Synthesis Of Different Proteins Research Articles

Tissue-specific temperature dependence of RNA editing levels in zebrafish

BackgroundRNA editing by adenosine deaminase acting on RNA (ADAR) occurs in all metazoans and fulfils several functions. Here, we examined effects of acclimation temperature (27 °C, 18 °C,13 °C) on editing patterns in six tissues of zebrafish (Danio rerio).ResultsSites and total amounts of editing differed among tissues. Brain showed the highest levels, followed by gill and skin. In these highly edited tissues, decreases in temperatures led to large increases in total amounts of editing and changes in specific edited sites. Gene ontology analysis showed both similarities (e.g., endoplasmic reticulum stress response) and differences in editing among tissues. The majority of edited sites were in transcripts of transposable elements and the 3′UTR regions of protein coding genes. By experimental validation, translation efficiency was directly related to extent of editing of the 3′UTR region of an mRNA.ConclusionsRNA editing increases 3′UTR polymorphism and affects efficiency of translation. Such editing may lead to temperature-adaptive changes in the proteome through altering relative amounts of synthesis of different proteins.

Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli, and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies.

Protein stable isotope probing with H218O differentiated cold stress response at permissive temperatures from general growth at optimal conditions in Escherichia coli K12

Tracing isotopically labeled water into proteins allows for the detection of species-specific metabolic activity in complex communities. However, a stress response may alter the newly synthesized proteins. We traced 18-oxygen from heavy water into proteins of Escherichia coli K12 grown from permissive to retardant temperatures. All samples were analyzed using UPLC/Orbitrap Q-Exactive-MS/MS operating in positive electrospray ionization mode. We found that warmer temperatures resulted in significantly (P-value < 0.05) higher incorporation of 18-oxygen as seen by both substrate utilization as relative isotope abundance (RIA) and growth as labeling ratio (LR). However, the absolute number of peptides with incorporation of 18-oxygen showed no significant correlation to temperature, potentially caused by the synthesis of different proteins at low temperatures, namely, proteins related to cold stress response. Our results unveil the species-specific cold stress response of E. coli K12 that could be misinterpreted as general growth; this is why the quantity as RIA and LR but also the quality as absolute number of peptides with incorporation (relative abundance, RA) and their function must be considered to fully understand the activity of microbial communities.

Proteome analysis of sesame leaves in response to waterlogging stress at vegetative and flowering stages

Waterlogging, a major environmental stress, impairs plant growth and development and induces synthesis of different proteins. To understand the molecular mechanisms coupled with morpho-physiological alterations underlying waterlogging tolerance, the LTQ-FTICR MS/MS technique was employed to map the proteomes of leaves of sesame grown under control and waterlogged conditions. The waterlogging treatment caused dramatic alterations in morphological and biochemical properties of the leaves of sesame. For proteome analysis, more than 75 reproducible protein spots were identified on 2-DE gels wherein 51 protein spots (≥ 1.5-fold change) were used for analysis by mass spectrometry. Among 51 differentially abundant proteins, 20 were specific to the 10-leaf stage and 31 were specific to the flowering stage. Most of the differentially abundant proteins were involved in group metabolism, and energy and stress defense. Oxygen-evolving enhancer protein 1, ATP synthase subunit, heat shock proteins, glutamine synthetase, glyceraldehyde-3-phosphate dehydrogenase, and superoxide dismutase were upregulated under waterlogging. However, the photosynthesis- and protein biosynthesis-related proteins (e.g., ribulose-1,5-bisphosphate carboxylase/oxygenase activase, and S-adenosylmethionine synthase 1) were down-regulated under waterlogging. The protein interaction network indicates that energy metabolism- and stress- and defense-related proteins were involved in the protein-protein interaction network, which could form an indispensable network in sesame leaves. To this end, physiological results highlighted the impairment of photosyntheis, which is consistent with results obtained at the proteome level. The upregulation of metabolism-, energy-, and stress defense-related proteins in response to waterlogging stress may provide new insights into the complex mechanisms underlying waterlogging tolerance in sesame.

