A nascent proteome study combining click chemistry with 2DE

To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified.     

Comparison of anti-aggregatory activities of 5-phenyl-3-(3-pyridyl)isoxazole and 5-phenyl-3-(3-pyridyl)-1,2,4-oxadiazole

Two bioisosteric analogs, 5-phenyl-3-(3-pyridyl)isoxazole and 5-phenyl-3-(3-pyridyl)-1,2,4-oxadiazole, were synthesized so as to compare their antiaggregatory activities, to determine a pharmacologically active fragment in molecules of this type, and to explore the mechanisms of action of potential antiag-gregatory compounds belonging to the class of 3,5-substituted isoxazoles. Antiaggregatory activities of these compounds were studied in vitro using three aggregation inducers, such as arachidonic acid, adenosine diphosphate, and adrenaline. It was shown that 5-phenyl-3-(3-pyridyl)-1,2,4-oxadiazole and 5-phenyl-3-(3-pyridyl)isoxazole completely suppressed platelet aggregation induced by arachidonic acid and the second wave of platelet aggregation induced by adrenaline or adenosine diphosphate. The antiaggregatory activity of substituted isoxasole was 1.1–1.5 times higher than that of substituted oxadiazole. In contrast to the isoxazole analog, 5-phenyl-3-(3-pyridyl)-1,2,4-oxadiazole in concentrations of 300–400 μM partially suppressed the first wave of aggregation induced by adenosine diphosphate. It was demonstrated that both compounds were not thrombin inhibitors in vitro at concentrations up to 250 μM. Thus, introduction of a nitrogen atom into the C4-position of the isoxazole ring changes the molecule properties. It suggests that the pharmacophoric fragment of the molecule should be the whole isoxazole or 1,2,4-oxadiazole ring but not a part of the ring as was supposed previously.     

T Cell Epitope Mapping of JC Polyoma Virus-Encoded Proteome Reveals Reduced T Cell Responses in HLA-DRB1*04:01+ Donors

JC polyomavirus (JCV) infection is highly prevalent and usually kept in a persistent state without clinical signs and symptoms. It is only during immunocompromise and especially impaired CD4⁺ T cell function in the brain, as seen in AIDS patients or natalizumab-treated multiple sclerosis patients, that JCV may cause progressive multifocal leukoencephalopathy (PML), an often life-threatening brain disease. Since CD4⁺ T cells likely play an important role in controlling JCV infection, we here describe the T cell response to JCV in a group of predominantly HLA-DR-heterozygotic healthy donors (HD) by using a series of overlapping 15-mer peptides spanning all JCV-encoded open reading frames. We identified immunodominant epitopes and compared T cell responses with anti-JCV VP1 antibody production and with the presence of urinary viral shedding. We observed positive JCV-specific T cell responses in 28.6% to 77.6%, humoral immune response in 42.6% to 89.4%, and urinary viral shedding in 36.4% to 45.5% of HD depending on the threshold. Four immunodominant peptides were mapped, and at least one immunogenic peptide per HLA-DRB1 allele was detected in DRB1*01⁺, DRB1*07⁺, DRB1*11⁺, DRB1*13⁺, DRB1*15⁺, and DRB1*03⁺ individuals. We show for the first time that JCV-specific T cell responses may be directed not only against JCV VP1 and large T antigen but also against all other JCV-encoded proteins. Heterozygotic DRB1*04:01⁺ individuals showed very low T cell responses to JCV together with normal anti-VP1 antibody levels and no urinary viral shedding, indicating a dominant-negative effect of this allele on global JCV-directed T cell responses. Our data are potentially relevant for the development of vaccines against JCV.     

Comparative proteomic characteristic of Mycoplasmas (Mollycutes)

Saturating proteome identification and the study of post-translational protein modifications of Acholeplasma laidlawii using combination of single- and two-dimension gel electrophoresis followed by mass-spectrometry analysis have been carried out. Results were compared to the earlier identified proteome of Mycoplasma gallisepticum. It was found that M. gallisepticum and A. laidlawii express 61 and 58% of the annotated ORFs respectively. All subunits of DNA-polymerase III were identified during our study which indicates that our methods can detect single copies of a given protein per cell. Metabolic pathways of the respective mycoplasmas were compared further in this work.     

Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.     

Poliomyelitis in Tajikistan. Protection of Russia from emergence and spread of wild poliomyelitis virus

Problem of emergence and spread of poliomyelitis in Russian Federation and neighboring states is examined. Measures taken in Russian Federation to prevent emergence of poliomyelitis cases caused by wild type virus are discussed, as well as treaties and agreements between Russia, Commonwealth of Independent States, Shanghai Cooperation Organization states regarding epidemiological control of poliomyelitis and acute flaccid paralysis. Measure planned by Federal Service for Surveillance for Protection of Consumers Rights and Human Welfare to prevent emergence of poliomyelitis and acute flaccid paralysis cases in Russian Federation and neighboring countries are presented.     

Complete genome and proteome of Acholeplasma laidlawii

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.     

Epidemiologic versions in investigation of Legionnaires' disease outbreak in town Verkhnyaya Pyshma

Materials of investigation of Legionnaires' disease outbreak in one of the town of Sverdlovsk region in summer 2007 are presented. Outbreak arose in situation of epidemiologic welfare on majority of registered infectious diseases. Epidemiologic diagnostics of the outbreak has been performed in maximal shorttime interval, during which it was necessary to investigate variety of miscellaneous versions. Active search of the source of the infection and its transmission factors resulted in desired result and allowed to terminate the outbreak in 14 days.     

Benzoylphenyl thiocyanates are new,effective inhibitors of the mycobacterial resuscitation promoting factor B protein

Background

Resuscitation promoting factors (Rpfs) are the proteins involved in the process of reactivation of the dormant cells of mycobacteria. Recently a new class of nitrophenylthiocyanates (NPTs), capable of inhibiting the biological and enzymatic activities of Rpfs has been discovered. In the current study the inhibitory properties of the compounds containing both nitro and thiocyanate groups alongside with the compounds with the modified number and different spatial location of the substituents are compared.

Methods

New benzoylphenyl thiocyanates alongside with nitrophenylthiocyanates were tested in the enzymatic assay of bacterial peptidoglycan hydrolysis as well as against strains of several actinobacteria (Mycobacterium smegmatis, Mycobacterium tuberculosis) on in-lab developed models of resuscitation of the dormant forms.

Results

Introduction of the additional nitro and thiocyanate groups to the benzophenone scaffold did not influence the inhibitory activity of the compounds. Removal of the nitro groups analogously did not impair the functional properties of the molecules. Among the tested compounds two molecules without nitro group: 3-benzoylphenyl thiocyanate and 4-benzoylphenyl thiocyanate demonstrated the maximum activity in both enzymatic assay (inhibition of the Rpf-mediated peptidoglycan hydrolysis) and in the resuscitation assay of the dormant M. tuberculosis cells.

Conclusions

The current study demonstrates dispensability of the nitro group in the NPT’s structure for inhibition of the enzymatic and biological activities of the Rpf protein molecules. These findings provide new prospects in anti-TB drug discovery especially in finding of molecular scaffolds effective for the latent infection treatment.