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Mapping of the influenza A hemagglutinin serotypes evolution by the ISSCOR method.
Get PDFAnalyses and visualizations by the ISSCOR method of influenza virus hemagglutinin genes of different A-subtypes revealed some rather striking temporal relationships between groups of individual gene subsets. Based on these findings we consider application of the ISSCOR-PCA method for analyses of large sets of homologous genes to be a worthwhile addition to a toolbox of genomics - allowing for a rapid diagnostics of trends, and ultimately even aiding an early warning of newly emerging epidemiological threats
Virus-like particles as vaccine.
Get PDFThis review presents data on commercial and experimental virus-like particle (VLP) vaccines, including description of VLP vaccines against influenza. Virus-like particles are multimeric, sometimes multiprotein nanostructures assembled from viral structural proteins and are devoid of any genetic material. VLPs present repetitive high-density displays of viral surface proteins. Importantly, they contain functional viral proteins responsible for cell penetration by the virus, ensuring efficient cell entry and thus tissue-specific targeting, determined by the origin of the virus. The foremost application of VLPs is in vaccinology, where they provide delivery systems that combine good safety profiles with strong immunogenicity and constitute a safe alternative to inactivated infectious viruses. These stable and versatile nanoparticles display excellent adjuvant properties capable of inducing innate and cognate immune responses. They present both, high-density B-cell epitopes, for antibody production and intracellular T- cell epitopes, thus inducing, respectively, potent humoral and cellular immune responses. Uptake of VLPs by antigen-presenting cells leads to efficient immune responses resulting in control of pathogenic microorganisms
Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma.
Get PDFAdenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth
Candida albicans cis-prenyltransferase Rer2 is required for protein glycosylation, cell wall integrity and hypha formation
Get PDFCis-prenyltransferase is the first enzyme of the mevalonate pathway committed to the biosynthesis of dolichol in eukaryotes. The RER2 gene encoding cis-prenyltransferase (Rer2p) in the human fungal pathogen Candida albicans was characterized. In addition, the ORF19.5236 encoding the second cis-prenyltransferase, which putatively is responsible for the synthesis of longer polyisoprenoids chains, was identified. When cultivated under repressive conditions, the conditional mutant strain expressing the RER2 gene from the regulatable MET3 promoter contained only 4% of cis-prenyltransferase activity and markedly diminished amounts of dolichols, as compared to the wild-type strain. Moreover, transcriptomal analyses revealed changes in the expression of 300 genes, mainly involved in transport, response to stress, filamentous growth and organelle organization. Growth of the conditional strain was blocked completely at 37 °C. The strain was hypersensitive to a wide range of inhibitors, which suggested glycosylation defects and compromised cell wall integrity. Moreover, the rer2 conditional mutant grown in the repressive conditions, unlike the same strain in the absence of repressor, failed to form hyphae. The results indicate that dolichols are essential not only for protein glycosylation and cell wall integrity but also for growth and development of C. albicans.
Keywords
protein glycosylation; dolichol; cis-prenyltransferase; Candida albicans; cell wall, hyph
Plant expression systems for production of hemagglutinin as a vaccine against influenza virus.
Get PDFMany examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity
8-Oxo-7,8-dihydroguanine and uric acid as efficient predictors of survival in colon cancer patients
Get PDFThe aim of this work was to answer the question whether the broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair are appropriate prognosis factors of colon cancer (CRC) patients survival? The following parameters were analyzed for 89 CRC patients: concentration of uric acid and vitamins A, E, C in plasma; levels of 8-oxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine) in DNA of leukocyte and colon tissues; urinary excretion rates of 8-oxodGuo and 8-oxoGua (8-oxo-7,8-dihydroguanine); the activity and mRNA or protein level of repair enzymes OGG1, APE1, ANPG, TDG and PARP1.
All DNA modifications and plasma antioxidants were analyzed using high performance liquid chromatography (HPLC) or HPLC/gas chromatography-mass spectrometry techniques. Expression of repair proteins was analyzed by QPCR, Western or immunohistochemistry methods.