Identification of insulin gene variants in neonatal diabetes

Objectives: Permanent neonatal diabetes (PNDM) is caused by mutations in the genes responsible for the synthesis of different proteins that are important for the normal behavior of beta cells in the pancreas. Mutations in the insulin gene (INS) are considered as one of the causes of diabetes in neonates. This study aimed to investigate the genetic variations in the INS gene in a group of Egyptian infants diagnosed with PNDM.Methods: We screened exons 2 and 3 with intronic boundaries of the INS gene by direct gene sequencing in 30 PNDM patients and 20 healthy controls. A detailed clinical phenotyping of the patients was carried out to specify the diabetes features in those found to carry an INS variant.Results: We identified five variants (four SNPs and one synonymous variant), c(0).187 + 11T > C, c.-17-6T > A, c.*22A > C, c.*9C > T, and c.36G > A (p.A12A), with allelic frequencies of 96.7%, 80%, 75%, 5%, and 1.7%, respectively. All showed no statistically significance difference compared with the controls, with the exception of c.*22A > C.Conclusion: Genetic screening for the INS gene did not reveal an evident role in the diagnosis of PNDM.

Exoskeletons across the Pancrustacea: Comparative Morphology, Physiology, Biochemistry and Genetics

The exoskeletons of pancrustaceans, as typified by decapod crustaceans and insects, demonstrate a high degree of similarity with respect to histology, ultrastructure, function, and composition. The cuticular envelope in insects and the outer epicuticle in crustaceans both serve as the primary barrier to permeability of the exoskeleton, preventing loss of water and ions to the external medium. Prior to and following ecdysis, there is a sequence of expression and synthesis of different proteins by the cuticular epithelium for incorporation into the pre-exuvial and post-exuvial procuticle of insects and the exocuticle and endocuticle of crustaceans. Both exhibit regional differences in cuticular composition, e.g., the articular (intersegmental) membranes of insects and the arthrodial (joint) membranes of crustaceans. The primary difference between these cuticles is the ability to mineralize. Crustaceans' cuticles express a unique suite of proteins that provide for the nucleation and deposition of calcium carbonate. Orthologs of genes discussed in the present review were mined from a recently completed cuticular transcriptome of the crayfish, Cherax quadricarinatus, providing new insights into the nature of these proteins.

Dual effects of noradrenaline on astroglial production of chemokines and pro-inflammatory mediators

BackgroundNoradrenaline (NA) is known to limit neuroinflammation. However, the previously described induction by NA of a chemokine involved in the progression of immune/inflammatory processes, such as chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein-1 (MCP-1), apparently contradicts NA anti-inflammatory actions. In the current study we analyzed NA regulation of astroglial chemokine (C-X3-C motif) ligand 1 (CX3CL1), also known as fractalkine, another chemokine to which both neuroprotective and neurodegenerative actions have been attributed. In addition, NA effects on other chemokines and pro-inflammatory mediators were also analyzed.MethodsPrimary astrocyte-enriched cultures were obtained from neonatal Wistar rats. These cells were incubated for different time durations with combinations of NA and lipopolysaccharide (LPS). The expression and synthesis of different proteins was measured by RT-PCR and enzyme-linked immunosorbent assay (ELISA) or enzyme immunoassays. Data were analyzed by one-way analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison tests.ResultsThe data presented here show that in control conditions, NA induces the production of CX3CL1 in rat cultured astrocytes, but in the presence of an inflammatory stimulus, such as LPS, NA has the opposite effect inhibiting CX3CL1 production. This inversion of NA effect was also observed for MCP-1. Based on the observation of this dual action, NA regulation of different chemokines and pro-inflammatory cytokines was also analyzed, observing that in most cases NA exerts an inhibitory effect in the presence of LPS. One characteristic exception was the induction of cyclooxygenase-2 (COX-2), where a summative effect was detected for both LPS and NA.ConclusionThese data suggest that NA effects on astrocytes can adapt to the presence of an inflammatory agent reducing the production of certain cytokines, while in basal conditions NA may have the opposite effect and help to maintain moderate levels of these cytokines.

Accelerated aging of ipê seeds under controlled conditions of storage

This research was aimed at studying effects of storage and accelerated aging on germination and profile of storage proteins in Handroanthus albus seeds. These were stored into a cold chamber (± 8 ºC; RH ± 40%) and after periods of 0, 3, 6, 9, and 12 months of storage, were subjected to accelerated aging for 0, 24, 48, 72, and 96 hours. Relationships between germination and proteins profile were assessed. Germination test was performed at 25 ºC, under constant light. For protein extraction, 125 mg of seeds were macerated in 2 mL of extraction buffer (1M Tris-HCl; pH 8.8) and applied to SDS-PAGE polyacrylamide gel at 80 V .15 h-1. Twelve month storage, combined with 72 hours accelerated aging have increased germination in approximately 65% when compared to non-aged seeds or to seeds with 24 h of accelerated aging. Besides beneficial effects, degradation and synthesis of different proteins were observed. It was concluded that germination of Handroanthus albus seeds, when not subjected to accelerated aging, is favored by storage in cold chamber during three to six months, or from nine to 12 months when subjected to accelerated aging process. Storage proteins may be associated to those increases, and hence further studies are needed.

