High Level Of Protein Synthesis Research Articles

The Up-Regulation of Ribosomal Proteins Further Regulates Protein Expression Profile in Female Schistosoma japonicum after Pairing

BackgroundPairing of Schistosoma males and females leads to and maintains female sexual maturation. However, the mechanism by which pairing facilitates sexual maturation of females is not clear. An increasing body of evidence suggests that ribosomal proteins have regulatory rather than constitutive roles in protein translation.Methodology/Principal FindingsTo investigate the effect of ribosome regulation on female sex maturation, Solexa and iTRAQ techniques were used to analyze the relationship between ribosomal gene or protein expression and sexual development of Schistosoma females. In the present study, considerably higher number of ribosomal genes or proteins were found to be differentially expressed in paired 23-day-old females. Moreover, mature female-specific proteins associated with egg production, such as ferritin-1 heavy chain and superoxide dismutase, were selectively highly expressed in paired females, rather than higher level of protein synthesis of all transcripts compared with those in unpaired 23-day-old females. Furthermore, other developmental stages were utilized to investigate different expression pattern of ribosomal proteins in females by analysing 18-day-old female schistosomula from single- or double-sex infections to determine the relationship between ribosomal protein expression pattern and development. Results showed that undeveloped 18-day-old females from single- and double-sex infections, as well as 23-day-old unpaired females, possessed similar ribosomal protein expression patterns, which were distinct from those in 23-day-old paired females.Conclusions/SignificanceOur findings reveal that the pairing of females and males triggers a specialized ribosomal protein expression profile which further regulates the protein profile for sexual maturation in Schistosoma japonicum, based on its gene expression profile.

The deadly connection between endoplasmic reticulum, Ca2+, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells

The endoplasmic reticulum (ER) is involved in Ca(2+) signaling and protein processing. Accumulation of unfolded proteins following ER Ca(2+) depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR. Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription-PCR, viability assays, and single cell Ca(2+) imaging. ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca(2+), activation of ER-associated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca(2+). Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca(2+) permeability causes opposite effects on ERSR deployment and death of malignant glioma cells. Excessive ER Ca(2+) loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies.

High performance CHO cell line development platform for enhanced production of recombinant proteins including difficult-to-express proteins

Background In an effort to improve product yield of mammalian cell lines, we have previously demonstrated that our proprietary DNA elements, Selexis Genetic Elements (SGEs), increase the transcription of a given transgene, thus boosting the overall expression of a therapeutic protein drug in mammalian cells [1]. However, there are additional cellular bottlenecks, notably in the molecular machineries of the secretory pathways. Most importantly, mammalian cells have some limitations in their intrinsic capacity to manage high level of protein synthesis as well as folding recombinant proteins. These bottlenecks often lead to increased cellular stress and, therefore, low production rates.

Excitation BolsTORs Motor Neurons in ALS Mice

It is unclear why motor neurons selectively degenerate in amyotrophic lateral sclerosis (ALS). Saxena etal. (2013) demonstrate that excitation of motor neurons can prevent their demise in a mouse model of inherited ALS by a mechanism involving the mTOR pathway.

Calcium Carbonate Mineralization: Involvement of Extracellular Polymeric Materials Isolated from Calcifying Bacteria

This study highlights the role of specific outer bacterial structures, such as the glycocalix, in calcium carbonate crystallization in vitro. We describe the formation of calcite crystals by extracellular polymeric materials, such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) isolated from Bacillus firmus and Nocardia calcarea. Organic matrices were isolated from calcifying bacteria grown on synthetic medium--in the presence or absence of calcium ions--and their effect on calcite precipitation was assessed. Scanning electron microscopy observations and energy dispersive X-ray spectrometry analysis showed that CPS and EPS fractions were involved in calcium carbonate precipitation, not only serving as nucleation sites but also through a direct role in crystal formation. The utilization of different synthetic media, with and without addition of calcium ions, influenced the biofilm production and protein profile of extracellular polymeric materials. Proteins of CPS fractions with a molecular mass between 25 and 70 kDa were overexpressed when calcium ions were present in the medium. This higher level of protein synthesis could be related to the active process of bioprecipitation.

