Molecular modeling study of the opioid receptor interactions with series of cyclic deltorphin analogues

In this study, ten tetra‐ and heptapeptide analogues of deltorphin containing the urea bridges between residues 2 and 4 have been docked into the δ‐ and µ‐opioid receptors to explain their different biological activities. The important factors explaining particular ligand activity such as free energy of binding, conformation of the ligand, its location inside the binding pocket as well as the number and strength of the receptor–ligand interactions have been discussed. Several different binding modes for investigated ligands have been proposed. It appears that the binding site is not identical even for very similar ligands. Results of this study help to explain the differences in biological activity of the deltorphin analogues, their interaction with the opioid receptors at the molecular level and support designing a new generation of potent opioid drugs with improved selectivity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Noninfectious X4 but not R5 human immunodeficiency virus type 1 virions inhibit humoral immune responses in human lymphoid tissue ex vivo

Ex vivo human immunodeficiency virus type 1 (HIV-1) infection of human lymphoid tissue recapitulates some aspects of in vivo HIV-1 infection, including a severe depletion of CD4(+) T cells and suppression of humoral immune responses to recall antigens or to polyclonal stimuli. These effects are induced by infection with X4 HIV-1 variants, whereas infection with R5 variants results in only mild depletion of CD4(+) T cells and no suppression of immune responses. To study the mechanisms of suppression of immune responses in this ex vivo system, we used aldrithiol-2 (AT-2)-inactivated virions that have functional envelope glycoproteins but are not infectious and do not deplete CD4(+) T cells in human lymphoid tissues ex vivo. Nevertheless, AT-2-inactivated X4 (but not R5) HIV-1 virions, even with only a brief exposure, inhibit antibody responses in human lymphoid tissue ex vivo, similarly to infectious virus. This phenomenon is mediated by soluble immunosuppressive factor(s) secreted by tissue exposed to virus.     

Comparative Genomics of Marine Mussels (Mytilus spp.) Gender Associated mtDNA: Rapidly Evolving atp8

The unusual mode of mitochondrial DNA inheritance, with two separate: maternal (F) and paternal (M) lineages, gives unique opportunities to study the evolution of the mitochondrial genome. This system was first discovered in the marine mussels Mytilus. The three related species: Mytilus edulis, Mytilus galloprovincialis and Mytilus trossulus form a complex in which the divergence of M and F lineages pre-dates the speciation. The complete mitochondrial genomes of both lineages were known for all species except Pacific M. trossulus. Here we report, for the first time, the complete sequences of both mitochondrial genomes of Pacific M. trossulus, filling the gap. While the reported M and F genomes are highly diverged (26%), they have similar organisation. The only difference is the translocation of one tRNA gene into the long, mosaic control region of the F genome. Consistent presence of an ORF which most likely represents the atp8 gene was confirmed in both genomes. The predicted protein has characteristics expected of the functional atp8 even though the M and F versions are markedly different in length. Comparative analysis involving all three species led to the conclusion that the cause of a faster evolution of atp8 and Mytilus mtDNA in general is most likely the Compensation-Draft Feedback process coupled with relatively relaxed selection in the M lineage. Thus, we postulate that the adaptive changes may have played a role in the emergence of highly diverged, barely recognizable atp8 in Mytilus mussels.     

Segmental motions of rat thymidylate synthase leading to half‐the‐sites behavior

Thymidylate synthase (TS) is a homodimeric enzyme with two equivalent active sites composed of residues from both subunits. Despite the structural symmetry of the enzyme, certain experimental results are consistent with half‐the‐sites activity, suggesting negative cooperativity between the active sites. To gain insight into the mechanism behind this phenomenon, we explore segmental motions of rat TS in the absence of ligands, with normal mode analysis as a tool. Using solvent accessible surface area of the active site pocket as a monitor of the degree of opening of the active sites, we classified the first 25 nontrivial normal modes, obtained from the web server of the program ElNémo, according to the behavior of the active sites. We found seven modes that open and close both sites symmetrically and nine that do so in an anticorrelated fashion. We characterized the motions of these modes by visual inspection and through measurement of distances between selected atoms lining the active site pockets. The segments that regulate access to the active site correspond to the loop containing R44, helix K, and a long loop containing residues 103–125, in agreement with a large body of crystallographic studies. These elements can be activated together or in isolation. There are more asymmetric modes than symmetric ones in the set we analyzed, probably accounting for the half‐the‐sites behavior of the enzyme. Three of the asymmetric modes result in changes at the dimer interface and indicate the endpoints of possible communication pathways between the active sites. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 549–559, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mutation in the Sp1 motif of the bovine leptin gene affects its expression

Leptin is expressed mainly by adipocytes and plays a crucial role in the regulation of energy expenditure, food intake, and adiposity. Using PCR-heteroduplex analysis and sequencing, we investigated a C/G substitution in the promoter region of the bovine leptin gene. Application of the electrophoretic mobility shift assay showed that the C→G transversion decreased the leptin gene promoter binding capacity for nuclear proteins. With real-time PCR and Western blotting, we showed that the leptin expression level was higher in cattle with the CC than with the GG genotype.     

