The maturation of HIV‐1 protease precursor studied by discrete molecular dynamics

The equilibrium properties of a HIV‐1‐protease precursor are studied by means of an efficient molecular dynamics scheme, which allows for the simulation of the folding of the protein monomers and their dimerization into an active form and compare them with those of the mature protein. The results of the model provide, with atomic detail, an overall account of several experimental findings, including the NMR conformation of the mature dimer, the calorimetric properties of the system, the effects of the precursor tail on the dimerization constant, the secondary chemical shifts of the monomer, and the paramagnetic relaxation enhancement data associated with the conformations of the precursor. It is found that although the mature protein can dimerize in a unique, single way, the precursor populates several dimeric conformations in which monomers are always native‐like, but their binding can be non‐native. Proteins 2014; 82:633–639. © 2013 Wiley Periodicals, Inc.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Comparative metabolomics charts the impact of genotype-dependent methionine accumulation in Arabidopsis thaliana

Methionine (Met) is an essential amino acid for all organisms. In plants, Met also functions as a precursor of plant hormones, polyamines, and defense metabolites. The regulatory mechanism of Met biosynthesis is highly complex and, despite its great importance, remains unclear. To investigate how accumulation of Met influences metabolism as a whole in Arabidopsis, three methionine over-accumulation (mto) mutants were examined using a gas chromatography–mass spectrometry-based metabolomics approach. Multivariate statistical analyses of the three mto mutants (mto1, mto2, and mto3) revealed distinct metabolomic phenotypes. Orthogonal projection to latent structures–discriminant analysis highlighted discriminative metabolites contributing to the separation of each mutant and the corresponding control samples. Though Met accumulation in mto1 had no dramatic effect on other metabolic pathways except for the aspartate family, metabolite profiles of mto2 and mto3 indicated that several extensive pathways were affected in addition to over-accumulation of Met. The pronounced changes in metabolic pathways in both mto2 and mto3 were associated with polyamines. The findings suggest that our metabolomics approach not only can reveal the impact of Met over-accumulation on metabolism, but also may provide clues to identify crucial pathways for regulation of metabolism in plants.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

Regulation of cholesterol synthesis in cultured mouse mammary carcinoma FM3A cells

Mouse mammary carcinoma FM3A cells, which are able to grow in a serum-free medium, have novel characteristics that could be valuable in biochemical and somatic cell genetic studies. In FM3A cells grown in the presence of serum, both sterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in the cholesterol biosynthetic pathway, were strongly suppressed by human low density lipoprotein (LDL). The addition of LDL (50 micrograms protein/ml) resulted in a 50% decrease in the reductase activity within 3 h and a 95% reduction after 24 h. Similarly, over 90% suppression of the reductase activity was obtained by the addition of LDL or mevalonolactone when the cells were grown on a serum-free medium. ML-236B (compactin), a specific inhibitor of HMG-CoA reductase, inhibited sterol synthesis from [14C]acetate by 80% at 1 microM. Reductase activity in FM3A cells was increased by 2.5- to 5-fold when the cells were treated with ML-236B (at 0.26-2.6 microM for 24 h). Thus, in FM3A cells, HMG-CoA reductase activity responded well to LDL, as is observed in human skin fibroblasts. Along with other novel features of this cell line, the present observations indicate that FM3A cells should be useful in biochemical and somatic cell genetic analysis of cholesterol metabolism, especially as regards the regulation of HMG-CoA reductase activity.     

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.     

Variability of the molecular mass of α-subunit in phycocyanins of colonial Microcystis aeruginosa f. aeruginosa in different eutrophic pond and lakes

Electrospray ionization mass spectra of phycocyaninsof eight samples from natural blooms of Microcystis aeruginosa f. aeruginosa collectedat different eutrophic pond and lakes indicated thatthe subunits of phycocyanins in the samespecies have different molecular masses, whereas the subunits have almost constant molecularmasses. A negative linear relationship between themolecular mass of subunit of phycocyanin andthe concentration of chlorophyll of natural andcolonial cyanobacterial samples was found. Aninterannual similarity of the subunitmolecular masses of phycocyanins was observed fromsamples collected at the same sampling site at LakeKasumigaura during 1994 and 1995. A locationalvariability of the subunit molecular massesof phycocyanins was also observed among samplescollected at three eutrophic pond and lakes.     

Characteristics and Pathogenicity of Non-Melibiose-Fermenting Strains of Yersinia pseudotuberculosis O3

The biological properties of non-melibiose-fermenting (NMF) strains of Yersinia pseudotuberculosis 03 were investigated. These strains were clearly distinguished from representative melibiose-fermenting (MF) strains of Y. pseudotuberculosis 03 by their pathogenicity in mice, sensitivity to some phages, production of catalase, restriction endonuclease analysis of virulence plasmid DNA with BamHI, detection of specific yersinia outer-membrane proteins with SDS-PAGE, antigenicity of the outer-membrane proteins and neutrophil resistance to phagocytosis. The pathogenicity of NMF strains was clearly less than that of MF strains. In addition, the resistance of NMF strains to phagocytosis and catalase activity was evidently weaker than that of MF strains. These results suggested that the difference of pathogenicity was due to the ability of catalase production. Although the relationship between the above characteristics and melibiose-fermentation was not analysed, the pathogenicity of Y. pseudotuberculosis 03 strains can probably be predicted by testing melibiose-fermentation and catalase production.     

Fine structure of isolated mesophyll protoplasts of tobacco

Summary Protoplasts of palisade cells isolated enzymatically from mature leaves of tobacco were studied with the electron microscope. A cell wall was completely absent, and the chloroplasts contained large inclusion bodies which were believed to be a crystalline form of fraction I protein. The fine structure of the protoplasts was otherwise that of healthy mesophyll cells, indicating that they are in a good physiological state. Some protoplasts were multinucleate as a result of fusion during the isolation process.