Ribosomal Protein L10a Research Articles

Discovery of new glomerular disease-relevant genes by translational profiling of podocytes in vivo.

Identifying new biomarkers and therapeutic targets for podocytopathies such as focal segmental glomerulosclerosis (FSGS) requires a detailed analysis of transcriptional changes in podocytes over the course of disease. Here we used translating ribosome affinity purification (TRAP) to isolate and profile podocyte-specific mRNA in two different models of FSGS. Expressed eGFP-tagged ribosomal protein L10a in podocytes under the control of the Collagen-1α1 promoter enabled podocyte-specific mRNA isolation in a one-step process over the course of disease. This TRAP protocol robustly enriched known podocyte-specific mRNAs. We crossed col1α1-L10a mice with the actn4−/− and actn4+/K256E models of FSGS and analyzed podocyte transcriptional profiles at 2, 6 and 44 weeks of age. Two upregulated podocyte genes in murine FSGS (CXCL1 and DMPK) were found to be upregulated at the protein level in biopsies from patients with FSGS, validating this approach. There was no dilution of podocyte-specific transcripts during disease. These are the first podocyte-specific RNA expression datasets during aging and in two models of FSGS. This approach identified new podocyte proteins that are upregulated in FSGS and help define novel biomarkers and therapeutic targets for human glomerular disease.

Stimulation of ovarian development in white shrimp, Fenneropenaeus merguiensis De Man, with a recombinant ribosomal protein L10a

Abstract Ribosomal protein L10a (RpL10a) is encoded by an essential gene related to the early stages of ovarian development. A recombinant RpL10a protein was produced and evaluated to determine its ability to stimulate ovarian development in the white shrimp Fenneropenaeus merguiensis De Man. The transportation of injected recombinant RpL10a to the ovary was monitored using FITC-conjugated rRpL10a in an in vivo assay. FITC-rRpL10a was imported into the ovarian tissue within 24 h after injection, observed with a fluorescence microscope. The signal was not detected in muscle tissue; thus, muscle was not a target tissue. A dose of 180 μg of rRpL10a/shrimp was effective for inducing the ovaries to reach stages I and II of vitellogenesis within 7 days after the first injection. Observations of the ovarian natural autofluorescence in tissue sections were performed to determine the stage of vitellogenesis in F. merguiensis and were shown to provide the same results as were obtained using sections stained according to the standard hematoxylin and eosin staining procedure, which spent more time. Methyl farnesoate (MF) levels were examined via a HPLC technique in different stages of ovarian development following RpL10a injection, and the observed MF levels indicated that the concentration of MF in the hemolymph increased in stage II of vitellogenesis.

Erythropoietin enhances mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia through Akt/eNOS signalling pathway

In the paper by Qin et al. (2014), there were errors in Figure 1. The correct figure is reproduced here. Effects of silencing endothelial nitric oxide synthase (eNOS) gene with shRNA plasmid (A) H9c2 cardiomyocytes were successfully transfected with shRNA plasmid of eNOS, which presented with green fluorescence (200x, fluorescence microscope). (B) Comparison of mtDNA copy number between the cells treated only with 20 U/mL recombinant human erythropoietin (rhEPO) (EPO control) and those treated with 20 U/mL rhEPO and shRNA plamid (EPOþshRNA), based on the cytochrome c/b-actin ratio. (C) Comparison of peroxisome profilerator activated receptor gamma coactivator alpha (PGC-1a) mRNA expression, normalized by ribosomal protein L10A (RPL10A) mRNA. (D) Comparison of the expression and phosphorylation of endothelial nitric oxide synthase (eNOS). p-eNOS: phosphorylated eNOS. #P < 0.05 versus cells treated only with 20 U/mL rhEPO.