CCL2/MCP-1 modulation of microglial activation and proliferation

BackgroundMonocyte chemoattractant protein (CCL2/MCP-1) is a chemokine that attracts cells involved in the immune/inflammatory response. As microglia are one of the main cell types sustaining inflammation in brain, we proposed here to analyze the direct effects of MCP-1 on cultured primary microglia.MethodsPrimary microglia and neuronal cultures were obtained from neonatal and embryonic Wistar rats, respectively. Microglia were incubated with different concentrations of recombinant MCP-1 and LPS. Cell proliferation was quantified by measuring incorporation of bromodeoxyuridine (BrdU). Nitrite accumulation was measured using the Griess assay. The expression and synthesis of different proteins was measured by RT-PCR and ELISA. Cell death was quantified by measuring release of LDH into the culture medium.ResultsMCP-1 treatment (50 ng/ml, 24 h) did not induce morphological changes in microglial cultures. Protein and mRNA levels of different cytokines were measured, showing that MCP-1 was not able to induce proinflammatory cytokines (IL-1β, IL6, MIP-1α), either by itself or in combination with LPS. A similar lack of effect was observed when measuring inducible nitric oxide synthase (NOS2) expression or accumulation of nitrites in the culture media as a different indicator of microglial activation. MCP-1 was also unable to alter the expression of different trophic factors that were reduced by LPS treatment. In order to explore the possible release of other products by microglia and their potential neurotoxicity, neurons were co-cultured with microglia: no death of neurons could be detected when treated with MCP-1. However, the presence of MCP-1 induced proliferation of microglia, an effect opposite to that observed with LPS.ConclusionThese data indicate that, while causing migration and proliferation of microglia, MCP-1 does not appear to directly activate an inflammatory response in this cell type, and therefore, other factors may be necessary to cause the changes that result in the neuronal damage commonly observed in situations where MCP-1 levels are elevated.

Changes in gene expression patterns during the sexual life cycle of Chlamydomonas reinhardtii

In the sexual life cycle of Chlamydomonas reinhardtii, three types of cells can be distinguished: vegetative cells, gametes, and zygotes. Using is vivo pulse labelling of proteins with 35S-sulfate, followed by two-dimensional separation of the polypeptides on polyacrylamide gels, we analyzed the patterns of protein synthesis typical for each cell type. Approximately 20% of the proteins detected were synthesized at very different rates when the pattern from vegetative cells was compared to that of gametes or zygotes. Gamete formation from vegetative cells is a two-step process controlled by two extrinsic signals: first, nitrogen-starvation induces the differentiation of vegetative cells to pregaroetes, which are then competent for a light–induced differentiation to mature gametes. The majority of the changes in rate of synthesis of different proteins during the switch from vegetative cells to gametes was observed already in pregametes. Between the stages of pregametes and gametes, changes in the rates of synthesis of four proteins were detected. Induction of zygote germination resulted in complex changes in the patterns of gene expression. Evidence is presented for three groups of proteins whose synthesis is turned on or turned off during zygote germination.

Tissue specificity of steroid hormone action: A new theory

A major problem still to be solved in endocrinology is that of tissue specificity of steroid (and other) hormone action. For example, ecdysone is known to co-induce the synthesis of different proteins in fat body, epidermis, salivary glands, etc. In flies the vitellogenin genes are only expressed in the fat body of adult females in response to 20-OH-ecdysone. This is a result of differentiation having occurred during development. The nature of differentiation is the generation of asymmetry. In somatic cells of developing animals and plants the asymmetry does not seem to reside in the DNA itself [e.g. experiments of Gurdon (1962) Devl Biol. 4, 256–273 and (1974) The Control of Gene Expression in Animal Development, p. 160. Clarendon Press, Oxford] but rather in the mechanisms which make use of genetic information. Control of gene expression is very complex: in addition to mechanisms operating at the level of DNA/RNA, there are also epigenetic phenomena, some of which are localized in the plasma-membrane. Asymmetry can only be introduced in a reproducible way by those molecular mechanisms which have a well defined and constant location in the cell and which are not subject to diffusion. A typical feature of differentiating cells is that they divide asymmetrically. Considering data which have recently become available, the plasma-membrane presents itself as a good candidate for the location of molecules contributing to this asymmetric development. In order to be of any importance for steroid hormone action the properties of the plasma-membrane (especially permeability) should differ between different cell types, should be influencible by steroids, and there should be a causal relationship between membrane permeability and nuclear activity. Besides receptors within the cell, some steroid hormones also seem to have plasma-membrane receptors. Tissue specificity of steroid hormone action may be determined largely by the properties of the cell membranes (plasma- and also, perhaps, nuclear membranes). Steroids may act at different levels within the cell.