Longer survival coincided with low levels of 8-oxodGuo/8oxoGua in urine and 8-oxodGuo in DNA as well as with high concentration of uric acid plasma level. In contrast to expectations, longer survival coincided with lower mRNA level in normal colon tissue of the main 8-oxoGua DNA glycosylase, OGG1, but no association was found for PARP-1 expression. When analyzing simultaneously two parameters the discriminating power increased significantly. Combination of low level of urinary 8-oxoGua together with low level of 8-oxodGuo in leukocyte (both below median value) or high concentration of plasma uric acid (above median value) have the best prediction power. Since prediction value of these parameters seems to be comparable to conventional staging procedure, they could possibly be used as markers to predict clinical success in CRC treatment
Identification of protein partners in Mycobacteria using a single step affinity purification method
Get PDFTuberculosis is a leading cause of death in developing countries. Efforts are being made
to both prevent its spreading and improve curability rates. Understanding the biology of
the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus
vital. We have implemented improved screening methods for protein-protein
interactions based on affinity purification followed by high-resolution mass
spectrometry. This method is attractively applicable to both medium- and highthroughput
studies aiming to characterize protein-protein interaction networks of
tubercle bacilli. From four tested epitopes, FLAG, eGFP, Protein A, and hemagglutinin,
the eGFP tag was found most useful based on easily monitored expression and as a
simultaneous tool for sub-cellular localization studies. It presents a relatively low
background with cost effective purification.
RNA polymerase subunit A (RpoA) was used as a model for investigation of a large
protein complex. When used as a bait, it co-purified with all remaining RNA polymerase
core subunits as well as many accessory proteins. The amount of RpoA strongly
correlated with the amount of quantification peptide used as part of the tagging system
in this study (SH), making it applicable for semi-quantification studies. Interactions
between the components of the RpoA-eGFP protein complex were further confirmed
using protein cross-linking.
Dynamic changes in the composition of protein complexes under induction of UV
damage were observed when UvrA-eGFP expressing cells, treated with UV light were
used to co-purify UvrA interaction partners
Cloning and Characterization of a Wheat Homologue of Apurinic/Apyrimidinic Endonuclease Ape1L
Get PDFBackground: Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3’-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized.
Methodology/Principal Findings: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3’-repair phosphodiesterase, 3’-phosphatase and 3’→5’ exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg2+ and Ca2+ (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn2+, Co2+ and Fe2+ cations (0.1-1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6–7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20°C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3’-blocking sugar-phosphate and 3’-phosphate groups with good efficiency (kcat/KM = 630 and 485 µM–1•min–1, respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1.
Conclusions/Significance: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors
Regulatory Mutations Affecting Sulfur Metabolism Induce Environmental Stress Response in Aspergillus nidulans
Get PDFMutations in the cysB, sconB and sconC genes affect sulfur metabolism in Aspergillus nidulans in different
ways. The cysB mutation blocks synthesis of cysteine by the main pathway and leads to a shortage of this
amino acid. The sconB and sconC mutations affect subunits of the SCF ubiquitin ligase complex, which
inactivates the MetR transcription factor in the presence of an excess of cysteine. In effect, both cysB
and scon mutations lead to permanent derepression of MetR-dependent genes. We compared transcriptomes
of these three mutants with that of a wild type strain finding altered expression of a few hundred
genes belonging to various functional categories. Besides those involved in sulfur metabolism, many
up-regulated genes are related to stress responses including heat shock and osmotic stress. However,
only the scon strains are more resistant to exogenous stress agents than the wild type strain while cysB
is more sensitive. The two-component signal transduction system is a functional category, which is most
enriched among genes up-regulated in the cysB, sconB and sconC mutants. A large group of up-regulated
genes are involved in carbohydrate and energy metabolism, including genes coding for enzymes of trehalose
and glycerol synthesis. The altered expression of these genes is accompanied by changes in sugar
and polyol accumulation in conidia of the mutants. Genes encoding enzymes of the glyoxylate bypass and
the GABA shunt are also up-regulated along with genes coding for enzymes of alcohol fermentation.
Among the down-regulated genes the most numerous are those encoding membrane proteins and
enzymes involved in secondary metabolism, including the penicillin biosynthesis cluster