Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation

Main Strategies for the Identification of Neoantigens

Genetic instability of tumors leads to the appearance of numerous tumor-specific somatic mutations that could potentially result in the production of mutated peptides that are presented on the cell surface by the MHC molecules. Peptides of this kind are commonly called neoantigens. Their presence on the cell surface specifically distinguishes tumors from healthy tissues. This feature makes neoantigens a promising target for immunotherapy. The rapid evolution of high-throughput genomics and proteomics makes it possible to implement these techniques in clinical practice. In particular, they provide useful tools for the investigation of neoantigens. The most valuable genomic approach to this problem is whole-exome sequencing coupled with RNA-seq. High-throughput mass-spectrometry is another option for direct identification of MHC-bound peptides, which is capable of revealing the entire MHC-bound peptidome. Finally, structure-based predictions could significantly improve the understanding of physicochemical and structural features that affect the immunogenicity of peptides. The development of pipelines combining such tools could improve the accuracy of the peptide selection process and decrease the required time. Here we present a review of the main existing approaches to investigating the neoantigens and suggest a possible ideal pipeline that takes into account all modern trends in the context of neoantigen discovery

Plant-made trastuzumab (herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth.

BACKGROUND: Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells. METHODOLOGY/PRINCIPAL FINDINGS: We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells. CONCLUSIONS/SIGNIFICANCE: We conclude that PMT is active in suppression of cell proliferation and tumor growth

Dietary Methanol Regulates Human Gene Activity

<div><p>Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, <i>HBA</i> and <i>HBB</i>, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, <i>GAPDH</i> and <i>SNX27</i>, and genes revealed in this study, including <i>MME</i>, <i>SORL1</i>, <i>DDIT4</i>, <i>HBA</i> and <i>HBB</i>. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.</p></div

Microarray analysis of differentially regulated murine brain mRNAs after mouse inhalation of methanol and wounded leaf vapors.

<p>(A) Experimental set-up for the inhalation of vapors from wounded leaves by the mice. (B) Venn diagram of the genes that are differentially expressed after the inhalation of methanol and wounded leaf vapors compared with those after the inhalation of water vapor. The genes were analyzed using the J-Express gene expression analysis software. The number of genes commonly regulated is indicated in the intersection of the circles. All the genes (with an average fold-change ≥1.3-fold) included in this analysis showed significant changes in their expression compared with the control, with a <i>Q</i>-value <0.05.</p

qRT-PCR analysis of WBC mRNA content after red wine intake.

<p>The relative mRNA quantities after red wine intake was normalized to the mRNA levels before red wine intake. Student’s <i>t</i>-test <i>P</i>-values were calculated by using triple bloods samples of three volunteers.</p

List of significantly down- and up-regulated genes in the WBCs of volunteers after citrus pectin intake.

<p>*KEGG, Kyoto Encyclopedia of Genes and Genomes (<a href="http://www.genome.jp/kegg/" target="_blank">http://www.genome.jp/kegg/</a>).</p><p>**The National Center for Biotechnology Information (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">http://www.ncbi.nlm.nih.gov</a>).</p>&<p>Genes selected for further analysis.</p

MeOH appearance in human blood is accompanied by the formation of FA and EtOH molecules.

<p>The dynamics of MeOH and FA changes in blood plasma after administering pectin are shown. Each volunteer took capsules containing citrus pectin (6 g). After 30, 60, 90 and 120 min, blood samples were obtained and analyzed for MeOH and FA content by GC and HPLC, respectively. The inset shows the ethanol concentrations after pectin administration. The standard error bars are indicated. ***<i>P</i><0.001 (Student’s <i>t</i>-test, n = 8); n.s., not significantly different.</p

The MeOH, EtOH and FA content of human blood plasma after red wine and alcohol intake.

<p>Each of the eight volunteers drank 150(13.731% EtOH and 0.0424% MeOH content, respectively) (A) or 40% v/v ethanol (1 ml per kg body weight) (B). After 15, 30, 60, 90 and 120 min, blood samples were collected and analyzed for MeOH, EtOH and FA contents by GC and HPLC, respectively. The data are presented as the means ± SE. Student’s <i>t</i>-test <i>P</i>-values were evaluated to determine the statistical significance of the MeOH, EtOH and FA content differences before and after alcohol intake. ***<i>P</i><0.001 (Student’s <i>t</i>-test, n = 8); n.s., not significantly different.</p

Verification of the microarray data for hemoglobin gene expression.

<p>(A) Experimental set-up for the MeOH inhalation by the mice. (B) qRT-PCR analysis of murine brain mRNAs for the hemoglobin genes after methanol inhalation. The data shown represent five independent experiments. ***<i>P</i><0.001 (Student’s <i>t</i>-test).</p