuronidase and hypoxanthine-guanine phosphoribosyltransferase were on the first rank order with the most stable expression among endogenous control genes analyzed and 60S acidic ribosomal protein P0. Besides this, the expression levels of 18S rRNA were revealed to be highly variable between coronary heart disease patients. We thus recommend the use of beta-glucuronidase and hypoxanthine-guanine phosphoribosyltransferase as reference genes for accurate normalization of relative quantities of gene expression levels in coronary artery disease patients using qPCR. Also the use of 18S rRNA as a control gene should be avoided.

Abstract 200: A transcribed BRCA1 pseudogene regulates gene expression and promotes breast cancer cell proliferation.

Abstract Background: Pseudogenes, originally derived from protein coding genes, have generally been considered to represent non-functional artifacts of evolutionary processes due to degenerative features such as the absence of promoters or the presence of premature stop codons. Pseudogenes, however, account for a significant fraction of the human genome with an estimated number of ∼20,000 comparable to the number of protein-coding genes (∼27,000). Mounting evidence indicates potential biological functions for pseudogenes as regulators of gene expression by generating siRNA, sequestering microRNAs, or regulating RNA stability. Despite the abundance of pseudogenes in the human genome, the pathophysiological roles of pseudogenes remain poorly understood. Methods: qRT-PCR was performed in 15 breast cancer cell lines with custom-designed SYBR assays. Somatic copy number alterations (SCNAs) were measured in 44 normal and breast tumour tissues from patients recruited from the U of Chicago Breast SPORE using custom-designed TaqMan CNV assays. Cell proliferation and invasion assays were performed in bioengineered breast cancer cells with BRCA1P1 over-expression. Affymetrix GeneChip U133 and miRNA 2.0 microarrays were utilized for gene and microRNA profiling. Results: We analyzed the structure of a pseudogene of breast cancer susceptibility protein1 (BRCA1P1) duplicated from the functional gene (BRCA1) with only three exons (exon 1a, 1b & 2). Comparison of DNA sequences revealed that BRCA1P1 is a fusion pseudogene with 343 bp sequences of 60S acidic ribosomal protein (RPLP1) inserted in the exon 1a. Because of a point mutation in the translation start site of exon 2, BRCA1P1 expresses noncoding RNAs (ncRNA) that accumulate in nuclei. BRCA1P1 is ubiquitously transcribed in human normal tissues and highly expressed in breast cancer cell lines. Frequent BRCA1P1 amplification events (44%) were observed in breast tumor DNAs whereas no amplification occurred in normal breast tissues. The data indicate a potential contribution of BRCA1P1 amplification to an increase in BRCA1P1 expression in breast cancer. A significant increase in cell proliferation was observed in cells with BRCA1P1 over-expression (2.82 ± 0.32 fold) compared to cells transfected with control vectors. We are currently performing microarray as well as investigating the pseudogene interaction with BRCA1, RPLP1, or other miRNAs to elucidate mechanisms of cell proliferation regulation by BRCA1P1. Conclusion: This novel study of BRCA1P1 suggests that pseudogenes may play critical roles in neoplastic processes of human cancer. A better understanding of the function of pseudogenes and elucidation of pathways driven by BRCA1P1 pseudogene may advance the field of cancer biology and could provide novel targets for preventive and therapeutic intervention of human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 200. doi:1538-7445.AM2012-200

Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors.

Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs.Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-011-1281-5) contains supplementary material, which is available to authorized users.

