Molecular and cellular mechanisms in the viral exacerbation of asthma

The aetiology of asthma associated with viral infection is complex. The dynamics that contribute to disease pathogenesis are multifactorial and involve overlapping molecular and cellular mechanisms, particularly the immune response to respiratory virus infection or allergen sensitization. This review summarizes the evidence associated with factors that may contribute to the development or exacerbation of asthma including age, host factors, genetic polymorphisms, altered immune responses, and aspects of viral antigen expression. This review also provides an important perspective of key events linked to the development of asthmatic disease and related pulmonary inflammation from human and animal studies, and discusses their relationship as targets for disease intervention strategies.     

Production of phenolic compounds,antioxidant and antimicrobial activities in hairy root and shoot cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.

Three hairy root clones of Hypericum perforatum (HR 2, HR 15 and HR 27) transformed with Agrobacterium rhizogenes A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-p-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-O-methylkaempferol-O-hexoside and quercetin 6-C-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-C-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa and Escherichia coli. Altogether, H. perforatum HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties.     

Entrapment of peptidoglycans and adamantyltripeptides into liposomes: an HPLC assay for determination of encapsulation efficiency

The encapsulation of different immunomodulating peptides, the peptidoglycan monomer, its semisynthetic derivatives (Adamant-1-yl)-acetyl-peptidoglycan monomer and Boc-Tyr-peptidoglycan monomer, respectively, and of two diastereoisomers of adamantyltripeptides into the large negatively charged multilamellar liposomes was investigated. The reproducible quantitative method using HPLC was established for the determination of the entrapped compounds. It was shown that the tested compounds could be efficiently incorporated into liposomes using either the film or modified film method. The results confirmed that the peptidoglycans with lipophilic substituents and particularly the adamantyltripeptides were incorporated into liposomes with higher efficiency than the peptidoglycan monomer using either of the described methods. Liposome preparations were stable at 4 degrees C up to seven days as shown by minimal leaking of the entrapped material.     

The repertoire of solute carriers of family 6: identification of new human and rodent genes

Tremendous amount of primary sequence information has been made available from the genome sequencing projects, although a complete annotation and identification of all genes is still far from being complete. Here, we present the identification of two new human genes from the pharmacologically important family of transporter proteins, solute carriers family 6 (SLC6). These were named SLC6A17 and SLC6A18 by HUGO. The human repertoire of SLC6 proteins now consists of 19 functional members and four pseudogenes. We also identified the corresponding orthologues and additional genes from mouse and rat genomes. Detailed phylogenetic analysis of the entire family of SLC6 proteins in mammals shows that this family can be divided into four subgroups. We used Hidden Markov Models for these subgroups and identified in total 430 unique SLC6 proteins from 10 animal, one plant, two fungi, and 196 bacterial genomes. It is evident that SLC6 proteins are present in both animals and bacteria, and that three of the four subfamilies of mammalian SLC6 proteins are present in Caenorhabditis elegans, showing that these subfamilies are evolutionary very ancient. Moreover, we performed tissue localization studies on the entire family of SLC6 proteins on a panel of 15 rat tissues and further, the expression of three of the new genes was studied using quantitative real-time PCR showing expression in multiple central and peripheral tissues. This paper presents an overall overview of the gene repertoire of the SLC6 gene family and its expression profile in rats.     

Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylation

A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate-polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N-glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N-glycans was compared. The most favourable results for native acidic N-glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N-glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

Novel desosamine-modified 14- and 15-membered macrolides without antibacterial activity

Novel modifications of the desosamine sugar of 14- and 15-membered antibacterial macrolides, in which the desosamine was fused with N-substituted-1,3-oxazolidin-2-ones, were developed in order to completely suppress antibacterial activity and make them promising agents for other biological targets. The synthesis of such bicyclic desosamine derivatives, especially 1,3-oxazolidin-2-one formation, was optimized and conducted under mild conditions without a need for protection/deprotection steps for other functional groups. A focused series of novel desosamine-modified macrolide derivatives was prepared and their antibacterial activities tested. It was shown that these macrolide derivatives do not possess any residual antibacterial activity.     

Proteogenomic Analysis to Identify Missing Proteins from Haploid Cell Lines

Chromosome‐centric Human Proteome Project aims at identifying and characterizing protein products encoded from all human protein‐coding genes. As of early 2017, 19 837 protein‐coding genes have been annotated in the neXtProt database including 2691 missing proteins that have never been identified by mass spectrometry. Missing proteins may be low abundant in many cell types or expressed only in a few cell types in human body such as sperms in testis. In this study, we performed expression proteomics of two near‐haploid cell types such as HAP1 and KBM‐7 to hunt for missing proteins. Proteomes from the two haploid cell lines were analyzed on an LTQ Orbitrap Velos, producing a total of 200 raw mass spectrometry files. After applying 1% false discovery rates at both levels of peptide‐spectrum matches and proteins, more than 10 000 proteins were identified from HAP1 and KBM‐7, resulting in the identification of nine missing proteins. Next, unmatched spectra were searched against protein databases translated in three frames from noncoding RNAs derived from RNA‐Seq data, resulting in six novel protein‐coding regions after careful manual inspection. This study demonstrates that expression proteomics coupled to proteogenomic analysis can be employed to identify many annotated and unannotated missing proteins.