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Simultaneous siRNA-mediated silencing of pairs of genes coding for enzymes involved in glycosaminoglycan synthesis
Get PDFIt has been demonstrated recently that it is possible to
decrease expression of genes coding for enzymes involved
in synthesis of glycosaminoglycans (GAGs) by
using specific siRNAs which interfere with stability of
particular mRNAs. This procedure has been proposed as
a potential treatment for patients suffering from mucopolysaccharidoses,
a group of inherited metabolic diseases
caused by dysfunction of enzymes required for
GAG degradation, and resultant storage of these compounds
in cells of affected persons. Here, we asked if the
simultaneous use two species of specific siRNAs aimed at
silencing two genes involved in particular steps of GAG
synthesis may be more effective than the use of single
siRNA. We found that inhibition of GAG synthesis in cells
treated with two siRNAs is generally more effective than
using single siRNAs. However, the differences were not
statistically significant, therefore the potential benefit
from the use of two siRNAs over the use of a single siRNA
is doubtful in the light of the cost-benefit ratio and possibly stronger side-effects of the putative therapy
Affinity of copper and zinc ions to proteins and peptides related to neurodegenerative conditions (Aβ, APP, α-synuclein, PrP).
Get PDFThe review describes the state of the art in the field of stability constant determination for Cu(II), Cu(I) and Zn(II) complexes of proteins and peptides involved in neurodegenerative diseases, α-Synuclein (aS), Prion Protein (PrP), Amyloid Precursor Protein (APP) and Amyloid β peptides (Aβ). The methodologies and results are critically analyzed and recommendations are formulated about possible systematic errors in these studies. The possibility of formation of ternary complexes with titration competitors is discussed
Synthesis and Physico-Chemical Properties in Aqueous Medium of All Possible Isomeric Bromo Analogues of Benzo-1H-Triazole, Potential Inhibitors of Protein Kinases.
Get PDFIn ongoing studies on the role of the individual bromine atoms of 4,5,6,7-tetrabromobenzotriazole (TBBt) in its relatively selective inhibition of protein kinase CK2α, we have prepared all the possible two mono-, four di-, and two tri- bromobenzotriazoles, and determined their physico-chemical properties in aqueous medium. They exhibited a general trend of a decrease in solubility with an increase in the number of bromines on the benzene ring, significantly modulated by the pattern of substitution. For a given number of attached bromines, this was directly related to the electronic effects resulting from different sites of substitution, leading to marked variations of pKa values for dissociation of the triazole proton. Experimental data (pKa, solubility) and ab initio calculations demonstrated that hydration of halogenated benzotriazoles is driven by a subtle balance of hydrophobic and polar interactions. The combination of QM-derived free energies for solvation and proton dissociations was found to be a reasonably good predictor of inhibitory activity of halogenated benzotriazoles vs CK2α. Since the pattern of halogenation of the benzene ring of benzotriazole has also been shown to be one of the determinants of inhibitory potency vs some viruses and viral enzymes, the present comprehensive description of their physico-chemical properties should prove helpful in efforts to elucidate reaction mechanisms, including possible halogen bonding, and the search for more selective and potent inhibitors
Maize calcium-dependent protein kinase (ZmCPK11): local and systemic response to wounding, regulation by touch and components of jasmonate signaling
Get PDFExpression of ZmCPK11, a member of the maize (Zea mays L.) Calcium-Dependent Protein Kinases (CDPKs) family, is induced by mechanical wounding. A rapid increase of the activity of a 56-kDa CDPK has been observed in damaged leaves. In the present work, it is shown that the 56-kDa CDPK, identified as ZmCPK11, is also activated in non-wounded leaves as an element of systemic wound response. Moreover, an increase of the enzyme’s activity and induction of ZmCPK11 expression was observed after touching the leaves. To study the role of ZmCPK11 in wound and touch signaling, transgenic Arabidopsis thaliana plants in which c-Myc-ZmCPK11 was expressed under control of the CaMV 35S promoter were generated. Analysis of the transgenic plants showed that c-Myc-ZmCPK11 was activated upon wounding and touching. Furthermore, pretreatment with acetylsalicylic acid (acSA), an inhibitor of jasmonic acid (JA)-dependent wound signaling, abolished the wound-induced activation of ZmCPK11 in maize and the transgenic A. thaliana plants. Methyl jasmonate (MeJA) and linolenic acid (LA) stimulated the activity of ZmCPK11 as well as induced the expression of ZmCPK11 and other wound-responsive genes, lipoxygenase 1 (ZmLOX1) and proteinase inhibitor 1 (ZmWIP1). These results indicate that ZmCPK11, regulated at the enzymatic and transcriptional level by LA and MeJA, is a component of touch- and wound-induced pathway(s), participating in early stages of local and systemic responses
Diaspores and phyto-remains accidentally transported to the Antarctic Station during three expeditions
Get PDFThe aim of the project was to assess the size and species range of alien plant diaspores and phyto-remains transported into the Polish Antarctic Station during three
Antarctic expeditions. Our study clearly demonstrates that many diaspores can be quite easily unintentionally transported in good conditions to the Antarctic. In the analyzed material there were present diaspores of invasive species. All identified species belong to 20 families. The most abundant were Asteraceae and Poaceae species. The most interesting finding was the presence of caryopses of Poa annua, the first alien angiosperm species which already established a stable breeding population in the Antarctic. Base on our results, we can predict that risk of establishment of anther alien plant species in the vicinity
of ‘‘Arctowski’’ Station is very high