Characterization of human melanoma cell lines and melanocytes by proteome analysis

We have analyzed the proteomes of two human melanoma cell lines (A375 and 526), and of the human melanocytes, (FOM 78), by two-dimensional electrophoresis (2D-PAGE) and liquid chromatography – tandem mass spectrometry (LC-MS/MS). Our comparative proteomic analysis revealed that six proteins were over-expressed in both melanoma cell lines as compared to melanocytes: galectin-1, inosine-5'-monophosphate dehydrogenase 2, serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform, protein DJ-1, cyclophilin A and cofilin-1. We show, for the first time, that only specific isoforms of these molecules are over-expressed in melanoma. Different protein profiles were also found between each individual melanoma cell line and the melanocytes. s-Methyl-5-thioadenosine phosphorylase, ubiquitin and ribosomal protein S27 a precursor, the basic form of protein DJ-1, annexin a1, proliferation associated protein 2g4, isoform alfa-enolase of alfa-enolase, protein disulfide-isomerase precursor, and elongation factor 2 were more strongly expressed in A375 cells compared to melanocytes. In 526 cells, 60s acidic ribosomal protein p1 and calreticulin precursor were more highly expressed than in melanocytes. These molecular differences may help in better understanding melanoma development and its different responsiveness to therapies. The identified proteins could be exploited as biomarkers or therapeutic targets for melanoma.

Influence of acute cadmium exposure on the liver proteome of a teleost fish, ayu (Plecoglossus altivelis)

Cadmium (Cd) is a toxic heavy metal that causes the disruption of a variety of physiological processes. In this study, the effect of Cd on liver proteome of ayu, Plecoglossus altivelis, was investigated by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Twenty-three altered protein spots were successfully identified. They were involved in oxidative stress response, metal metabolism, methylation, and so on. The mRNA expression of 60S acidic ribosomal protein P0, heat shock protein 70, apolipoprotein A-I, betaine-homocysteine S-methyltransferase, parahox cluster neighbor, and transferrin was subsequently determined by real-time PCR. The mRNA expression of these genes was consistent with proteomic results. These findings enrich our knowledge on the influence of Cd toxicity to teleost fish, and may be worthy of further investigation to develop biomarkers.

Nitration of tryptophan in ribosomal proteins is a novel post-translational modification of differentiated and naïve PC12 cells

Neuron growth factor (NGF) signaling in PC12 cell, which is derived from pheochromocytoma of rat adrenal medulla, induces expression of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) production. Subsequently, NO causes differentiation of PC12 cell to neuronal cell with morphological changes, such as neurite extension. In this study, we showed that 6-nitrotryptophan-containing proteins were produced in PC12 cell (naïve PC12 cell) and/or NGF-induced PC12 cell (differentiated PC12 cell). Western blot analysis of the protein extract of naïve PC12 cell and differentiated PC12 cell using anti 6-nitrotryptophan antibody showed several immunoreactive bands, which were subsequently subjected to trypsin digestion and LC–ESI–MS–MS analysis. The peptides from five ribosomal proteins, namely, 60S ribosomal protein L7 (Trp154), 60S acidic ribosomal protein P1 (Trp43), 40S ribosomal protein S2 (Trp60), 40S ribosomal protein S6 (Trp45), and 40S ribosomal protein S19 (Trp52), were identified as nitrotryptophan residue-containing proteins with significant ion score levels ( p < 0.05). Among these, tryptophan nitration was observed only in differentiated PC12 cell for S19 protein, and only in naïve PC12 cell for L7 protein. Tryptophan nitration of the other ribosomal proteins P1, S2, and S6 was observed in both naive and differentiated PC12 cells. The positive signal of nitrotryptophan-containing proteins in the Western blotting around 16 kDa (Band 1), which includes 40S ribosomal protein S19, was suppressed by treatment with NOS inhibitor, L-NAME. The tryptophan nitration of 40S ribosomal protein was not observed by LC–ESI–MS–MS analysis of this sample. This is the first study to identify several specific sites of nitrated tryptophan on proteins not only in viable culture cells but also in a physiological process: cell differentiation.