A test of alternate models for increased tissue nitrogen isotope ratios during fasting in hibernating arctic ground squirrels

We describe two models explaining the increase in tissue nitrogen isotope ratios (δ(15)N) that occurs during fasting in animals. The catabolic model posits that protein breakdown selectively removes the lighter isotope of nitrogen ((14)N) from catabolized tissues, causing an increase in the proportion of heavy nitrogen isotope ((15)N). The anabolic model posits that protein synthesis during fasting results in elevated δ(15)N values, as the unreplaced loss of (14)N to urea results in a higher proportion of (15)N in plasma amino acids used for protein synthesis. We effected a range of lean mass loss in arctic ground squirrels (Urocitellus parryii) fasting during hibernation and then collected organ and muscle tissues for analysis of δ(15)N values. The catabolic model predicts increased δ(15)N values in both liver and muscle, as these tissues undergo significant catabolism during hibernation. The anabolic model predicts no change in muscle, but an increase in δ(15)N values in liver, which has high levels of protein synthesis during euthermic phases of hibernation. We found a significant increase in liver δ(15)N values and no change in muscle δ(15)N values with lean mass loss, which supports the anabolic model. Heart, small intestine and brown adipose tissue also showed an increase in δ(15)N values, indicating protein synthesis in these organ tissues during hibernation. Urine was 3.8% lighter than plasma, and both urine and plasma increased in δ(15)N values with lean mass loss. This study helps clarify the mechanisms causing δ(15)N change during nutritional stress, thus increasing its utility for physiological research and reconciling previously contradictory results.

Physiological correlates of seasonal growth patterns in lake trout Salvelinus namaycush

Physiological correlates of seasonal growth patterns were measured in lake trout Salvelinus namaycush from two populations with contrasting diets (zooplankton-dominated diet in Louisa Lake; fish-dominated diet in Opeongo Lake). Fish in Opeongo Lake grew faster and were in better condition than fish in Louisa Lake. The most prominent biochemical difference between populations was higher citrate synthase (CS) and cytochrome c oxidase activity in the white muscle of fish from Opeongo Lake, indicating greater sustained swimming activity in this lake. In contrast, lactate dehydrogenase (LDH) activity in white muscle, an indicator of capacity for burst swimming, was similar between lakes. Nucleoside diphosphate kinase (NDPK) activity in white muscle, an indicator of protein synthesis, was higher in Opeongo Lake than in Louisa Lake but only in the autumn. In both lakes, protein concentration and therefore nutritional status increased as the growing season progressed from spring to summer to autumn. Biochemical indicators of growth and activity showed similar seasonal patterns in the two lakes with the spring characterized by high NDPK, high CS and high LDH activities (i.e. high levels of protein synthesis in association with high aerobic and anaerobic activities). These results suggest high foraging effort and allocation to growth early in the growing season in both lakes.

Identification of Differentially Expressed Sequence Tags in Rapidly Elongating Phyllostachys pubescens Internodes by Suppressive Subtractive Hybridization

Bamboo shoots grow quickly through the rapid elongation of internodes, but the precise molecular mechanisms underlying this process remain unknown. We used a combination of suppressive subtractive hybridization (SSH), dot blotting, sequencing and bioinformatics to identify Phyllostachys pubescens genes that are differentially expressed in rapidly elongating vs. static internodes (SIs). We isolated 1020 expressed sequence tags (ESTs) by SSH, 173 of which were shown to be differentially expressed by dot blotting. We then sequenced the 20 ESTs showing the greatest difference in expression, 13 of which were preferentially expressed in elongating internodes and seven in SIs. Functional characterization of the ESTs showed that rapid internode elongation requires meristem initiation and proliferation, high-level protein synthesis, cellular respiration, and cell wall synthesis, as well as the regulation of the activated methyl cycle, gibberellin and brassinosteroid biosynthesis, and their signal transduction pathways.