Recurrent horizontal transfer of bacterial toxin genes to eukaryotes
No full textIn this work, we report likely recurrent horizontal (lateral) gene transfer events of genes encoding pore-forming toxins of the aerolysin family between species belonging to different kingdoms of life. Clustering based on pairwise similarity and phylogenetic analysis revealed several distinct aerolysin sequence groups, each containing proteins from multiple kingdoms of life. These results strongly support at least six independent transfer events between distantly related phyla in the evolutionary history of one protein family and discount selective retention of ancestral genes as a plausible explanation for this patchy phylogenetic distribution. We discuss the possible roles of these proteins and show evidence for a convergent new function in two extant species. We hypothesize that certain gene families are more likely to be maintained following horizontal gene transfer from commensal or pathogenic organism to its host if they 1) can function alone; and 2) are immediately beneficial for the ecology of the organism, as in the case of pore-forming toxins which can be utilized in multicellular organisms for defense and predation
Novel broad-host-range vehicles for cloning and shuffling of gene cassettes
Get PDFNovel vectors for cloning and shuffling of gene cassettes based on minireplicon of broadhost-
range RA3 plasmid from IncU incompatibility group were constructed. A series of
minireplicon variants were prepared with copy number ranging from low (1-2 copies per
chromosome), medium (10-15 copies per chromosome) to high copy number (80-90 copies per
chromosome). The new cloning vectors are relatively small in size (4.5-5.4 kb) and carry various
resistance determinants: kanamycin (KmR), tetracycline (TcR) or chloramphenicol (CmR). The
vectors were engineered to facilitate cloning and shuffling of the functional modules with or
without transcriptional terminators. Using the described strategy, a bank of functional modules,
ready for exchange, has been initiated
Synthetic genistein derivatives as modulators of glycosaminoglycan synthesis
Get PDFBackground: Mucopolysaccharidoses (MPS) are severe metabolic disorders caused by
26 accumulation of undegraded glycosaminoglycans (GAGs) in lysosomes due to defects in certain
27 lysosomal hydrolases. Substrate reduction therapy (SRT) has been proposed as one of potential
28 treatment procedures of MPS. Importantly, small molecules used in such a therapy might
29 potentially cross the blood-brain barrier (BBB) and improve neurological status of patients, as
30 reported for a natural isoflavone, 5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one,
31 also known as genistein. Although genistein is able to cross BBB to some extent, its delivery to
32 the central nervous system is still relatively poor (below 10% efficiency). Thus, we aimed to
33 develop a set of synthetically modified genistein molecules and characterize physicochemical as
34 well as biological properties of these compounds. Methods: Following parameters were
35 determined for the tested synthetic derivatives of genistein: cytotoxicity, effects on cell
36 proliferation, kinetics of GAG synthesis, effects on epidermal growth factor (EGF) receptor’s
37 tyrosine kinase activity, effects on lysosomal storage, potential ability to cross BBB. Results: We
38 observed that some synthetic derivatives inhibited GAG synthesis similarly to, or more
39 efficiently than, genistein and were able to reduce lysosomal storage in MPS III fibroblasts. The
40 tested compounds were generally of low cytotoxicity and had minor effects on cell proliferation.
41 Moreover, synthetic derivatives of genistein revealed higher lipophilicity (assessed in silico) than
42 the natural isoflavone. Conclusion: Some compounds tested in this study might be promising
43 candidates for further studies on therapeutic agents in MPS types with neurological symptoms
A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life.
Get PDFSelenoproteins serve important functions in many organisms, usually providing essential oxidoreductase enzymatic activity, often for defense against toxic xenobiotic substances. Most eukaryotic genomes possess a small number of these proteins, usually not more than 20. Selenoproteins belong to various structural classes, often related to oxidoreductase function, yet a few of them are completely uncharacterised.Here, the structural and functional prediction for the uncharacterised selenoprotein O (SELO) is presented. Using bioinformatics tools, we predict that SELO protein adopts a three-dimensional fold similar to protein kinases. Furthermore, we argue that despite the lack of conservation of the "classic" catalytic aspartate residue of the archetypical His-Arg-Asp motif, SELO kinases might have retained catalytic phosphotransferase activity, albeit with an atypical active site. Lastly, the role of the selenocysteine residue is considered and the possibility of an oxidoreductase-regulated kinase function for SELO is discussed.The novel kinase prediction is discussed in the context of functional data on SELO orthologues in model organisms, FMP40 a.k.a.YPL222W (yeast), and ydiU (bacteria). Expression data from bacteria and yeast suggest a role in oxidative stress response. Analysis of genomic neighbourhoods of SELO homologues in the three domains of life points toward a role in regulation of ABC transport, in oxidative stress response, or in basic metabolism regulation. Among bacteria possessing SELO homologues, there is a significant over-representation of aquatic organisms, also of aerobic ones. The selenocysteine residue in SELO proteins occurs only in few members of this protein family, including proteins from Metazoa, and few small eukaryotes (Ostreococcus, stramenopiles). It is also demonstrated that enterobacterial mchC proteins involved in maturation of bactericidal antibiotics, microcins, form a distant subfamily of the SELO proteins.The new protein structural domain, with a putative kinase function assigned, expands the known kinome and deserves experimental determination of its biological role within the cell-signaling network