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.     

Molecular Diagnostics of the Involvement of Visually Normal Mucosa in the Malignancy Process in Urothelial Bladder Cancer

Molecular Biology - Immunofluorescent method by flow cytometry was used to quantify the expression of the tumor-associated protein βIII-tubulin (TUBB3) in the tissue of urothelial bladder...     

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.     

Synthesis of biologically active human interferon α-2b in Nicotiana benthamiana

A method was developed for the production and purification of biologically active recombinant human interferon α-2b (rhIFN α-2b) synthesized by expression in Nicotiana benthamiana plants. A gene construct containing a modified hIFN α-2b gene was cloned in two vectors based on tobacco mosaic virus driven by an actin promoter from Arabidopsis thaliana (pA-IFN-A) and cauliflower mosaic virus driven by a 35S promoter (pA-IFN-S). The expression vectors were introduced into the plant cells by agroinfiltration. The maximum rates of synthesis achieved in the case of pA-IFN-A and pA-IFN-S 5 days after agroinfiltration were determined to be 200 and 20 mg per 1 kg of fresh leaves, respectively. The recombinant hIFN α-2b synthesized in the plant showed high antiviral and antitumor activity comparable with that of commercial drug.     

Functional role of carbohydrate residues in human immunoglobulin G and therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies (TMA) provide an important means for treating diseases that were previously considered untreatable. Currently more than 40 full-size TMAs created primarily based on immunoglobulin G1 are widely used for treating various illnesses. Glycosylation of TMA is among other numerous factors that affect their biological activity, effector functions, immunogenicity, and half-life in the patient’s serum. The importance of carbohydrate residues for activity of human serum immunoglobulin and TMA produced in animal cells is considered in this review, with emphasis given to N-glycosylation of the Fc fragment of the antibody.     

Trastuzumab and pertuzumab plant biosimilars: Modification of Asn297-linked glycan of the mAbs produced in a plant with fucosyltransferase and xylosyltransferase gene knockouts

Plant biosimilars of anticancer therapeutic antibodies are of interest not only because of the prospects of their practical use, but also as an instrument and object for study of plant protein glycosylation. In this work, we first designed a pertuzumab plant biosimilar (PPB) and investigated the composition of its Asn297-linked glycan in comparison with trastuzumab plant biosimilar (TPB). Both biosimilars were produced in wild-type (WT) Nicotiana benthamiana plant (PPBWT and TPB-WT) and transgenic ΔXTFT N. benthamiana plant with XT and FT genes knockout (PPB-ΔXTFT and TPBΔXTFT). Western blot analysis with anti-α1,3-fucose and anti-xylose antibodies, as well as a test with peptide-N-glycosidase F, confirmed the absence of α1,3-fucose and xylose in the Asn297-linked glycan of PPB-ΔXTFT and TPB-ΔXTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that PPB-WT and TPB-WT Asn297-linked glycans are mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297linked GnGn-type glycan contains α1,3-fucose and β1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguishes these plant biosimilars from human IgG. Analysis of TPB-ΔXTFT total carbohydrate content indicates the possibility of changing the composition of the carbohydrate profile not only of the Fc, but also of the Fab portion of an antibody produced in transgenic ΔXTFT N. benthamiana plants. Nevertheless, study of the antigen-binding capacity of the biosimilars showed that absence of xylose and fucose residues in the Asn297-linked glycans does not affect the ability of the glycomodified antibodies to interact with HER2/neu positive cancer cells.     

Dietary Methanol Regulates Human Gene Activity

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, HBA and HBB, 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, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.     

Overview of fusion tags for recombinant proteins

Virtually all recombinant proteins are now prepared using fusion domains also known as “tags”. The use of tags helps to solve some serious problems: to simplify procedures of protein isolation, to increase expression and solubility of the desired protein, to simplify protein refolding and increase its efficiency, and to prevent proteolysis. In this review, advantages and disadvantages of such fusion tags are analyzed and data on both well-known and new tags are generalized. The authors own data are also presented.     

Methanol may function as a cross-kingdom signal

Recently, we demonstrated that leaf wounding results in the synthesis of pectin methylesterase (PME), which causes the plant to release methanol into the air. Methanol emitted by a wounded plant increases the accumulation of methanol-inducible gene mRNA and enhances antibacterial resistance as well as cell-to-cell communication, which facilitates virus spreading in neighboring plants. We concluded that methanol is a signaling molecule involved in within-plant and plant-to-plant communication. Methanol is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of methanol into toxic formaldehyde. However, recent data showed that methanol is a natural compound in normal, healthy humans. These data call into question whether human methanol is a metabolic waste product or whether methanol has specific function in humans. Here, to reveal human methanol-responsive genes (MRGs), we used suppression subtractive hybridization cDNA libraries of HeLa cells lacking ADH and exposed to methanol. This design allowed us to exclude genes involved in formaldehyde and formic acid detoxification from our analysis. We identified MRGs and revealed a correlation between increases in methanol content in the plasma and changes in human leukocyte MRG mRNA levels after fresh salad consumption by volunteers. Subsequently, we showed that the methanol generated by the pectin/PME complex in the gastrointestinal tract of mice induces the up- and downregulation of brain MRG mRNA. We used an adapted Y-maze to measure the locomotor behavior of the mice while breathing wounded plant vapors in two-choice assays. We showed that mice prefer the odor of methanol to other plant volatiles and that methanol changed MRG mRNA accumulation in the mouse brain.We hypothesize that the methanol emitted by wounded plants may have a role in plant-animal signaling. The known positive effect of plant food intake on human health suggests a role for physiological methanol in human gene regulation.     

Destabilization of emulsions during extraction of antibiotics from natural solutions]

Various means for changing the aggregative stability of emulsions in the system of benzylpenicillin fermentation broth filtrate and butyl acetate were tested. It was shown that the use of desemulgators was more efficient than the use of some means for removing the desemulgating admixtures. Moreover, addition of surface active substances after the preliminary thermocoagulation resulted in maximum decreasing of the emulsion aggregative stability.