A New Strategy to Identify and Annotate Human RPE-Specific Gene Expression

BackgroundTo identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa.MethodologyRPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44 k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software.Principal FindingsOur previous 22 k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways.ConclusionsIn this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE.

Functional annotation of the human retinal pigment epithelium transcriptome

BackgroundTo determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63–78 years) were laser dissected and used for 22k microarray studies (Agilent technologies). Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software.ResultsIn total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194) expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776) genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194) of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM) composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover.ConclusionOur study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the RPE, and is useful for candidate retinal disease gene identification or gene dose-dependent therapeutic studies.

Overexpression of eukaryotic elongation factor eEF2 in gastrointestinal cancers and its involvement in G2/M progression in the cell cycle

A high level protein synthesis is one of the characteristics of cancer cells. The aim of this study is to show the contribution of eukaryotic elongation factor 2 (eEF2), which plays an essential role in the polypeptide chain elongation step, in the tumorigenesis of gastrointestinal cancers. In the present study, we demonstrated by using immunohistochemistry that eEF2 protein was overexpressed in 92.9% (13 of 14) of gastric and 91.7% (22 of 24) of colorectal cancers. No mutations were found in any of the exons of the eEF2 gene in six gastric and six colorectal cancers. Knockdown of eEF2 by eEF2-specific short-hairpin RNA (shEF2) inhibited cancer cell growth in two gastric cancer cell lines, AZ-521 and MKN28, and one colon cancer cell line, SW620. Flow cytometric analysis showed that knockdown of eEF2 induced G2/M arrest and resulted in inactivation of Akt and cdc2 (a G2/M regulator) and activation of eEF2 kinase (a negative regulator of eEF2) in these cancer cells. Conversely, forced expression of eEF2 in AZ-521 cells significantly enhanced the cell growth through promotion of G2/M progression in cell cycle, activated Akt and cdc2, and inactivated eEF2 kinase. Furthermore, forced expression of eEF2 in these cancer cells enhanced in vivo tumorigenicity in a mouse xenograft model. These results showed that overexpressed eEF2 in gastrointestinal cancers promoted G2/M progression and enhanced their cell growth in vitro and in vivo. These results also suggested a novel linkage between translational elongation and cell cycle mechanisms, implying that the linkage might play an important role to orchestrate the deregulated translation and cell cycle mechanisms for promotion of the development of gastrointestinal cancers.

High level protein expression in mammalian cells using a safe viral vector: Modified vaccinia virus Ankara

Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of β-galactosidase, about 15 mg protein could be produced from 10 8 BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production.

Heat-Shock Response of the Upper Intertidal BarnacleBalanus glandula:Thermal Stress and Acclimation

In the intertidal zone in the Pacific Northwest, body temperatures of sessile marine organisms can reach 35 degrees C for an extended time during low tide, resulting in potential physiological stress. We used immunochemical assays to examine the effects of thermal stress on endogenous Hsp70 levels in the intertidal barnacle Balanus glandula. After thermal stress, endogenous Hsp70 levels did not increase above control levels in B. glandula exposed to 20 and 28 degrees C. In a separate experiment, endogenous Hsp70 levels were higher than control levels when B. glandula was exposed to 34 degrees C for 8.5 h. Although an induced heat-shock response was observed, levels of conjugated ubiquitin failed to indicate irreversible protein damage at temperatures up to 34 degrees C. With metabolic labeling, we examined temperature acclimation and thermally induced heat-shock proteins in B. glandula. An induced heat-shock response of proteins in the 70-kDa region (Hsp70) occurred in B. glandula above 23 degrees C. This heat-shock response was similar in molting and non-molting barnacles. Acclimation of B. glandula to relatively higher temperatures resulted in higher levels of protein synthesis in the 70-kDa region and lack of an upward shift in the induction temperature for heat-shock proteins. Our results suggest that B. glandula may be well adapted to life in the high intertidal zone but may lack the plasticity to acclimate to higher temperatures.