Ammonia affects the activity and expression of soluble and particulate GC in cultured rat astrocytes

Neurotoxic effects of ammonia are mediated by increased accumulation of nitric oxide (NO), which combines with free radicals to form a highly toxic compound, peroxynitrite. Previous experiments in vivo and in vitro have suggested that this phenomenon engages neuron-derived NO and is coupled to changes in the accumulation of cGMP. The present study accounted for the facts that: (i) astrocytes, not neurons are the morphological target of ammonia, and (ii) both NO-dependent, soluble (sGC) and NO-independent, particulate guanylate cyclase (pGC) mediate cGMP production in the cells. Neocortical rat astrocytes were treated for 1 or 24 h with 5 mM ammonium chloride ("ammonia") and then subjected to: (i) cGMP measurement, and (ii) mRNA and/or protein expression analysis of alpha1 and beta1 subunits of sGC and two pGC forms: pGC-A and pGC-B. Treatment with ammonia for 1h increased accumulation of cGMP and sGCbeta1 mRNA expression, without producing significant changes in the protein expression. This was followed by a decrease of cGMP level at 24 h treatment, associated with a decreased expression of sGCbeta1 and sGCalpha1 mRNA and sGCbeta1 protein. Expression of pGC-A and pGC-B mRNA was elevated in ammonia-treated astrocytes after 24 h. Accordingly, increased cGMP accumulation was noted in the presence of a specific sGC inhibitor (ODQ). The results show that ammonia affects cGMP production in astrocytes, and that this may involve not only sGC but also pGC.     

Toll-like receptor gene polymorphism and its relationship with somatic cell concentration and natural bacterial infections of the mammary gland in sheep

Possible correlation between Toll-like receptor (TLR)-gene mutations and the susceptibility of the mammary gland to bacterial infections and also the associate breed-dependent aspects of somatic cell concentration (SCC), bacterial infection and TLR-gene mutations in sheep are described. In Polish Lowland Sheep (PLS), milk samples exceeding the level of 500/microL (i.e. 5 x 10(5) per mL) of SCC were recorded almost twice more frequently than in Polish Heath Sheep (PHS) (40 and 22.3%, respectively). The frequency of bacterial infections was also found in a similar ratio (20 and 12.7%, respectively). During detection of the TLR-gene mutation we recorded 2 alleles of TLR1, 6 alleles of TLR2 and 10 alleles of TLR4 genes in PHS sheep, while PLS sheep possessed 2, 4 and 6 alleles, respectively. Statistical analyses revealed a relationship between the specified TLR alleles, SCC and the frequency of incidence of bacterial inflammations of mammary gland. The data may serve as a benchmark for further study of TLR-gene mutation-dependent predisposition of mammary gland defensive cells to recognize the pathogen properly and initiate the immunological response, and may help in identifying one of the markers of natural resistance against sheep mastitis.     

Investment in radiotherapy infrastructure positively affected the economic status of an oncology hospital

BackgroundRadiotherapy is among the most efficient treatment methods of cancer. However, a radiotherapy base needs a substantial financial investment, especially before the beginning of its operation, and in some cases, in developing countries such a huge investment may cause some financial disturbances for a hospital concerned.AimTo assess the influence of investments modernizing the radiotherapy base in the period between 2000 and 2007 on the financial condition of the oncology hospital in the region with population of about 3 million.Material and methodsFinancial reports and medical statistics for the period between 2000 and 2007 from the studied oncology hospital and a recognized staffing model, as well as data on epidemiological situation of the region have been used to calculate the economic effects of financial investment in the radiotherapy base.ResultsThe growth of RT therapeutic potential has been driven by two cost-effective investment programmes. The total amount invested in both programmes was PLN 127,191,000.The number of radiotherapy patients treated in the hospital increased from 2301 in 2000 to 4799 in 2007 with a the same number of five therapeutic machines, although all five of them were replaced over that period. Investments modernizing the radiotherapy base lead to a significant increase in depreciation and operating costs, which adversely affects financial results of the hospital.ConclusionLong term trends showed that investments had positive influence on hospital performance shown both in increased income and larger number of patients treated.     

Changes in reproductive behavior in adult damselfly Calopteryx splendens (Odonata: Calopterygidae) in response to flood

The reproductive behavior of adult Calopteryx splendens males and females inhabiting the Nida River, south Poland, was studied and compared during a pre‐flood and a post‐flood year. The flood disturbance in 2010 caused a decrease in aquatic macrophytes, thus reducing availability of potential territories and consequently, significantly influencing male behavior towards a frequent non‐territorial strategy. Many males in the post‐flood population had damaged wings due to extremely aggressive contests. Male–male tandems were commonly observed; this is an uncommon behavior in C. splendens. Although the sex ratio was male‐biased throughout the whole study, we observed more males in the post‐flood year. We also observed less‐frequent copulations and ovipositions during the post‐flood year. The only unchanged characteristic was population density, which did not differ before and after the flood disturbance. Floods have significant impact on damselfly reproductive sites and this, due to changes in behavior and sex ratio, may result in further consequences on population dynamics.     

Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids

An efficient method for the heteroconjugation of biomolecules carrying free amino groups was reported previously, where mixed polyfluorophenyl diesters of dicarboxylic acids with varied aliphatic chain length were shown to be efficient reagents for the conjugation of a variety of model biomolecules. The concept was based on the differential reactivity of the esters towards amines. The concept has now been further optimized, and a 2,6‐difluorophenyl‐pentafluorophenyl diester combination has been demonstrated to be the most efficient, both with respect to selectivity and to reaction rate. A pentafluorophenyl ester reacts faster with an amino group and requires a weaker base than a 2,6‐difluorophenyl ester that requires a stronger base and longer reaction time. With the use of this combination of esters, we obtained considerably shortened reaction times compared with those reported previously, yet still retaining the desired selectivity in heteroconjugation. The increased reactivity of the bifunctional reagent allowed the construction of sophisticated peptide heteroconjugates from peptides, carbohydrates and proteins, showing a wide scope of applicability in the field of assembling functional bioconjugates. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.