The steady-state kinetics of isotope exchange at equilibrium: one substrate–one product enzymic mechanisms where two molecules of substrate or product are bound to an enzyme molecule (Short Communication)

The conclusion that the steady-state kinetics of isotope exchange at equilibrium do not show first-order behaviour for some one substrate-one product enzymic mechanisms in which two molecules of substrate or product can be combined with an enzyme molecule at the one time was shown to be erroneous.     

A new multicolor-FISH approach for the characterization of marker chromosomes: centromere-specific multicolor-FISH (cenM-FISH)

Centromere-specific multi-color FISH (cenM-FISH) is a new multicolor FISH technique that allows the simultaneous characterization of all human centromeres by using labeled centromeric satellite DNA as probes. This approach allows the rapid identification of all human centromeres by their individual pseudo-coloring in one single step and is therefore a powerful tool in molecular cytogenetics. CenM-FISH fills a gap in multicolor karyotyping using WCP probes and distinguishes all centromeric regions apart from the evolutionary highly conserved regions on the chromosomes 13 and 21. The usefulness of the cenM-FISH technique for the characterization of small supernumerary marker chromosomes with no (or nearly no) euchromatin and restricted amounts of available sample material is demonstrated in prenatal, postnatal, and tumor cytogenetic cases. In addition, rarely described markers with the involvement of heterochromatic material inserted into homogeneously staining regions could be identified and characterized by using the cenM-FISH technique.     

Evolution and functional characterization of the RH50 gene from the ammonia-oxidizing bacterium Nitrosomonas europaea

The family of ammonia and ammonium channel proteins comprises the Amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous Rh proteins (Rh50 and Rh30) that have been described thus far only in eukaryotes. The existence of an RH50 gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium Nitrosomonas europaea. Here we have used a phylogenetic approach to study the evolution of the N. europaea RH50 gene, and we show that this gene, probably as a component of an integron cassette, has been transferred to the N. europaea genome by horizontal gene transfer. In addition, by functionally characterizing the Rh50_Ne protein and the corresponding knockout mutant, we determined that NeRh50 can mediate ammonium uptake. The RH50_Ne gene may thus have replaced functionally the AMT gene, which is missing in the genome of N. europaea and may be regarded as a case of nonorthologous gene displacement.     

The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758)

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species’ unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.     

Differential effects of N-glycans on surface expression suggest structural differences between the acid-sensing ion channel (ASIC) 1a and ASIC1b

ASICs (acid-sensing ion channels) are H(+)-gated Na(+) channels with a widespread expression pattern in the central and the peripheral nervous system. ASICs have a simple topology with two transmembrane domains, cytoplasmic termini and a large ectodomain between the transmembrane domains; this topology has been confirmed by the crystal structure of chicken ASIC1. ASIC1a and ASIC1b are two variants encoded by the asic1 gene. The variable part of the protein includes the cytoplasmic N-terminus, the first transmembrane domain and approximately the first third of the ectodomain. Both variants contain two consensus sequences for N-linked glycosylation in the common, distal part of the ectodomain. In contrast with ASIC1a, ASIC1b contains two additional consensus sequences in the variable, proximal part of the ectodomain. Here we show that all the extracellular asparagine residues within the putative consensus sequences for N-glycosylation carry glycans. The two common distal glycans increase surface expression of the channels, but are no absolute requirement for channel activity. In sharp contrast, the presence of at least one of the two proximal glycans, which are specific to ASIC1b, is an absolute requirement for surface expression of ASIC1b. This result suggests substantial differences in the structure of the proximal ectodomain between the two ASIC1 variants.     

Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation

Although much physiology in resistance vessels has been attributed to the cytoplasmic connection between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), little is known of the protein expression between the two cell types. In an attempt to identify the proteins between ECs and VSMCs, mouse cremaster arterioles were stained with phalloidin-Alexa 594 and viewed on a confocal microscope that resolved "actin bridges" within the internal elastic lamina between ECs and VSMCs. To determine the incidence of protein, the pixel intensity from the antibodies on actin bridges were compared with the pixel intensity from antibodies within ECs or VSMCs. N-cadherin, desmin, connexin (Cx)40, and Cx43 and phosphorylated Cx43 at serine-368 were identified on actin bridges, but NG2, CD31, and Cx45 were not evident. Cx37 expression was more variable than the other connexins examined. Using this method on rat mesentery, we confirm the previously published predominance of Cx37 and Cx40 at the myoendothelial junction that was determined using electron microscopy. We conclude that this new method represents an important screening mechanism in which to rapidly test for protein expression between ECs and VSMCs and possibly a first-step in quantifying protein expression at the myoendothelial junction.     

Hypermutable Non-Synonymous Sites Are under Stronger Negative Selection

Mutation rate varies greatly between nucleotide sites of the human genome and depends both on the global genomic location and the local sequence context of a site. In particular, CpG context elevates the mutation rate by an order of magnitude. Mutations also vary widely in their effect on the molecular function, phenotype, and fitness. Independence of the probability of occurrence of a new mutation''s effect has been a fundamental premise in genetics. However, highly mutable contexts may be preserved by negative selection at important sites but destroyed by mutation at sites under no selection. Thus, there may be a positive correlation between the rate of mutations at a nucleotide site and the magnitude of their effect on fitness. We studied the impact of CpG context on the rate of human–chimpanzee divergence and on intrahuman nucleotide diversity at non-synonymous coding sites. We compared nucleotides that occupy identical positions within codons of identical amino acids and only differ by being within versus outside CpG context. Nucleotides within CpG context are under a stronger negative selection, as revealed by their lower, proportionally to the mutation rate, rate of evolution and nucleotide diversity. In particular, the probability of fixation of a non-synonymous transition at a CpG site is two times lower than at a CpG site. Thus, sites with different mutation rates are not necessarily selectively equivalent. This suggests that the mutation rate may complement sequence conservation as a characteristic predictive of functional importance of nucleotide sites.     

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.     

Neopterin kinetics after cardiac surgery with or without cardiopulmonary bypass

Cardiac surgery (CS) with cardiopulmonary bypass (CPB) induces systemic inflammatory response by activating plasma proteins and blood cells. Activated monocytes/macrophages produce inflammatory marker neopterin (NP). The aim was to explore the NP kinetics in first 24 hours after CS according to the CPB use. Significant difference between groups was found for NP levels 12 and 24 hrs after CS, being higher in on-pump group. Strong association was found between NP levels 12 hrs after CS and the length of ICU stay for on-pump group (r=0.744, p<0.001). Strong association was found between preoperative NP levels and the length of ICU stay for those on-pump patients with elevated preoperative NP (r=0.855, p=0.001; linear regression equation y=0.50x-5.14, p<0.001). Preoperative NP levels higher than 10 nmol/L in on-pump group could predict prolonged ICU stay and outpoint patients at higher risk for developing postoperative complications and, therefore, help to determine the necessary therapeutic interventions.