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A contribution to identification of novel regulators of plant response to sulfur deficiency: characteristics of a tobacco gene UP9, its protein product and the effects of UP9 silencing
No full textExtensive changes in plant transcriptome and metabolome have been observed by numerous research groups after transferring plants from optimal conditions to sulfur (S) deficiency. Despite intensive studies and recent important achievements, like identification of SLIM1/EIL3 as a major transcriptional regulator of the response to S-deficiency, many questions concerning other elements of the regulatory network remain unanswered. Investigations of genes with expression regulated by S-deficiency stress encoding proteins of unknown function might help to clarify these problems. This study is focused on the UP9C gene and the UP9-like family in tobacco. Homologues of these genes exist in other plant species, including a family of four genes of unknown function in Arabidopsis thaliana (LSU1-4), of which two were reported as strongly induced by S-deficit and to a lesser extent by salt stress and nitrate limitation. Conservation of the predicted structural features, such as coiled coil region or nuclear localization signal suggests that these proteins might have important functions possibly mediated by interactions with other proteins. Analysis of transgenic tobacco plants with silenced expression of UP9-like genes strongly argues for their significant role in regulation of plant response to S-deficit. Although our study shows that the UP9-like proteins are important components of such response and they might be also required during other stresses, their molecular functions remain a mystery
Reversal of a mutator activity by a nearby fidelity-neutral substitution in the RB69 DNA polymerase binding pocket
Get PDFPhage RB69 family-B DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only small impacts on DNA replication fidelity. When the Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that of Y567A alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A alone, the double mutant mainly reduced base substitution mutations and to a lesser extent frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity and of mismatch insertion and extension, we propose that the recovered fidelity of the double mutant may result in part from increased dissociation of the enzyme from DNA followed by the binding of the same or another polymerase molecule in either the exonuclease or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in the following report
Novel endoribonucleases as central players in various pathways of eukaryotic RNA metabolism
Get PDFFor a long time it has been assumed that the decay of RNA in eukaryotes is mainly carried out by exoribonucleases, which is in contrast to bacteria, where endoribonucleases are well documented to initiate RNA degradation. In recent years, several as yet unknown endonucleases have been described, which has changed our view on eukaryotic RNA metabolism. Most importantly, it was shown that the primary eukaryotic 3' --> 5' exonuclease, the exosome complex has the ability to endonucleolytically cleave its physiological RNA substrates, and novel endonucleases involved in both nuclear and cytoplasmic RNA surveillance pathways were discovered concurrently. In addition, endoribonucleases responsible for long-known processing steps in the maturation pathways of various RNA classes were recently identified. Moreover, one of the most intensely studied RNA decay pathways--RNAi--is controlled and stimulated by the action of different endonucleases. Furthermore, endoribonucleolytic cleavages executed by various enzymes are also the hallmark of RNA degradation and processing in plant chloroplasts. Finally, multiple context-specific endoribonucleases control qualitative and/or quantitative changes of selected transcripts under particular conditions in different eukaryotic organisms. The aim of this review is to discuss the impact of all of these discoveries on our current understanding of eukaryotic RNA metabolism
Involvement of oxidatively damaged DNA and repair in cancer development and aging
Get PDFDNA damage and DNA repair may mediate several cellular processes, like replication and transcription, mutagenesis and apoptosis and thus may be important factors for the organism development and pathology, including cancer.
DNA is constantly damaged by reactive oxygen species (ROS) and reactive nitrogen species (RNS) directly and also by products of lipid peroxidation (LPO), which form exocyclic adducts to DNA bases. A wide variety of oxidatively-generated DNA lesions are present in living cells. 8-oxoguanine (8-oxoGua) is one of the best known DNA damage due to its mutagenic properties. Among LPO-derived DNA base modifications the most intensively studied are ethenoadenine and ethenocytosine, highly miscoding DNA lesions considered as markers of oxidative stress and promutagenic DNA damage. Although at present it is impossible to answer directly the question concerning involvement of oxidatively damaged DNA in cancer etiology, it is likely that oxidatively modified DNA bases may serve as a source of mutations that initiate carcinogenesis and are involved in aging (i.e. they may be causal factors responsible for the processes). To counteract the deleterious effect of oxidatively damaged DNA, all organisms developed several DNA repair mechanisms. It was observed that the repair efficiency of oxidatively damaged DNA is frequently decreased in cancer patients.
The present work reviews the basis for the biological significance of DNA damage, particularly effects of 8-oxoGua and ethenoadducts occurrence in DNA in aspect of cancer development, drawing attention to the multiplicity of proteins with repair activities
The Frequency of Selected Polymorphic Variants of the RET Gene in Patients with Medullary Thyroid Carcinoma and in the General Population of Central Poland.
Get PDFThe object of this work was to compare the frequency of three polymorphic changes in the RET proto-oncogene: L769L, S836S, and S904S in patients with medullary thyroid carcinoma (MTC; n = 246) and in the general population (n = 420 for single-nucleotide polymorphism [SNP] L769L and S904S; n = 411 for SNP 836). We tried to investigate how the harbored SNPs affect the age at onset of sporadic medullary thyroid carcinoma (sMTC) and MTC in carriers of known pathogenic mutations at codons 634 and 791 of the RET gene. A statistically significant difference was found in the frequency of the heterozygous change L769L in patients with sMTC (48.3%) and in unaffected individuals (39.5%). The presence of the polymorphic change L769L in the RET gene predisposes to the development of sMTC and also lowers the age of onset of MTC in carriers of the homozygous polymorphic variant L769L. The presence of this polymorphic change in MTC patients carrying, at the same time, the RET codon 634 mutation lowers the age of onset of MTC in this group
Three Members of the 6-cys Protein Family of Plasmodium Play a Role in Gamete Fertility
Get PDFThe process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates
Relative role of halogen bonds and hydrophobic interactions in inhibition of human protein kinase CK2alpha by tetrabromobenzotriazole and some C(5)-substituted analogues.
