Comparative genomics indicates the mammalian CD33rSiglec locus evolved by an ancient large-scale inverse duplication and suggests all Siglecs share a common ancestral region

The CD33-related sialic acid binding Ig-like lectins (CD33rSiglecs) are predominantly inhibitory receptors expressed on leukocytes. They are distinguishable from conserved Siglecs, such as Sialoadhesin and MAG, by their rapid evolution. A comparison of the CD33rSiglec gene cluster in different mammalian species showed that it can be divided into subclusters, A and B. The two subclusters, inverted in relation to each other, each encode a set of CD33rSiglec genes arranged head-to-tail. Two regions of strong correspondence provided evidence for a large-scale inverse duplication, encompassing the framework CEACAM-18 (CE18) and ATPBD3 (ATB3) genes that seeded the mammalian CD33rSiglec cluster. Phylogenetic analysis was consistent with the predicted inversion. Rodents appear to have undergone wholesale loss of CD33rSiglec genes after the inverse duplication. In contrast, CD33rSiglecs expanded in primates and many are now pseudogenes with features consistent with activating receptors. In contrast to mammals, the fish CD33rSiglecs clusters show no evidence of an inverse duplication. They display greater variation in cluster size and structure than mammals. The close arrangement of other Siglecs and CD33rSiglecs in fish is consistent with a common ancestral region for Siglecs. Expansion of mammalian CD33rSiglecs appears to have followed a large inverse duplication of a smaller primordial cluster over 180 million years ago, prior to eutherian/marsupial divergence. Inverse duplications in general could potentially have a stabilizing effect in maintaining the size and structure of large gene clusters, facilitating the rapid evolution of immune gene families. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multiplex detection of surface molecules on colorectal cancers

A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was captured on a CD antibody microarray. The cancer cells were detected using a fluorescently tagged antibody for carcinoembryonic antigen (CEA-Alexa647) or epithelial cell adhesion marker (EpCAM-Alexa488). Using this multiplexing procedure, dot patterns from colorectal cancers were distinct from those of adjacent normal tissue. Subtraction of the expression levels for each antigen from normal tissue from those for the cancer shows differential expression in the cancer of CD66c, CD15s, CD55, CD45, CD71, CD45RO, CD11b and CEA, in descending order. Cells captured on the same microarray were also labelled with fluorescent CD3-phycoerythrin antibody revealing the presence of tumour-infiltrating lymphocytes. The immunophenotypes of T lymphocytes from the tumour samples showed differential expression of HLA-DR, TCR alpha/beta, CD49d, CD52, CD49e, CD5, CD95, CD28, CD38 and CD71, in descending order. Fluorescence multiplexing of mixed cell populations captured on a single antibody microarray enables expression profiling of multiple sub-populations of cells within a tumour sample.     

The submolecular mechanism of the initial stage of the electron coupling in the water oxidizing center of higher plants

The results of quantum chemistry calculations of the total energy and proton position in tyrosine-histidine system in a water-oxidizing complex are presented. On the basis of these calculations, the submolecular mechanism of the initial stage of the electron coupling was proposed.     

Immunoglobulin genetics of Ornithorhynchus anatinus (platypus) and Tachyglossus aculeatus (short-beaked echidna)

In this paper, we review data on the monotreme immune system focusing on the characterisation of lymphoid tissue and of antibody responses, as well the recent cloning of immunoglobulin genes. It is now known that monotremes utilise immunoglobulin isotypes that are structurally identical to those found in marsupials and eutherians, but which differ to those found in birds and reptiles. Monotremes utilise IgM, IgG, IgA and IgE. They do not use IgY. Their IgG and IgA constant regions contain three domains plus a hinge region. Preliminary analysis of monotreme heavy chain variable region diversity suggests that the platypus primarily uses a single VH clan, while the short-beaked echidna utilises at least 4 distinct VH families which segregate into all three mammalian VH clans. Phylogenetic analysis of the immunoglobulin heavy chain constant region gene sequences provides strong support for the Theria hypothesis. The constant region of IgM has proven to be a useful marker for estimating the time of divergence of mammalian lineages.     

Isolation and characteristics of the lipid granules of Candida tropicalis yeasts

Preparative isolation of lipid granules from the protoplasts of Candida tropicalis was conducted by a technique of flotation in a stepwise density gradient. Parameters were selected for decomposing the protoplasts under hypotonic and isotonic conditions which made it possible to preserve the lipid granules being isolated intact, as well as parameters of a density gradient and centrifugation. The specific content of lipids, proteins and low molecular weight compounds was assayed using labeled compounds in the lipid granules which were isolated from yeast cells cultivated on various carbon substrates (1-6(-14C)-glucose and 1(-14C-octadecane). The lipid composition of the spherosomes was determined. If the yeast was grown on glucose, lipids localized in the lipid granules were represented mainly by triglycerides whose carbon constituted 69 per cent of the total lipid carbon. If it was cultivated on n-octadecane, these lipids were represented by hydrocarbons (51 per cent) and triglycerides (22 per cent). The structures isolated possessed a small lipase activity. The specific lipase activity of the lipid granules was lower by 16 per cent than that of the cell protoplast.     

Prothymosin alpha fragmentation in apoptosis

We observed fragmentation of an essential proliferation-related human nuclear protein prothymosin alpha in the course of apoptosis induced by various stimuli. Prothymosin alpha cleavage occurred at the DDVD(99) motif. In vitro, prothymosin alpha could be cleaved at D(99) by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin alpha and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin alpha fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin alpha in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein.     

A comparative evaluation of the pharmacokinetics of different forms of benzathine benzylpenicillin]

Comparative randomized opened pharmacokinetic evaluation of benzathine benzylpenicillin in three dosage forms was performed. Benzathine benzylpenicillin was used as extencilline (2.4 million U or 1.2 million U, "Rh?ne-Poulenc Rorer", France) and as bicillin-5 (1.5 million U, "Synthesis" Russia). 33 patients were included in investigation (23 women and 10 men aged 16-60 years). 25 persons had verified rheumatism without blood circulation failure signs, 4--had chronic tonsillitis and 4 were healthy volunteers. Benzylpenicillin concentration was estimated by microbiology test in blood samples taken at 1, 3, 24 hours and 7, 14 and 21 days after intramuscular drug injection. After 2.4 million U extencilline injection (12 patients) its concentration, was at the inhibition level for beta-hemolytic streptococcus group A (25 ng/ml) for 3 weeks-period in 83.3 per cent of patients. After 1.2 million U extencilline injection (10 patients) or 1.5 million U bicillin-5 injection (12 patients) the above mentioned concentration was achieved on the 21st day in 30 and 0 per cent of patients respectively. Thus the treatment with benzathine benzylpenicillin at the 1.2 million U dose in the form of extencilline or bicillin-5 doesn't provide adequate antistreptococcal concentration in blood in prolonged period and is not suitable for correct rheumatism prophylaxis in adult patients.     

Poliovirus proteins induce membrane association of GTPase ADP-ribosylation factor

Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication.