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Biosynthesis of a water-soluble lipid I analogue and a convenient assay for translocase I
Get PDFTranslocase I (MraY/MurX) is an essential enzyme in growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park’s nucleotide) to prenyl-MurNAc-pentapeptide (lipid I), the first membrane-anchored peptidoglycan precursor. MurX has received considerable attention in the development of new tuberculosis (TB) drugs due to the fact that the MurX inhibitors kill exponentially growing Mycobacterium tuberculosis (Mtb) much faster than clinically used TB drugs. Lipid I isolated from Mtb contains the C50-prenyl unit that shows very poor water solubility; thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays.Wehave established a scalable chemical synthesis of Park’s nucleotide-Ne-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of the minimumstructure requirement of the prenyl phosphate in the MraY/MurX-catalyzed lipid I analogue synthesis with 2,we found that neryl phosphate (C10 phosphate) can be recognized by MraY/MurX to generate the water-soluble lipid I analogue in quantitative yield under the optimized conditions. Here, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecule
Expression of avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) under control of the ptcB promoter in Lactococcus lactis.
Get PDFGram-positive and nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of new, safe systems of heterologous protein expression. Recombinant LAB has been shown to induce specific local and systemic immune response against selected pathogens, and could be a good alternative to classical attenuated carriers. The main goal of our study was to express the avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) in Lactococcus lactis. Results of this study were anticipated to lead to construction of lactococcal strain(s) with potential vaccine properties against the avian influenza A (H5N1) virus. Expression of the cloned H5 gene, its His-tagged variant and chIL-2 gene, under the control of the ptcB gene promoter was attested by RT-PCR on transcriptional level and Western or dot blot analysis on translational level, demonstrating that system can be an attractive solution for production of heterologous proteins. The results of the preliminary animal trial conducted in mice are a promising step toward development of a vaccine against avian bird flu using Lactococcus lactis cells as antigen carriers
Genetic diversity ofSCN5A gene and its possible association with the concealed form 2 of Brugada Syndrome development in Polish group of patients
Get PDFBrugada Syndrome (BS) is an inherited channelopathy associated with a high incidence of sudden cardiac death. The article presents the discovery of new genetic variants at SCN5A gene which might be associated with the development of concealed form of Brugada Syndrome. The study involved a group of 59 patients (37 men) with suspected concealed form of Brugada Syndrome. Pharmacological provocation with intravenous ajmaline administration was performed. Six patients with positive test result were subjected to molecular analysis of SCN5A gene with MSSCP method. Additionally, MSSCP genotyping was performed for samples obtained from the family members with Brugada Syndrome, despite they had negative ajmaline challenge test results.Genetic examinations of the SCN5A gene at 6 positive patients showed 6 known polymorphisms, 8 new single nucleotide point (SNP) variants located at exons and 12 new single nucleotide point variants at introns. Among detected in exons SNP, two of them were synonymous SNPs (no change in the coded amino acids), whereas 3 represented non-synonymous SNPs and affected the protein sequences
Study of beef blade muscles’ differentiation depending on conformation and fat class
Get PDFThe object of this study was to identify variation of the intramuscular fat and connective tissue content in different blade muscles in carcasses characterized by various quality grades. It was found that there is a cumulative impact of muscle and conformation class on intramuscular fat in blade muscle (P = 0.0330), as well as type of muscle and fat class (P = 0.0424), but there is no cumulative impact of conformation class and fat class (P = 0.1788). There is no cumulative influence of muscle type, conformation class, and fat class on amount of connective tissue in blade muscle, but the infraspinatus muscle was characterized by the highest quantity of connective tissue. The differences in the content of intramuscular fat in blade muscles depend on type of muscle as well as fat or conformation class,
but there is no cumulative effect of fat and conformation class
Cold stress effects on organelle ultrastructure in polar Caryophyllaceae species
Get PDFAbstract: This study investigated leaf mesophyll cells of Caryophyllaceae plants growing in
polar regions – Cerastium alpinum and Silene involucrata from the Hornsund region of
Spitsbergen island (Svalbard Archipelago, Arctic), and Colobanthus quitensis from the Ad−
miralty Bay region on King George Island (South Shetland Islands, West Antarctic). Ultra−
structural changes were analyzed in mesophyll protoplasts of plants growing in natural Arctic
and Antarctic habitats and plants grown in a greenhouse, including plants exposed to
short−term cold stress under semi−controlled conditions. Cell organelles of plants growing in
natural polar habitats and greenhouse−grown plants were characterized by significant mor−
phological plasticity.Chloroplasts of plants studied in this work formed variously shaped pro−
trusions and invaginations that visibly increased the contact area between adjacent cell com−
partments and reduced the distance between organelles. S. involucrata plants grown under
greenhouse conditions, tested by us in this work, were characterized by highly dynamic cell
nuclei with single or multiple invaginations of the nuclear membrane and the presence of
channels and cisternae filled with cytoplasm and organelles. Crystalline inclusion proteins
were observed in the cell nuclei of C. quitensis between nuclear membranes and in the direct
proximity of heterochromatin. Our study revealed significant conformational dynamics of
organelles, manifested by variations in the optical density of matrices, membranes and envelopes, in particular in C. quitensis, which could suggest that the analyzed Caryophyllaceae
taxa are well adapted to severe climate and changing conditions in polar regions
Changes in soluble carbohydrates in polar Caryophyllaceae and Poaceae plants in response to chilling
Get PDFAbstract Four species of flowering plants comprising
Arctic populations of Cerastium alpinum and Poa arctica
var. vivipara and indigenous Antarctic species Colobanthus
quitensis and Deschampsia antarctica were investigated.
