Alkylcobalt chelates with Schiff bases derived from a β-diketone bearing both alkyl and aryl groups

Organocobalt(III) complexes with Schiff bases derived from a β-diketone bearing both an alkyl and an aryl group have been prepared. The template syntheses using benzoylacetone and ethylenediamine as complexing agents provide a route to alkylcobalt chelates with the corresponding tri- and tetradentate Schiff bases. However, if a β-diketone with two aryl groups, e.g. dibenzoylmethane, was employed as the starting ketoenol component, no organometallic products were detected; a new mixed-ligand ‘inorganic’ chelate of cobalt(II), [Co{O=C(Ph)CH=C(Ph)O}₂(en)], was isolated instead. Its structure as well as that of one of the alkylcobalt complexes with a tridentate Schiff base composed of benzoylacetone and ethylenediamine have been established by X-ray techniques. The current scope of the template synthesis of alkylcobalt complexes with Schiff bases is summarized.     

Mathematical frameworks for phenotypical selection and epistasis

A mathematical approach to interactions between genotypes and phenotypes in a multilocus multiallele population is developed. No a priori information on a fitness function is required. In particular, some structural definitions of epistasis and the position effect are given in terms of a decomposition of phenotypical structures. On this base a distance to the additive non-epistasis is introduced and an explicit formula for it is obtained. A class of phenotypical structures including multilocus dominance is described in terms of directed graphs. The evolutionary equations are adjusted to a fitness function compatible with a phenotypical structure. Some results on the finiteness of the equilibria set are presented.     

Isolation and characterization of high affinity aptamers against DNA polymerase iota

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.     

Role of thrombospondin-1 in T cell response to ocular pigment epithelial cells

Ocular pigment epithelium (PE) cells promote the generation of T regulators (PE-induced Treg cells). Moreover, T cells exposed to PE acquire the capacity to suppress the activation of bystander T cells via TGFbeta. Membrane-bound TGFbeta on iris PE cells interacts with TGFbeta receptors on T cells, leading to the conversion of T cells to CD8(+) Treg cells via a cell contact-dependent mechanism. Conversely, soluble forms of TGFbeta produced by retinal PE cells can convert CD4(+) T cells into Treg cells in a manner that is independent of cell contact. In this study, we looked at the expression of immunoregulatory factors (TGFbeta, thrombospondins, CD59, IL-1 receptor antagonist, etc.) in PE cells as identified via an oligonucleotide microarray. Several thrombospondin-binding molecules were detected, and thus we focused subsequent analyses on thrombospondins. Via the conversion of latent TGFbeta to an active form that appears to be mediated by thrombospondin 1 (TSP-1), cultured iris PE and retinal PE cells induce a PE-induced Treg cell fate. After conversion, both ocular PE and PE-induced Treg cells express TSP-1. Regulatory T cell generation was amplified when the T cells also expressed TSP-1. In addition, PE-induced Treg cells significantly suppressed activation of bystander T cells via TSP-1. These results strongly suggest that the ability of ocular PE and PE-induced Treg cells to suppress bystander T cells depends on their capacity to produce TSP-1. Thus, intraocular TSP-1 produced by both ocular parenchymal cells and regulatory T cells is essential for immune regulation in the eye.     

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.     

Do microbes have macroecology? Large‐scale patterns in the diversity and distribution of marine benthic ciliates

Aim The biogeography and global distribution of protists has long been disputed, with two primary, opposing views. To test these two sets of views in greater detail, we have compiled the available data for marine benthic ciliates and assessed the general patterns of their diversity and distribution compared with Metazoa. Location World‐wide. Methods A comprehensive database (1342 species, over 350 sources) was used to analyse the diversity, distribution, species occurrences and range size distribution of free‐living ciliates that inhabit marine sediments in 17 geographical regions. Results Twenty‐five per cent of the species have been found in a single region only, whereas 18% are widespread (they occur in more than half the regions covering both hemispheres). Only 5–7% of regional faunas are endemic, which is much lower than for macroorganisms. Regional diversity depends neither on total area nor on coastline length and does not show any obvious latitudinal trends, but correlates highly with the investigation effort expended in a region and (negatively) with the average salinity. A comparison of species composition reveals distinctions between the Arctic Area (the White, Barents and Kara seas), Laurasian Area (north Atlantic, north Pacific and European seas), Gondwanian Area (Southern Ocean) and the Antarctic. No clear geographical correlations are found for faunistic composition at the genus or family levels. There is the tendency to narrow the latitudinal ranges for species found at high latitudes (reversal of Rapoport's rule). Main conclusions Undersampling and data insufficiency are the key factors affecting the observed diversity and distribution of microorganisms. Nevertheless, marine benthic ciliates demonstrate certain patterns that generally agree with the ‘moderate endemicity’ model ( Foissner, 2004, 2008 ), but consistently contradict the regularities commonly observed for multicellular taxa. Thus, ciliates do have a biogeography, but their macroecological patterns may be different in some respects from that of macroorganisms.     

Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.     

Plant-made trastuzumab (herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth

Background

Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells.

Methodology/Principal Findings

We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells.

Conclusions/Significance

We conclude that PMT is active in suppression of cell proliferation and tumor growth.