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The effects of temperature on the behaviour of the Antarctic sea star Odontaster validus
Get PDFMany Antarctic marine benthic invertebrates are adapted to specific environ−
mental conditions (e.g. low stable temperatures, high salinity and oxygen content). Changes
caused by global climatic shifts can be expected to have significant impact on their physiol−
ogy and distribution. Odontaster validus, an ubiquitous, omnivorous sea star is one of the
“keystone species” in the Antarctic benthic communities. Laboratory experiments were car−
ried out to study the effect of temperature rise (from 0 to 5C) on some vital biological func−
tions that sea stars must perform in order to survive in their environment. Parameters such as
behavioural reaction of sea stars to food and food odour, locomotory performance and abil−
ity to right were measured. Temperature increase significantly impaired the ability of
O. validusto perform these functions (e.g. lowering the number of sea stars able to right, in−
creasing time−to−right, reducing locomotory activity, weakening chemosensory reaction to
food and food odour). At temperatures of 4 and 5C a loss of motor coordination was ob−
served, although at all tested temperatures up to 5C there were single individuals perform−
ing successfully
Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein.
Get PDFIn Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), alpha-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3-->Arg(+) revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB(-)) strain in comparison with the AB1157 (alkB(+)) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd(+), and no repair in BS87 mfd(-) cells. Determination of specificity of Arg(+) revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT-->TA transversions by supL suppressor formation (1meA) and GC-->AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB(-)mfd(-) bacteria but not in the alkB(+)mfd(-) counterpart
Regulation of sporulation in the yeast Saccharomyces cerevisiae.
Get PDFSporulation of the budding yeast Saccharomyces cerevisiae — equivalent to gametogenesis in higher organisms, is a complex differentiation program induced by starvation of cells for nitrogen and carbon. Such environmental conditions activate coordinated, sequential changes in gene expression leading to production of haploid, stress-resistant spores. Sporulation comprises two rounds of meiosis coupled with spore morphogenesis and is tightly controlled to ensure viable progeny. This review concerns the regulation of differentiation process by nutritional and transcriptional signals
Prostaglandin-endoperoxide synthase genes COX1 and COX2 - novel modifiers of disease severity in cystic fibrosis patients.
No full textCystic fibrosis (CF) is one of the most common autosomal recessive diseases among Caucasians caused by a mutation in the CFTR gene. However, the clinical outcome of CF pulmonary disease varies remarkably even in patients with the same CFTR genotype. This has led to a search for genetic modifiers located outside the CFTR gene. The aim of this study was to evaluate the effect of functional variants in prostaglandin-endoperoxide synthase genes (COX1 and COX2) on the severity of lung disease in CF patients. To the best of our knowledge, it is the first time when analysis of COX1 and COX2 as potential CF modifiers is provided. The study included 94 CF patients homozygous for F508del mutation of CFTR. To compare their clinical condition, several parameters were recorded, e.g. a unique clinical score: disease severity status (DSS). To analyse the effect of non-CFTR genetic polymorphisms on the clinical course of CF patients, the whole coding region of COX1 and selected COX2 polymorphisms were analysed. Statistical analysis of genotype-phenotype associations revealed a relationship between the heterozygosity status of identified polymorphisms and better lung function. These results mainly concern COX2 polymorphisms: -765G>C and 8473T>C. The COX1 and COX2 polymorphisms reducing COX protein levels had a positive effect on all analysed clinical parameters. This suggests an important role of these genes as protective modifiers of pulmonary disease in CF patients, due to inhibition of arachidonic acid conversion into prostaglandins, which probably reduces the inflammatory process
Phosphorylation of maize eif5a by ck2: identification of phosphorylated residue and influence on intracellular localization of eif5a.
Get PDFMaize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with catalytic subunit of protein kinase CK2 and is phosphorylated by this enzyme. Phosphorylated ZmeIF5A was also identified after separation of maize leaf proteins by two-dimensional electrophoresis. Multiple sequence alignment of eIF5A proteins showed that in monocots, in contrast to other eukaryotes, there are two serine/threonine residues that could potentially be phosphorylated by CK2. To identify the phosphorylation site(s) of ZmeIF5A, the serine residues potentially phosphorylated by CK2 were mutated. ZmeIF5A and its mutated variants Ser2Ala and Ser4Ala were expressed in E. coli and purified. Of these recombinant proteins, only ZmeIF5ASer2Ala was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A Ser2Ala mutants were not phosphorylated despite effective phosphorylation of wild-type variants. A newly developed method exploiting the specificity of thrombin cleavage was used to confirm that Ser2 in ZmeIF5A is indeed phosphorylated. To find a role of the Ser2 phosphorylation, ZmeIF5A and its variants mutated at Ser2 (Ser2Ala and Ser2Asp) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. While wild-type ZmeIF5A and its Ser2Ala variant were distributed evenly between the nucleus and the cytoplasm, the variant with Ser2 replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser2 plays a role in the regulation of nucleocytoplasmic shuttling of eIF5A in plant cells
CATALYTIC PROPERTIES OF THE EUKARYOTIC EXOSOME
Get PDFThe eukaryotic exosome complex is built around the backbone of a 9‑subunit ring similar to phosporolytic ribonucleases such as RNase PH and polynucleotide phosphorylase (PNPase). Unlike those enzymes, the ring is devoid of any detectable catalytic activities, with the possible exception of the plant version of the complex. Instead, the essential RNA decay capability is supplied by associated hydrolytic ribonucleases belonging to the Dis3 and Rrp6 families. Dis3 proteins are endowed with two different activities: the long known processive 3′‑5′ exonucleolytic one and the recently discovered endonucleolytic one. Rrp6 proteins are distributive exonucleases. This chapter will review the current knowledge about the catalytic properties of theses nucleases and their interplay within the exosome holocomplex
Double Selective Synthetic Approach to the N-Functionalized 1,4,7-Triazacyclononane Derivatives: Chelating Compounds for Controllable Protein Orientation
No full textThe double selective synthetic approach is proposed for easy preparation of asymmetrically N-substituted derivatives of 1,4,7-triazacyclononane (tacn, [9]aneN(3)) with the same or different pendant arms. The synthetic routes simplify the synthesis of "tacn" ligands for functionalization of other carboxy terminated molecules/supports and also facilitate access to azamacrocycle functionalized self-assembled monolayer (SAMs) or nanopatterns for specific and stable Histidine-tagged protein immobilization
Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering: reaction conditions and molecular mechanism.