Antigen-subtracted 2-DE/MS strategy, a novel proteomic analysis platform

In the present study, we developed a novel proteomic research strategy named antigen-subtracted 2-DE/MS strategy and applied it to comparative proteomic analysis of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-transformed human bronchial epithelial cell line (16HBE-C) and its parental cell line (16HBE) G0/G1 cells. Following pre-purification by ammonium sulfate precipitation, rabbit antibodies against 16HBE G0/G1 cells were coupled with protein A/G PLUS-agarose under the maximal coupling rate of about 50%. The agarose-antibody complex was then used in immunoprecipitation known as antigen subtraction. When the mass ratio of antibody to the sample was 2.5-3:1, the subtraction rates for 16HBE and 16HBE-C G0/G1 cell proteins were 44 and 34%, respectively. Both subtracted and unsubtracted samples were then subjected to the 2-DE resolution. In 16HBE-subtracted 2-DE maps, 315 protein spots were subtracted and 49 new protein spots were detected, whereas in 16HBE-C-subtracted 2-DE maps, 287 protein spots were subtracted and 33 new protein spots were detected. Taken together, antigen subtraction results in 65 new protein spots being allowed to be detected, therefore, makes it possible to get more information of the samples. Finally, 4 protein spots only detected in 16HBE-C-subtracted 2-DE maps were analyzed by the Q-TOF MS/MS, and successfully identified as U6 snRNA-associated Sm-like protein LSm3, 60S acidic ribosomal protein P1, Peroxiredoxin-6 and 60S acidic ribosomal protein P2. These proteins are involved in carcinogenesis, oxidation stress and protein synthesis. In conclusion, the antigen-subtracted 2-DE/MS strategy could reduce the complexities of protein samples and therefore, improve the resolution for the sample analysis.

Cloning and Characterization of the Acidic Ribosomal Protein P2 ofCryptosporidium parvum, a New 17-Kilodalton Antigen

Cryptosporidium infection is commonly observed among children and immunocompromised individuals in developing countries, but large-scale outbreaks of disease among adults have not been reported. In contrast, outbreaks of cryptosporidiosis in the United States and Canada are increasingly common among patients of all ages. Thus, it seems likely that residents of regions where Cryptosporidium is highly endemic acquire some level of immunity, while residents of the developed world do not. A new immunodominant Cryptosporidium parvum antigen in the 15- to 17-kDa size range was identified as the Cryptosporidium parvum 60S acidic ribosomal protein P2 (CpP2). We developed a recombinant protein-based enzyme-linked immunosorbent assay for serologic population surveillance for antibodies that was 89% sensitive and 92% specific relative to the results of the large-format Western blot assay. The human IgG response is directed almost exclusively toward the highly conserved, carboxy-terminal 15 amino acids of the protein. Although IgG antibody cross-reactivity was documented with sera from patients with acute babesiosis, the development of an anti-CpP2 antibody response in our Peru study population correlated better with Cryptosporidium infection than with infection by any other parasitic protozoan. In Haiti, the prevalence of antibodies to CpP2 plateaus at 11 to 20 years of age. Because anti-CpP2 IgG antibodies were found only among residents of countries in the developing world where Cryptosporidium infection occurs early and often, we propose that this response may be a proxy for the intensity of infection and for acquired immunity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin up-regulates stathmin 1 in the eye: Suggestive evidence for the involvement of stathmin 1 in accelerated eye opening in mice induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

When pregnant mice were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the average time to eye opening in the offspring was shortened by about a day. How acceleration of eye opening by TCDD occurs remains unknown. To reveal the underlying mechanisms of the accelerated eye opening, pregnant mice were intraperitoneally injected with corn oil or TCDD at GD (gestation day) 11, and tissues around the eye of neonatal mice were subject to proteome analysis and RT-PCR. Upon TCDD administration, translationally controlled tumor protein (TCTP) and 60S acidic ribosomal protein p2 (RLA2) were reduced, while stathmin 1(STMN1) was increased, at both protein and mRNA levels. One hypothetical mechanism for eye opening is the proliferation of corneal epithelial cells before eye opening. STMN1, but not TCTP and RLA2, was up-regulated in immortalized human corneal epithelial cells (HCE-T) by TCDD, which promoted proliferation of HCE-T probably by accelerating the G1/S transition. Down-regulation of STMN1 by the antisense oligonucleotide technology inhibited proliferation of HCE-T, suggesting that STMN1, of which expression is enhanced by TCDD, may be involved in accelerated eye opening, probably by stimulating proliferation of corneal epithelial cells.