Diversity and Genetic Relationships among Seven West African Goat Breeds

This study was carried out to determine the genetic relationships among seven west African goat breeds: Casamance Goat (Kolda, Senegal), Labe Goat (Fouta Djallon, Guinea), three Sahel Goat (Djoloff, Senegal; Maradi, Niger; Gorgol, Mauritania) red Sokoto Goat (Maradi, Niger) and Guera goat (Atar, Mauritania).The polymorphism of six microsatellites and the α s1 -casein locus was analysed. The six microsatellite loci were polymorphic with a mean number of alleles ranging from 2.71 to 4.0. At the α s1 -casein locus, A and B were the most frequent alleles, which are known to be associated with a high level of protein synthesis. A neighbour-joining tree and a Principal Component Analysis were performed and the reliability of both methods was tested. Our study shows that the genetic relationships among the breeds analysed correspond to their geographical distribution and in addition, that the Labe Goat is strongly separated from the other breeds. Among the seven markers used, four have an effect on the distribution of breeds while three seem to be non-informative.

Proteomic Profiling of Escherichiacoli Proteins under High Cell Density Fed‐Batch Cultivation with Overexpression of Phosphogluconolactonase

In this study, we used proteomics to better understand the growth on glucose of Escherichia coli in high cell density, fed-batch cultures and the response to overexpression of plasmid-encoded 6-phosphogluconolactonase (PGL). Using liquid chromatography coupled to electrospray mass spectrometry, at least 300 proteins were identified in the cytosolic fraction of the six time points used to monitor the fermentation. The relative abundance changes of selected proteins were obtained by comparing the peak area of the corresponding peptides at a particular m/z (mass over charge ratio) value. During the time course of samples collected during the rapid growth achieved under batch and fed-batch conditions, both the control and recombinant E. coli strains showed up-regulation of proteins participating in the tricarboxylic acid (TCA) cycle, particularly acetyl-CoA synthetase (AcCoAS), malate dehydrogenase (MDH), and succinyl-CoA synthetase (SuccCoAS). In the recombinant strain culture, fumarase was up-regulated until 35 h after inoculation but was not in the control strain culture. In addition, the proteomic measurement detected up-regulation of three well-characterized binding transport proteins in both control and recombinant strains. The up-regulation of TCA cycle enzymes is consistent with the increase in growth rate observed in the cell culture. In addition, up-regulation of these proteins demonstrated the importance of both the pentose-phosphate shunt and TCA cycle to the increased biosynthetic activity required by a high level protein synthesis. This study shows the potential of proteomics using shotgun sequencing (LC/MS of tryptic digests) to measure global changes in protein abundance during a fermentation process and will facilitate the development of robust manufacturing systems.

A natural vaccinia virus promoter with exceptional capacity to direct protein synthesis

A survey of vaccinia virus promoters, through a reporter gene approach, has identified the viral I1L promoter as having exceptional activity. The I1L promoter exhibited over 10 times the activity of other vaccinia promoters and even rivaled the activity of the bacteriophage T7 promoter in the hybrid vaccinia/T7 expression system. The I1L promoter had high activity in both transient transfection experiments and in the context of recombinant viruses. The I1L promoter should be useful for high-level protein synthesis and poxvirus studies in general.