Plants derived from natural origins were grown in an
experimental greenhouse in Poland (53�470N and 20�300E
latitude). Plants for experiment were collected during spring
of 2010. Soluble carbohydrates in the intact shoots of C.
alpinum and C. quitensis, polar plants of the family Caryophyllaceae,
and D. antarctica and P. arctica var. vivipara,
representatives of the family Poaceae, were analyzed by gas
chromatography, and their involvement in the plants’
response to chilling stress was examined. Plant tissues of the
examined families growing in a greenhouse conditions
(18–20 �C, short day 10/14 h light/darkness) differed in the
content and composition of soluble carbohydrates. In addition
to common monosaccharides, myo-inositol and sucrose,
Caryophyllaceae plants contained raffinose family oligosaccharides
(RFOs), D-pinitol and mono-galactosyl pinitols.
RFOs and D-pinitol were not detected in plants of the family
Poaceae which contain 1-kestose, a specific tri-saccharide.
The accumulation of significant quantities of sucrose in all
investigated plants, RFOs in Caryophyllaceae plants and
1-kestose in Poaceae plants in response to chilling stress(4 �C for 48 h with a long day photoperiod, 20/4 h) indicates
that those compounds participate in the stress response. The
common sugar accumulating in cold stress response and
probably most important for chilling tolerance of four
investigated plants species seems to be sucrose. On the other
hand, the accumulation of above-mentioned carbohydrates
during chilling stress can be a return to sugars metabolism,
occurring in natural environmental conditions. No changes
in D-pinitol concentrations were observed in the tissues of C.
alpinum and C. quitensis plants subjected to both low and
elevated temperatures, which probably rules out the protective
effects of D-pinitol in response to cold stress
Proteins contribute insignificantly to the intrinsic buffering capacity of yeast cytoplasm.
Get PDFIntracellular pH is maintained by a combination of the passive buffering of cytoplasmic dissociable compounds and several active systems. Over the years, a large portion of and possibly most of the cell's intrinsic (i.e., passive non-bicarbonate) buffering effect was attributed to proteins, both in higher organisms and in yeast. This attribution was not surprising, given that the concentration of proteins with multiple protonable/deprotonable groups in the cell exceeds the concentration of free protons by a few orders of magnitude. Using data from both high-throughput experiments and in vitro laboratory experiments, we tested this concept. We assessed the buffering capacity of the yeast proteome using protein abundance data and compared it to our own titration of yeast cytoplasm. We showed that the protein contribution is less than 1% of the total intracellular buffering capacity. As confirmed with NMR measurements, inorganic phosphates play a crucial role in the process. These findings also shed a new light on the role of proteomes in maintaining intracellular pH. The contribution of proteins to the intrinsic buffering capacity is negligible, and proteins might act only as a recipient of signals for changes in pH
Application of 13C–13C Spin–Spin Couplings in Structural Studies on Organic Compounds
No full textIt is already well recognized that crucial information on the electron structure of a chemical bond and the structure of organic compounds including those of biological origin is encoded in the Js. Therefore, the knowledge of trends governing them provides a unique opportunity for the creative designing of new chemical reactions, leading to unknown molecular structures and/or the elucidation of biosynthetic pathways. This chapter is devoted to the CC couplings across one, two, and three bonds. However, it is not our goal to provide a comprehensive review on all couplings published in the literature; the references to the relevant reviews published so far on this topic are included in the list of the literature [1–14]. We shall rather concentrate our attention on the factors which influence the magnitude of a given coupling. The J data presented in the subsequent parts of the chapter have been collected and arranged with the thought of showing how hybridization of the orbitals of the atoms involved
in the coupling, substituent electronegativity, the complex and hydrogen bond formation and geometry of the compound bear on the JCC magnitude and which range of changes can be expected for a given type of coupling when all these effects are taken into account. The changes caused by the
abovementioned factors are in many cases very strong and by no means can be neglected when either structural studies or measurements of Js are carried out. With the introduction of high magnetic fields and of cryogenic probes, the measurements of JCCs became routine even for comparatively
large molecules and for a small amount of the sample. The latter is especially important in the case of compounds of biological origin which are often only accessible in the quantity of several milligrams. Another valuable source of information on Js is provided by ab initio and density functional theory (DFT) quantum mechanical calculations. In particular, DFT calculations of spin–spin couplings for molecules of moderate sizes can currently be performed on modest computer systems, providing an access to large sets