No full textRecently we screened a combinatorial library of R(1)-(Ser/Thr)-Xaa-His-Zaa-R(2) peptides (Xaa = 17 common alpha-amino acids, except Asp, Glu, and Cys; Zaa =19 common alpha-amino acids, except Cys; R(1) = CH(3)CO-Gly-Ala, R(2) = Lys-Phe-Leu-NH(2)) and established criteria for selecting Ser/Thr, Xaa, and Zaa substitutions optimal for specific R(1)-Ser/Thr peptide bond hydrolysis in the presence of Ni(II) ions (Krezel, A.; Kopera, E.; Protas, A. M.; Poznanski, J.; Wysłouch-Cieszynska, A.; Bal, W. J. Am. Chem. Soc. 2010, 132, 3355-3366). The screening results were confirmed by kinetic studies of hydrolysis of seven peptides: R(1)-Ser-Arg-His-Trp-R(2), R(1)-Ser-Lys-His-Trp-R(2), R(1)-Ser-Ala-His-Trp-R(2), R(1)-Ser-Arg-His-Ala-R(2), R(1)-Ser-Gly-His-Ala-R(2), R(1)-Thr-Arg-His-Trp-R(2), and R(1)-Thr-His-His-Trp-R(2). In this paper, we used the same seven peptides to investigate the molecular mechanism of the hydrolysis reaction. We studied temperature dependence of the reaction rate at temperatures between 24 and 75 degrees C, measured stability constants of Ni(II) complexes with hydrolysis substrates and products, and studied the course of R(1)-Ser-Arg-His-Trp-R(2) peptide hydrolysis under a broad range of conditions. We established that the specific square planar complex containing the Ni(II) ion bonded to the His imidazole nitrogen and three preceding peptide bond nitrogens (4N complex) is required for the reaction to occur. The reaction mechanism includes the N-O acyl shift, yielding an intermediate ester of R(1) with the Ser/Thr hydroxyl group. This ester hydrolyzes spontaneously, yielding final products. The Ni(II) ion activates the R(1)-Ser peptide bond by destabilizing it directly through peptide nitrogen coordination and, indirectly, by imposing a strain in the peptide chain
Benefits and risks of iron supplementation in anemic neonatal pigs.
Get PDFIron deficiency is a common health problem. The most severe consequence of this disorder is iron deficiency anemia (IDA), which is considered the most common nutritional deficiency worldwide. Newborn piglets are an ideal model to explore the multifaceted etiology of IDA in mammals, as IDA is the most prevalent deficiency disorder throughout the early postnatal period in this species and frequently develops into a critical illness. Here, we report the very low expression of duodenal iron transporters in pigs during the first days of life. We postulate that this low expression level is why the iron demands of the piglet body are not met by iron absorption during this period. Interestingly, we found that a low level of duodenal divalent metal transporter 1 and ferroportin, two iron transporters located on the apical and basolateral membrane of duodenal absorptive enterocytes, respectively, correlates with abnormally high expression of hepcidin, despite the poor hepatic and overall iron status of these animals. Parenteral iron supplementation by a unique intramuscular administration of large amounts of iron dextran is current practice for the treatment of IDA in piglets. However, the potential toxicity of such supplemental iron implies the necessity for caution when applying this treatment. Here we demonstrate that a modified strategy for iron supplementation of newborn piglets with iron dextran improves the piglets' hematological status, attenuates the induction of hepcidin expression, and minimizes the toxicity of the administered iron
A Comprehensive, Quantitative, and Genome-Wide Model of Translation
Get PDFTranslation is still poorly characterised at the level of individual proteins and its role in regulation of gene expression has
been constantly underestimated. To better understand the process of protein synthesis we developed a comprehensive and
quantitative model of translation, characterising protein synthesis separately for individual genes. The main advantage of
the model is that basing it on only a few datasets and general assumptions allows the calculation of many important
translational parameters, which are extremely difficult to measure experimentally. In the model, each gene is attributed with a set of translational parameters, namely the absolute number of transcripts, ribosome density, mean codon translation
time, total transcript translation time, total time required for translation initiation and elongation, translation initiation rate,
mean mRNA lifetime, and absolute number of proteins produced by gene transcripts. Most parameters were calculated
based on only one experimental dataset of genome-wide ribosome profiling. The model was implemented in
Saccharomyces cerevisiae, and its results were compared with available data, yielding reasonably good correlations. The
calculated coefficients were used to perform a global analysis of translation in yeast, revealing some interesting aspects of
the process. We have shown that two commonly used measures of translation efficiency – ribosome density and number of
protein molecules produced – are affected by two distinct factors. High values of both measures are caused, i.a., by very
short times of translation initiation, however, the origins of initiation time reduction are completely different in both cases.
The model is universal and can be applied to any organism, if the necessary input data are available. The model allows us to
better integrate transcriptomic and proteomic data. A few other possibilities of the model utilisation are discussed
concerning the example of the yeast system