No full textTo examine the relative role of halogen bonding and hydrophobic interactions in the inhibition of human CK2alpha by 4,5,6,7-tetrabromobenzotriazole (TBBt), we have synthesized a series of 5-substituted benzotriazoles (Bt) and the corresponding 5-substituted 4,6,7-tribromobenzotriazoles (Br3Bt) and examined their inhibition of human CK2alpha relative to that of TBBt. The various C(5) substituents differ in size (H and CH3), electronegativity (NH2 and NO2), and hydrophobicity (COOH and Cl). Some substituents were halogen bond donors (Cl, Br), while others were fluorine bond donors (F and CF3). Most of the 5-substituted analogues of Br3Bt (with the exception of COOH and NH2) exhibited inhibitory activity comparable to that of TBBt, whereas the 5-substituted analogues of the parent Bt were only weakly active (Br, Cl, NO2, CF3) or inactive. The observed effect of the volume of a ligand molecule pointed to its predominant role in inhibitory activity, indicating that presumed halogen bonding, identified in crystal structures and by molecular modeling, is dominated by hydrophobic interactions. Extended QSAR analysis additionally pointed to the monoanion and a preference for the N(1)-H protomer of the neutral ligand as parameters crucial for prediction of inhibitory activity. This suggests that the monoanions of TBBt and its congeners are the active forms that efficiently bind to CK2alpha, and the binding affinity is coupled with protomeric equilibrium of the neutral ligand
Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity
Get PDFSeveral studies focused on elucidating the mechanism of nitric oxide (NO) signalling in plant cells highlighted that its biological effects are partly mediated by protein kinases. Identity of these kinases and details of how NO modulates their activities, however, remain poorly investigated. Here, we have attempted to clarify the mechanisms underlying NO action in regulation of Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SNF1-related protein kinase 2 family. We found that in tobacco BY-2 cells exposed to salt stress, NtOSAK is rapidly activated partly through a NO-dependent process. This activation, as well as the one observed following treatment of BY-2 cells with the NO donor DEA/NO, involved the phosphorylation of two residues located in the kinase activation loop, one being identified as Ser-158. Our results indicate that NtOSAK does not undergo the direct chemical modifications of its Cys residues by S-nitrosylation. Using a co-immunoprecipitation-based strategy, we identified several proteins present in immunocomplex with NtOSAK in salt-treated cells including the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Our results indicate that NtOSAK directly interacts with GAPDH in planta. Furthermore, in response to salt, GAPDH showed a transient increase of its S-nitrosylation level which was correlated with the time course of NtOSAK activation. However, GADPH S-nitrosylation did not influence its interaction with NtOSAK and did not impact the activity of the protein kinase. Taken together, the results support the hypothesis that NtOSAK and GAPDH form a cellular complex and that both proteins are regulated directly or indirectly by NO
Oxidative stress and 8-oxoguanine repair are enhanced in colon adenoma and carcinoma patients.
Get PDFOxidative stress is involved in the pathogenesis of colon cancer. We wanted to elucidate at which stage of the disease this phenomenon occurs. In the examined groups of patients with colorectal cancer (CRC, n = 89), benign adenoma (AD, n = 77) and healthy volunteers (controls, n = 99), we measured: vitamins A, C and E in blood plasma, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in leukocytes and urine, leukocyte 8-oxoGua excision activity, mRNA levels of APE1, OGG1, 8-oxo-7,8-dihydrodeoxyguanosine 5'-triphosphate pyrophosphohydrolase (MTH1) and OGG1 polymorphism. The vitamin levels decreased gradually in AD and CRC patients. 8-OxodG increased in leukocytes and urine of CRC and AD patients. 8-OxoGua was higher only in the urine of CRC patients. 8-OxoGua excision was higher in CRC patients than in controls, in spite of higher frequency of the OGG1 Cys326Cys genotype, encoding a glycosylase with decreased activity. mRNA levels of OGG1 and APE1 increased in CRC and AD patients, which could explain increased 8-oxoGua excision rate in CRC patients. MTH1 mRNA was also higher in CRC patients. The results suggest that oxidative stress occurs in CRC and AD individuals. This is accompanied by increased transcription of DNA repair genes, and increased 8-oxoGua excision rate in CRC patients, which is, however, insufficient to counteract the increased DNA damage
Influence of the Escherichia coli oxyR gene function on lambda prophage maintenance
Get PDFIn Escherichia coli hosts, hydrogen peroxide is
one of the factors that may cause induction of lambda prophage.
Here, we demonstrate that H2O2-mediated lambda prophage
induction is signiWcantly enhanced in the oxyR mutant host.
The mRNA levels for cI gene expression were increased in
a lambda lysogen in the presence of H2O2. On the other hand,
stimulation of the pM promoter by cI857 overproduced
from a multicopy plasmid was decreased in the lambda oxyR
mutant in the presence of H2O2 but not under normal
growth conditions. The puriWed OxyR protein did bind
speciWcally to the pM promoter region. This binding
impaired eYciency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and
OR1 sites, in the regulatory region of the pM promoter. We
propose that changes in cI gene expression, perhaps in
combination with moderately induced SOS response, may
be responsible for enhanced lambda prophage induction by
hydrogen peroxide in the oxyR mutant. Therefore, OxyR
seems to be a factor stimulating lambda prophage maintenance
under conditions of oxidative stress. This proposal is discussed in the light of eYciency of induction of lambdoid
prophages bearing genes coding for Shiga toxins