Nafoxidine-responsive uterine protein: Further characterization and distinction from the “estrogen-induced” protein (IP)

In a previous paper, we reported that nafoxidine (UA) stimulated the synthesis of a uterine protein showing the same electrophoretic mobility as the “estrogen-induced protein” (E 2-IP) first described by Notides and Gorski. In the present work, we analyzed the IP-containing electrophoretic zone by SDS polyacrylamide-gel electro phoresis, and found that estradiol-17β (E 2) and nafoxidine stimulated the synthesis of different proteins. As expected, estradiol-17β stimulated the synthesis of the E 2-IP of 46000 Mr. On the other hand, UA stimulated the synthesis of 27 000 and 30000 Mr proteins (UA-IP). These UA-IP were not precipitated by an antiserum raised against E 2-IP. Therefore, UA-IP appear to be independent entities and not degradation or precursor products of E 2-IP. Both UA-IP are constitutively present in the uterus and even in higher relative amounts in rat brain. The present finding, that an “anti-estrogen”, such as nafoxidine, stimulates the synthesis of different proteins than estrogen, provides a new approach to the study of the molecular mechanism of estrogen action.

The Mechanism of Evagination of Imaginal Discs ofDrosophila melanogaster

The evagination of imaginal leg discs to produce legs is a useful model for studying morphogenesis. Evagination of imaginal leg discs occurs in vitro in defined culture media in the presence of the molting hormone β-ecdysone. Evagination involves limited, organized movement of imaginal disc cells. The movement appears to be a result of contractile activity, coordinated with the presence of appropriate structural and surface properties of disc cells. However, ecdysone does not produce its effects directly, but acts through the genome to cause evagination. Evagination is a result then of increased synthesis of different proteins, one of which is myosin. If the results on discs are generalizable they indicate that similar morphogenetic processes are the direct result of the readout of the specific genetic programs.

The effect of β-ecdysone on protein synthesis in imaginal discs of Drosophila melanogaster cultured in vitro: II. Effects on synthesis in specific cell fractions

The effect of β-ecdysone on the synthesis of different proteins in imaginal discs of Drosophila melanogaster was investigated using double-label experiments and SDS acrylamide gel electrophoresis. Four different cellular fractions were analyzed (12K pellet, cytosol, core ribosomes, and KCl-soluble ribosomal proteins). β-Ecdysone alters the net synthesis of proteins in all of these fractions, with the most dramatic effects occurring in the ribosomal protein fractions. The maintenance of the altered pattern of synthesis of proteins in these fractions depends on the continued presence of β-ecdysone in the culture medium. The data suggest that β-ecdysone alters the synthesis of different proteins by acting on the genome. The apparent mechanisms by which β-ecdysone increases total protein synthesis and alters the net synthesis of different proteins in discs are discussed.

Hypercholesterolemia in experimental and human hepatic porphyria

Administration of the porphyria-inducing chemicals allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1, 4-dihydrocollidine (DDC) to rabbits, results in the development of marked elevation of serum cholesterol, total lipids and phospholipids. The liver total lipids, cholesterol lipid phosphorus and water concentrations remain unchanged, but the organ becomes greatly enlarged, reflecting active synthesis of new protoplasm. It is suggested that these compounds affect the lipoprotein transport of serum cholesterol in a manner inducing sequestration of the synthesized cholesterol in the plasma and preventing the accumulation of cholesterol in the liver cells hence, the activation of the hepatic feed-back control for cholesterol synthesis. The possibility also exists that AIA and DDC enhances the production of one or more enzymes of cholesterol synthesis, in a way analogous to what Granick and Urata have demonstrated to occur for the enzyme δ-aminolevulinic acid synthetase of the porphyrin synthetic pathway. The chemicals, in fact, may have their primary effect on the genetic control of the rate of synthesis of different proteins by the liver. A review of the records of 51 patients with hepatic porphyria, in whom serum cholesterol determinations were recorded, reveals that hypercholesterolemia, although inconstant, is of common occurrence in the “acute intermittent” and “mixed” forms of the disease.