Expression profile of ribosomal protein L10a throughout gonadal development in rainbow trout (Oncorhynchus mykiss)

Ribosomal protein L10a (RpL10A) has been previously established as a stimulator during the early stages of ovarian development in both the banana prawn and the fruit fly. In order to develop a greater understanding of the role of this protein in vertebrates, the present study aimed to characterize the expression profile of rpl10a during gonadal development in fish. It was determined that the expression of rpl10a within genital ridges increased during embryonic development. Although rpl10a expression was observed in both gonadal somatic cells and primordial germ cells, higher levels of both transcript and protein expression were detected in somatic cells. rpl10a transcripts were observed in all of the adult tissues examined. Cellular level expression of rpl10a was subsequently characterized across various maturational stages using in situ hybridization and immunohistochemistry of both testes and ovaries. Analysis of tissue derived from the testis showed high levels of rpl10a expression within spermatogonia and the Sertoli cells attached to them. In ovarian tissue, rpl10a was strongly expressed in chromatin-nucleolus-stage and peri-nucleolus-stage oocytes. The relationship between rpl10a expression and regulation of gonadal development was confirmed using real-time PCR, which was performed in order to analyze rpl10a expression in testicular and ovarian tissues subsequent to incubation with salmon pituitary extract and various sex steroids for 24h. Among them, 11-ketotestosterone at 100ng/mL effectively up-regulated expression of rpl10a in testicular tissues, while 17β-estradiol down-regulated rpl10a expression in ovarian tissues. These results suggested that rpl10a played a role in the regulation of gonadal development in fish.

Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons

Drugs of abuse, such as cocaine, induce changes in gene expression and epigenetic marks including alterations in histone posttranslational modifications in striatal neurons. These changes are thought to participate in physiological memory mechanisms and to be critical for long-term behavioral alterations. However, the striatum is composed of multiple cell types, including two distinct populations of medium-sized spiny neurons, and little is known concerning the cell-type specificity of epigenetic modifications. To address this question we used bacterial artificial chromosome transgenic mice, which express EGFP fused to the N-terminus of the large subunit ribosomal protein L10a driven by the D1 or D2 dopamine receptor (D1R, D2R) promoter, respectively. Fluorescence in nucleoli was used to sort nuclei from D1R- or D2R-expressing neurons and to quantify by flow cytometry the cocaine-induced changes in histone acetylation and methylation specifically in these two types of nuclei. The two populations of medium-sized spiny neurons displayed different patterns of histone modifications 15 min or 24 h after a single injection of cocaine or 24 h after seven daily injections. In particular, acetylation of histone 3 on Lys 14 and of histone 4 on Lys 5 and 12, and methylation of histone 3 on Lys 9 exhibited distinct and persistent changes in the two cell types. Our data provide insights into the differential epigenetic responses to cocaine in D1R- and D2R-positive neurons and their potential regulation, which may participate in the persistent effects of cocaine in these neurons. The method described should have general utility for studying nuclear modifications in different types of neuronal or nonneuronal cell types.

Molecular mechanism of serotonin via methyl farnesoate in ovarian development of white shrimp: Fenneropenaeus merguiensis de Man

Ovarian maturation in shrimp broodstock is a complex process controlled by several factors. There is evidence that 5-HT (5-Hydroxytryptamine) can stimulate maturation. The mechanism of this effect was investigated by both in vitro and in vivo assays. Ovarian explants of Fenneropenaeus merguiensis de Man incubated with 5-HT had a significantly higher expression of the RPL10a (ribosomal protein L10a), SOP (shrimp ovarian peritrophin) and TCTP (translational controlled tumor protein) genes up to 4 h after being incubated in the presence of 1 μg/mL 5-HT, while there was no upregulation of the expression of these genes in 5-HT-treated muscle explants at any point of their incubation. The results of an in vivo assay indicated that 5-HT injection stimulated ovarian development to stage 3 (mature) of vitellogenesis. The transcripts of the RPL10a, SOP and TCTP in the ovaries at days 5 and 10 were significantly higher in the shrimp injected with 1 μg 5-HT/g B.W. compared to their controls. However, the RPL10a transcript was the only one that had a statistically significant increase from day 5 to day 10 ( P ≤ 0.05) after injection with 50 μg 5-HT/g B.W. 5-HT at 1 μg/g B.W. induced the presence of MF (methyl farnesoate) in the hemolymph and this increased to its highest level (1.1 ± 0.05 ng/mL) at day 5, but there was no significant increase observed in shrimp that received 50 μg/g B.W. of 5-HT at day 5. Although, ovarian development reached a mature stage of 29% after injection of both levels of 5-HT but the ovaries of shrimp injected with 50 μg 5-HT/g B.W. were observed to have 14% undeveloped ovaries. However, no undeveloped ovaries were detected in the group injected with 1 μg 5-HT/g B.W. Moreover, second stage ovaries were found at 29% and 14% in shrimp injected with 1 μg 5-HT/g B.W. and 50 μg 5-HT/g B.W., respectively. It was also shown that injection of 5-HT released MF into the hemolymph, and MF has been reported to be involved in the ovarian development in shrimp. There was a significant correlation observed between the ovarian development stimulated by 5-HT, the upregulation of the RPL10a, SOP and TCTP genes and the hemolymph MF level.

