Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

192 Computer-aided search for novel anti-HIV-1 agents presenting peptidomimetics of broadly neutralizing antibodies against the virus envelope GP120 V3 loop

Computer-aided search for novel anti-HIV-1 agents that are able to imitate the pharmacophore properties of the antigen-binding site of a broadly neutralizing mAb 3074 against the envelope gp120 V3 loop was carried out followed by evaluation of their potential inhibitory activity by molecular modeling. In doing so, the following problems were solved: (1) the mAb 3074 amino acid residues responsible for specific binding to the HIV-1 V3 loop were identified from the X-ray structures of this antibody Fab in complexes with the MN, UR29, and VI191 V3 peptides (Jiang et al., 2010); (2) using these data, 2039 possible mAb-3074 peptidomimetics were found by pepMMsMIMIC presenting a public, web-oriented virtual screening platform (Floris et al., 2011); (3) the complexes of these compounds with the above V3 peptides were built by molecular docking and, based on their analysis, the four molecules exhibiting a high affinity to V3 in the in silico studies were selected as the most probable peptidomimetics of mAb 3074 (Figure 1); and (4) stability of the complexes of these molecules with the MN, UR29, and VI191 V3 peptides was estimated by molecular dynamics and free energy simulations. As a result, a key role in specific binding of the selected compounds to the V3 loop was shown to belong to π-π interactions between their aromatic rings and the conserved Phe20 and/or Tyr21 of the V3 immunogenic crown. Similarly to mAb 3074, these compounds were found to block the tip of the V3 loop forming its invariant structural motif, which contains residues critical for cell tropism (Andrianov et al., 2011; Andrianov et al., 2012). In addition, the complexes of interest do not undergo significant changes within the molecular dynamics calculations, exhibiting the low values of free energy of their formation. In this context, the compounds given in Figure 1 are considered as the promising basic structures for the design of novel, potent, and broad anti-HIV-1 drugs.     

Rotamer libraries and probabilities of transition between rotamers for the side chains in protein-protein binding

Conformational changes in the side chains are essential for protein-protein binding. Rotameric states and unbound- to-bound conformational changes in the surface residues were systematically studied on a representative set of protein complexes. The side-chain conformations were mapped onto dihedral angles space. The variable threshold algorithm was developed to cluster the dihedral angle distributions and to derive rotamers, defined as the most probable conformation in a cluster. Six rotamer libraries were generated: full surface, surface noninterface, and surface interface-each for bound and unbound states. The libraries were used to calculate the probabilities of the rotamer transitions upon binding. The stability of amino acids was quantified based on the transition maps. The noninterface residues' stability was higher than that of the interface. Long side chains with three or four dihedral angles were less stable than the shorter ones. The transitions between the rotamers at the interface occurred more frequently than on the noninterface surface. Most side chains changed conformation within the same rotamer or moved to an adjacent rotamer. The highest percentage of the transitions was observed primarily between the two most occupied rotamers. The probability of the transition between rotamers increased with the decrease of the rotamer stability. The analysis revealed characteristics of the surface side-chain conformational transitions that can be utilized in flexible docking protocols.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Structural analysis of the envelope gp120 V3 loop for some HIV-1 variants circulating in the countries of Eastern Europe

The V3 loop on gp120 from human immunodeficiency virus type 1 (HIV-1) is a focus of many research groups involved in anti-AIDS drug development because this region of the protein is a principal target for neutralizing antibodies and a major determinant for cell tropism and syncytium formation. In this study, the nucleotide sequences of the env gene region coding the V3 loop were determined by DNA sequencing methods for four novel HIV-1 strains that circulate in the countries of Eastern Europe, such as Russia, Belarus, Ukraine, etc. Based on the empirical data obtained, the 3D structures of the V3 loops associated with these viral modifications were generated by computer modeling and then compared to discover similarities in the spatial arrangement of this functionally important site of gp120. Despite the HIV-1 genetic variety, several regions of the V3 loop that contain residues critical for cell tropism were shown to be structurally invariant, which may explain its exceptional role in a co-receptor usage. These data together with those on the biological activity of the V3 individual residues clearly show that these conserved structural motifs of gp120 represent potential HIV-1 weak points most suitable for therapeutic intervention.     

The role of hydrophobic interactions in catalysis of RNA cleavage by 1,4-diazabicyclo[2.2.2]-octane based artificial ribonucleases

Molecular interactions of RNA cleaving compounds-conjugates of 1,4-diazabicyclo[2.2.2.]octane substituted at the bridge position with tetradecamethylene fragment and imidazole were investigated using light scattering and small angle x-ray scattering methods. The compounds are known to efficiently cleave RNA and one source of the activity could result from micellar catalysis. It was found that the compounds indeed are capable of forming complex aggregates in solution. However, maximal efficacy of RNA cleavage by the conjugates is observed at concentrations well below the concentration required for micelle formation.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.