Proteomic analysis of EZH2 downstream target proteins in hepatocellular carcinoma

Enhancer of zeste homolog 2 (EZH2) is suggested to be a potential therapeutic target and a diagnostic marker for cancer. Our previous study also showed the critical role of EZH2 in hepatocellular carcinoma (HCC) tumorigenesis. The present study is aimed at revealing the comprehensive downstream pathways of EZH2 by functional proteomic profiling. Lentivirus mediated RNA interference (RNAi) was employed to knockdown EZH2 in HCC cells. The 2-DE was employed to compare the expression profile difference between parental and EZH2-knockdown HCC cells. In total, 28 spots were differentially expressed during EZH2 inhibition. Among all, 18 proteins were identified by PMF with MALDI-TOF MS. Western blotting further validated upregulation of 60S acidic ribosomal protein P0 (L10E), and downregulation of two proteins with EZH2 inhibition: stathmin1 and probable protein disulfide isomerase (PDI) ER-60 precursor (ERp57). Moreover, L10E was downregulated with overexpression of EZH2 in hepatocytes, and L10E reversed the effect of EZH2 on cell proliferation, suggesting it a downstream target of EZH2. The comprehensive and comparative analyses of proteins associated with EZH2 could further our understanding on the downstream signal cascade of EZH2 leading to tumorigenesis.

Analysis of Ras‐induced oncogenic transformation of NIH‐3T3 cells using differential‐display 2‐DE proteomics

Ras proteins control at least three crucial signalling networks responsible for several cellular processes including anchorage independence, survival, and proliferation. Point mutations in one of the three ras genes are frequent in human tumours. In these tumours, Ras oncoproteins contribute significantly to the malignant phenotype, including deregulation of tumour-cell growth, apoptosis and invasiveness, and the ability to induce angiogenesis. Although significant strides have been made in understanding Ras biology, the collaborative actions of Ras effectors are still poorly understood. Here, we describe a proteomics approach to study global changes in protein expression in Ras-transformed NIH3T3 cells. We exploited 2-D difference gel electrophoresis (DIGE) for pre-separation fluorescent protein labelling with three separate dyes to reduce gel-to-gel variability, to increase sensitivity and dynamic range of protein detection, and to enhance quantification of dysregulated proteins. Proteins dysregulated (> 1.5-fold) by oncogenic Ras transformation reported to be implicated in Ras-regulated pathways include S-methyl-5-thioadenosine phosphorylase, stress-induced-phosphoprotein 1, galectin-1, annexin A7 (synexin), 60S acidic ribosomal protein P0, serine/threonine protein phosphatase type 1 (PP1alpha) and prohibitin. Significantly, we report for the first time the expression of the newly discovered cytokine IL-25 (or IL-17E) in mouse embryonic fibroblast cells and its down-regulation (2.1-fold) upon Ras-induced oncogenic transformation.

Nuclear protein profiling of Jurkat cells during heat stress‐induced apoptosis by 2‐DE and MS/MS

Heat stress causes severe constraints on numerous physiological functions of cells, such as the repression of splicing of mRNA precursors. In this study, we performed proteomic profiling of a nuclear fraction of Jurkat cells during heat stress using 2-DE and LC-MS/MS. We found 10 protein spots whose expression had changed after heat stress at 43 degrees C for 30 min. Seven of those protein spots, periodic tryptophan protein 1 homolog (PWP1), importin beta-1 subunit, sumoylated protein, splicing factor 3a subunit 3 (SF3a3), TAR DNA-binding protein 43, U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit (U2AF35) and small ubiquitin-related modifier-1 (SUMO-1) were downregulated, while three other protein spots, Protein SET, 40S ribosomal protein SA and 60S acidic ribosomal protein P0 were upregulated by the heat stress. We focused on the downregulation of two splicing factors, which might participate in the repression of pre-mRNA processing by heat stress, leading to cell apoptosis.