Evolution of Rhizaria: new insights from phylogenomic analysis of uncultivated protists

BackgroundRecent phylogenomic analyses have revolutionized our view of eukaryote evolution by revealing unexpected relationships between and within the eukaryotic supergroups. However, for several groups of uncultivable protists, only the ribosomal RNA genes and a handful of proteins are available, often leading to unresolved evolutionary relationships. A striking example concerns the supergroup Rhizaria, which comprises several groups of uncultivable free-living protists such as radiolarians, foraminiferans and gromiids, as well as the parasitic plasmodiophorids and haplosporids. Thus far, the relationships within this supergroup have been inferred almost exclusively from rRNA, actin, and polyubiquitin genes, and remain poorly resolved. To address this, we have generated large Expressed Sequence Tag (EST) datasets for 5 species of Rhizaria belonging to 3 important groups: Acantharea (Astrolonche sp., Phyllostaurus sp.), Phytomyxea (Spongospora subterranea, Plasmodiophora brassicae) and Gromiida (Gromia sphaerica).Results167 genes were selected for phylogenetic analyses based on the representation of at least one rhizarian species for each gene. Concatenation of these genes produced a supermatrix composed of 36,735 amino acid positions, including 10 rhizarians, 9 stramenopiles, and 9 alveolates. Phylogenomic analyses of this large dataset revealed a strongly supported clade grouping Foraminifera and Acantharea. The position of this clade within Rhizaria was sensitive to the method employed and the taxon sampling: Maximum Likelihood (ML) and Bayesian analyses using empirical model of evolution favoured an early divergence, whereas the CAT model and ML analyses with fast-evolving sites or the foraminiferan species Reticulomyxa filosa removed suggested a derived position, closely related to Gromia and Phytomyxea. In contrast to what has been previously reported, our analyses also uncovered the presence of the rhizarian-specific polyubiquitin insertion in Acantharea. Finally, this work reveals another possible rhizarian signature in the 60S ribosomal protein L10a.ConclusionsOur study provides new insights into the evolution of Rhizaria based on phylogenomic analyses of ESTs from three groups of previously under-sampled protists. It was enabled through the application of a recently developed method of transcriptome analysis, requiring very small amount of starting material. Our study illustrates the potential of this method to elucidate the early evolution of eukaryotes by providing large amount of data for uncultivable free-living and parasitic protists.

Characterization and Biological Activity of the Ribosomal Protein L10a of the White Shrimp: Fenneropenaeus merguiensis De Man During Vitellogenesis