Preparation of Recombinant Mannan-Binding Protein with a Native Oligomeric Structure

This chapter investigates the preparation of recombinant mannan-binding protein (MBP) with a native oligomeric structure. Hepatoma cells using a vaccinia virus expression system and the recombinant protein have been demonstrated to be structurally and functionally similar to native human MBP. Vaccinia virus that has been used as a vector to introduce cDNAs into mammalian cells has several useful advantages, including a relatively high level of protein synthesis, proper folding, disulfide bond formation, glycosylation, and other posttranslational modifications. So, a vaccinia virus expression system seems to be useful for the recombinant preparation of highly assembled macromolecules such as MBP. The complement activation activity of MBP is assayed by a passive hemolysis using mannan-coated-sheep erythrocytes in the presence of a complement. The binding of MBP to the surfaces of mannan-coated erythrocytes triggers the complement cascade, resulting in the formation of membrane-attack complexes and cell lysis.

Overview of the Vaccinia Virus Expression System

The vaccinia virus expression system differs from others in that transcription occurs in the cytoplasm of mammalian cells rather than in the nucleus. As a vector, vaccinia virus has a number of useful characteristics, including a capacity that permits cloning large fragments of foreign DNA (20+ kbp) with retention of infectivity, a wide host range, a relatively high level of protein synthesis, and "appropriate" transport, secretion, processing, and posttranslational modifications as dictated by the primary structure of the expressed protein and the cell type used. This overview discusses the life cycle of the vaccinia virus along with effects of vaccinia infection. The vaccinia vector expression system is described along with specific steps for expressing genes using these vectors. Important safety considerations are also presented.

A six-nucleotide segment within the 3' untranslated region of hibiscus chlorotic ringspot virus plays an essential role in translational enhancement.

RNA plant viruses use various translational regulatory mechanisms to control their gene expression. Translational enhancement of viral mRNAs that leads to higher levels of protein synthesis from specific genes may be essential for the virus to successfully compete for cellular translational machinery. The control elements have yet to be analyzed for members of the genus Carmovirus, a small group of plant viruses with positive-sense RNA genomes. In this study, we examined the 3' untranslated region (UTR) of hibiscus chlorotic ringspot virus (HCRSV) genomic RNA (gRNA) and subgenomic RNA (sgRNA) for its role in the translational regulation of viral gene expression. The results showed that the 3' UTR of HCRSV significantly enhanced the translation of several open reading frames on gRNA and sgRNA and a viral gene in a bicistronic construct with an inserted internal ribosome entry site. Through deletion and mutagenesis studies of both the bicistronic construct and full-length gRNA, we demonstrated that a six-nucleotide sequence, GGGCAG, that is complementary to the 3' region of the 18S rRNA and a minimal length of 180 nucleotides are required for the enhancement of translation induced by the 3' UTR.

Immunohistochemical Analysis of Endothelial-Monocyte-Activating Polypeptide-II Expression in Vivo

Endothelial-monocyte activating polypeptide (EMAP)-II is a novel molecule with cytokine-like pro-inflammatory properties, inducing procoagulant activity on the surface of endothelial cells and monocyte/macrophages in vitro, as well as up-regulating E- and P-selectin expression. EMAP-II is chemotactic for monocytes/macrophages and neutrophils, and stimulates myeloperoxidase release from neutrophils. Injection of EMAP-II into the mouse footpad induces an acute inflammatory response, although some regression occurs in response to direct injection of EMAP-II into murine tumors. Very little is known about the expression of EMAP-II in normal tissues of mice or humans, or about its function in vivo. We developed polyclonal antibodies against EMAP-II using recombinant protein produced in Escherichia coli, and used these antibodies to carry out an immunohistochemical study of the occurrence and distribution of EMAP-II in human tissues. The distribution of EMAP-II protein is relatively restricted, occurring primarily in endocrine organs, in cells of neuroendocrine origin, but also in tissues with high turnover. EMAP-II is strongly expressed in secretory epithelial cells of the thyroid, pancreas, adrenal and salivary glands, among others, as well as in neurons and subsets of monocytes/macrophages. It is also found in the epithelium of the small and large intestines. We conclude that EMAP-II expression is usually, but not always, associated with tissues that display high turnover and high levels of protein synthesis.