Bioprocessing of avian eggshells and eggshell membranes using lactic acid bacteria

Lactic acid fermentation combined with chemical treatments is a new alternative to chemical extraction of by-products from organic wastes such as prawn and krill shells. This study investigated the potential of whole eggshells (WES) and eggshell membranes (ESM) fermentation to provide a multi-product process, thereby eliminating waste and generating revenue. Dry WES and dry ESM were treated separately in a 2-L reactor using a glucose solution, with and without citric acid solution (CA) and commercial lactic acid bacteria as inoculants, to determine the protein and carbohydrate contents from the final products. Through this process, protein and calcium removal is achieved by enzymatic action on the tissues and by dissolving of calcium by organic acids, respectively. The best fermentation condition was obtained by including CA in the process. Mass balances from the WES and ESM treatments were carried out to identify the phases with major amount of protein, carbohydrate and calcium. In the WES treatments with/without CA, the liquid phase turned out to contain the major amount of protein (85.4 and 73.2%) from the final product. On the other hand, the solid phase turned out to contain mainly ash (80.47 and 80.49%), with high calcium content, from the final product. The high-solubility dialysed liquid phase (LP1) from the ESM treatment turned out to contain the major amount both of soluble protein (88.3%) and of exopolysaccharides expressed as carbohydrates (4.3%) and the low-solubility dialysed liquid phase (LP2) turned out to contain the major amount of insoluble protein (60.2%) from the final product. In addition, the first-precipitation solid phase (SP1) from the ESM treatment turned out to contain the major amount of ash (60.5%) from the final product. The foamy phase and the liquid phase from the WES treatment using CA were analysed by SDS–PAGE, two-dimensional electrophoresis and electrospray ionisation ion-trap mass spectrometry. Four proteins related to the hen eggshells were identified from these phases: chicken heat shock protein (Gallus gallus), glucose-regulated protein precursor, calcyclin and 60s acidic ribosomal protein p2 were identified in the liquid phase and a protein homologous with calmodulin, a ubiquitous intracellular Ca2+ receptor, was identified in the foamy phase. Copyright © 2006 Society of Chemical Industry

Isolation and Characterization of a cDNA Clone Encoding One IgE-Binding Fragment of Penicillium brevicompactum

Background:The abundance of allergenic Penicillium species has been associated with an increased incidence of childhood asthma and pulmonary bleeding. Penicillium brevicompactum has been identified as the most prevalent indoorspecies of this genus. However, detailed studies on the allergens of the ubiquitous Penicillium species are still lacking. For the characterization of allergens of prevalent Penicillium species, molecular cloning of the allergen genes of P. brevicompactum was performed in the present study. Methods:A phage cDNA library of P. brevicompactum was constructed in Uni-ZAP^® XR vector using mRNA isolated from the organism. The cDNA library of P.brevicompactum was screened using pooled atopic sera. Results: Screening of P. brevicompactum cDNA library resulted in one positive clone encoding an estimated molecular weight of 11 kDa polypeptide, rich in acidic residues (>20%), with a pI of 3.87. This clone was designated as Pen b 26 and found to be reactive only against the atopic sera obtained from individuals sensitive to P. brevicompactum. The amino acid sequence analysis of Pen b 26 revealed that it had strong homology to the 60S acidic ribosomal protein P1 family from different eukaryotic sources, predominantly fungal aero-allergens. Other features of Pen b 26 including having high alpha-helical content (>50%), alanine-rich residues (>20%), and a well-conserved C-terminal epitope region fits well into the common properties of 60S acidic ribosomal proteins. Conclusions:The results obtained suggest that the allergenic clone, Pen b 26 is a 60S acidic ribosomal protein P1 of P. brevicompactum and shows strong similarity to other P1 family proteins.