The molecular events in the ovaries of Fenneropenaeus merguiensis De Man during vitellogenesis were investigated. The ribosomal protein L10a (RPL10a) was characterized and cloned. It consisted of 669 bp and the deduced polypeptide had 217 amino acids (GeneBank/EBI accession number FJ623402). The calculated molecular mass and pI were 25.7 kDa and 10.06, respectively. An immunohistochemical technique showed that RPL10a was located in the cytoplasm and nucleus of developing oocytes and follicle cells. Treatment of undeveloped ovarian explant cultures with recombinant histidine (His)-RPL10a stimulated the expression of translationally controlled tumor protein (TCTP), heat shock protein 70 (HSP70), and shrimp ovarian peritrophin (SOP) genes, previously shown to be involved in ovarian maturation. The transcripts of all three genes in the ovarian explants showed their highest expression after 4 h incubation with the His-RPL10a at 37 degrees C. The TCTP and HSP70 transcripts declined after 12 h, while the transcript of SOP remained high until 24 h. The His-RPL10a did not stimulate the expression of the TCTP, SOP, and HSP70 genes in shrimp muscle tissue. The information on the molecular behavior of the RPL10a in this study may, in the future, lead to new methods to stimulate ovarian development in shrimp.

Dynamic changes in gene expression during vitellogenic stages of the white shrimp:Fenneropenaeus merguiensisde Man

Ovarian maturation is a crucial step for shrimp brood stock. A suppressive subtractive hybridization was used to identify differentially expressed genes in the ovaries during vitellogenesis of Fenneropenaeus merguiensis. Three- to sevenfold up-regulated genes were selected. A blast search identified nine unique genes. The genes that may be involved in ovarian maturation, namely translationally controlled tumour protein (TCTP), heat shock protein 70 (HSP70), H-L(3)MBT-LIKE, shrimp ovarian peritrophin (SOP), vitellin (Vn), thrombospondin (TSP) and ribosomal protein L10a (RPL10a), were further studied. The transcripts of HSP70, TCTP, SOP and RPL10a in the ovary showed their highest expression in the early stage and declined in the later stages. In contrast, the transcripts of the H-L(3)MBT-LIKE, TSP and Vn genes increased from the early stage to be significantly up-regulated during the late stage. A comparison of gene expression among organs during the vitellogenesis showed that the transcripts of HSP70, SOP, H-L(3)MBT-LIKE and TSP were down-regulated in the brain, intestine, hepatopancreas and lymphoid (except for TSP) when compared with their expression in shrimp with non-developed ovaries. The mRNA of TCTP and RPL10a was significantly over-expressed in the lymphoid and heart, whereas TCTP transcripts were significantly down-regulated in the brain during the vitellogenesis. The molecular behaviour of the transcripts in this study may, in the future, lead to an ability to stimulate the ovarian development in shrimp.

A Translational Profiling Approach for the Molecular Characterization of CNS Cell Types

The cellular heterogeneity of the brain confounds efforts to elucidate the biological properties of distinct neuronal populations. Using bacterial artificial chromosome (BAC) transgenic mice that express EGFP-tagged ribosomal protein L10a in defined cell populations, we have developed a methodology for affinity purification of polysomal mRNAs from genetically defined cell populations in the brain. The utility of this approach is illustrated by the comparative analysis of four types of neurons, revealing hundreds of genes that distinguish these four cell populations. We find that even two morphologically indistinguishable, intermixed subclasses of medium spiny neurons display vastly different translational profiles and present examples of the physiological significance of such differences. This genetically targeted translating ribosome affinity purification (TRAP) methodology is a generalizable method useful for the identification of molecular changes in any genetically defined cell type in response to genetic alterations, disease, or pharmacological perturbations.

Early gene expression in human lymphocytes after gamma-irradiation–a genetic pattern with potential for biodosimetry