of data in a reasonable time [15,16]. A comparison of the experimental couplings with calculated ones is very interesting and useful since, on the one hand, one can evaluate the validity of the theoretical approach employed and, on the other hand, it provides a deeper insight into the coupling mechanism. It is also important from a practical point of view since it allows one to trace possible errors in the experimental JCCs reported. However, it should be stressed at this point that it is crucial to have large sets of data for such comparisons, as otherwise any linear regressions of J(expl.) versus J(calcd.) obtained are likely to be accidental. We would like to mention at the end that generally, the IUPAC nomenclature
has been applied throughout the whole chapter. However, in some cases, the arbitrary numbering of the substituents has been necessary in order to account for their strong effect
A kinetic model of the evolution of a protein interaction network
Get PDFAbstract
Background: Known protein interaction networks have very particular properties. Old proteins tend to have more
interactions than new ones. One of the best statistical representatives of this property is the node degree
distribution (distribution of proteins having a given number of interactions). It has previously been shown that this
distribution is very close to the sum of two distinct exponential components. In this paper, we asked: What are the
possible mechanisms of evolution for such types of networks? To answer this question, we tested a kinetic model
for simplified evolution of a protein interactome. Our proposed model considers the emergence of new genes and
interactions and the loss of old ones. We assumed that there are generally two coexisting classes of proteins.
Proteins constituting the first class are essential only for ecological adaptations and are easily lost when ecological
conditions change. Proteins of the second class are essential for basic life processes and, hence, are always
effectively protected against deletion. All proteins can transit between the above classes in both directions. We also
assumed that the phenomenon of gene duplication is always related to ecological adaptation and that a new copy
of a duplicated gene is not essential. According to this model, all proteins gain new interactions with a rate that
preferentially increases with the number of interactions (the rich get richer). Proteins can also gain interactions
because of duplication. Proteins lose their interactions both with and without the loss of partner genes.
Results: The proposed model reproduces the main properties of protein-protein interaction networks very well. The
connectivity of the oldest part of the interaction network is densest, and the node degree distribution follows the
sum of two shifted power-law functions, which is a theoretical generalization of the previous finding. The above
distribution covers the wide range of values of node degrees very well, much better than a power law or
generalized power law supplemented with an exponential cut-off. The presented model also relates the total
number of interactome links to the total number of interacting proteins. The theoretical results were for the
interactomes of A. thaliana, B. taurus, C. elegans, D. melanogaster, E. coli, H. pylori, H. sapiens, M. musculus, R.norvegicus and S. cerevisiae.
Conclusions: Using these approaches, the kinetic parameters could be estimated. Finally, the model revealed the
evolutionary kinetics of proteome formation, the phenomenon of protein differentiation and the process of gaining
new interactions
Distinct protein classes in human red cell proteome revealed by similarity of phylogenetic profiles.
No full textThe minimal set of proteins necessary to maintain a vertebrate cell forms an interesting core of cellular machinery. The known proteome of human red blood cell consists of about 1400 proteins. We treated this protein complement of one of the simplest human cells as a model and asked the questions on its function and origins. The proteome was mapped onto phylogenetic profiles, i.e. vectors of species possessing homologues of human proteins. A novel clustering approach was devised, utilising similarity in the phylogenetic spread of homologues as distance measure. The clustering based on phylogenetic profiles yielded several distinct protein classes differing in phylogenetic taxonomic spread, presumed evolutionary history and functional properties. Notably, small clusters of proteins common to vertebrates or Metazoa and other multicellular eukaryotes involve biological functions specific to multicellular organisms, such as apoptosis or cell-cell signaling, respectively. Also, a eukaryote-specific cluster is identified, featuring GTP-ase signalling and ubiquitination. Another cluster, made up of proteins found in most organisms, including bacteria and archaea, involves basic molecular functions such as oxidation-reduction and glycolysis. Approximately one third of erythrocyte proteins do not fall in any of the clusters, reflecting the complexity of protein evolution in comparison to our simple model. Basically, the clustering obtained divides the proteome into old and new parts, the former originating from bacterial ancestors, the latter from inventions within multicellular eukaryotes. Thus, the model human cell proteome appears to be made up of protein sets distinct in their history and biological roles. The current work shows that phylogenetic profiles concept allows protein clustering in a way relevant both to biological function and evolutionary history