Purpose: Identification of early radiation response genes (ERG) in human lymphocytes after γ-irradiation by using the whole-human-genome DNA-microarrays and the evaluation of their possible role in rapid radiation biodosimetry by applying real-time quantitative polymerase chain reaction (RT-qPCR) methodology for validation in a small group of human individuals.Materials and methods: Whole blood from a healthy human donor was exposed at 37°C to 137Cs γ-radiations (absorbed dose: 1–4 Gy). Fifteen minutes following irradiation the lymphocytes were isolated from the blood (for 2 h at 20°C) and their gene expression was investigated using the DNA-microarrays. Subsequently, 14 genes were selected and validated using the TaqMan™ probes based upon the RT-qPCR assay within a group of 6 human donors.Results: A dose-related relative change in quantitative gene expression using the DNA-microarray assay was demonstrated in 24 of 102 genes. Up-regulation of expression was observed in 15 genes: CD69 (CD69 molecule), CDKN1A (cyclin-dependent kinase inhibitor 1A), EGR1 (early growth response 1), EGR4 (early growth response 4), FLJ35725 (chromosome 4 ORF 23), hCG2041177 (hCG – human Celera® Genome), hCG1643466.2, IFN-γ (interferon-γ), ISG20L (interferon stimulated exonuclease gene 20 kDa – like 1), c-JUN (jun oncogene), MDM2 (mouse double minute 2), MUC5B (mucine), PLK2 (polo-like kinase 2), RND1 (rho-family GTPase 1) and TNFSF9 (tumour necrosis factor superfamily member 9). Down-regulation of expression was found in the remaining nine genes: GRIK3 (glutamate receptor ionotropic kainate 3), hCG1985174, hCG1998530, hCG2038519, OCLN (occludin), RPL10A (ribosomal protein L10a), SERHL2 (serine hydrolase-like 2), SGK3 (serum/glucocorticoid regulated kinase 3) and STARD13 (START domain containing 13).Conclusion: A significant correlation between absorbed radiation dose and change in relative gene expression was particularly evident for EGR1, EGR4, IFN-γ, c-JUN and TNFSF9 (p ≤ 0.05). Results warrant the further investigation of these ERG as potential biodosimetric markers.

Ribosomal protein L10a, a bridge between trichosanthin and the ribosome

Trichosanthin is a type I ribosome-inactivating protein with many pharmacological activities. The trichosanthin-coupled Sepharose affinity purification revealed a protein, which was identified by mass spectrometry as the ribosomal protein L10a. The interaction between trichosanthin and recombinant L10a was further confirmed by in vitro binding assay. Kinetic analysis by surface plasmon resonance technology revealed that L10a had a high affinity to trichosanthin with a K D of 7.78 nM. The study with mutated forms of trichosanthin demonstrated that this specific association correlates with the ribosome-inactivating activity of trichosanthin. This finding might provide insight into the mechanisms by which trichosanthin inactivates ribosome and that underlies its pharmacological effect.

The Oxidative Stress-Sensitive yap1 Null Strain of Saccharomyces cerevisiae Becomes Resistant Due to Increased Carotenoid Levels upon the Introduction of the Chlamydomonas reinhardtii cDNA, Coding for the 60S Ribosomal Protein L10a

The Saccharomyces cerevisiae yap1 null strain was transformed with a Chlamydomonas reinhardtii cDNA expression library. A 688-bp cDNA fragment, coding for the 60S ribosomal protein L10a (RPL10a), restored the capacity of the S. cerevisiae yap1 null strain to resist oxidative stress. The rpl10a gene is a single-copy gene in C. reinhardtii and encodes a constitutively produced 1.35-kb mRNA. The deduced 214-residue amino acid sequence was highly related with RPL10a proteins from eukarya (between 46.1 and 63.7% identity) and archaea (between 24.5 and 29.2% identity). Resistant transformants were pink, due to increased carotenoid levels, with the same chemical structure as torularhodin, the main carotenoid of the pink yeast Rhodotorula mucilaginosa. The pink transformants showed high resistance levels against H2O2, paraquat, menadione, and UV light. Partial inhibition of the carotenoid synthesis by diphenylamine reduced the resistance levels, demonstrating the role of excess carotenoid synthesis in the resistance mechanism.

The Primary Structure of Rat Ribosomal Protein L10a

The amino acid sequence of the rat 60S ribosomal subunit protein L10a was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L10a has 217 amino acids and has a molecular weight of 24,815. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 10 copies of the L10a gene. The mRNA for the protein is about 760 nucleotides in length. Rat L10a is related to ribosomal proteins from other eukaryotes and from archaebacteria.