Dynamical characterization of the heme NO oxygen binding (HNOX) domain. Insight into soluble guanylate cyclase allosteric transition

Since the discovery of soluble guanylate cyclase (sGC) as the mammalian receptor for nitric oxide (NO), numerous studies have been performed in order to understand how sGC transduces the NO signal. However, the structural basis of sGC activation is still not completely elucidated. Spectroscopic and kinetic studies showed that the key step in the activation mechanism was the NO-induced breaking of the iron proximal histidine bond in the so-called 6c-NO to 5c-NO transition. The main breakthrough in the understanding of sGC activation mechanism came, however, from the elucidation of crystal structures for two different prokaryotic heme NO oxygen (HNOX) domains, which are homologues to the sGC heme domain. In this work we present computer simulation results of Thermoanaerobacter tencogensis HNOX that complement these structural studies, yielding molecular explanations to several poorly understood properties of these proteins. Specifically, our results explain the differential ligand binding patterns of the HNOX domains according to the nature of proximal and distal residues. We also show that the natural dynamics of these proteins is intimately related with the proposed conformational dependent activation process, which involves mainly the alphaFbeta1 loop and the alphaA-alphaC distal subdomain. The results from the sGC models also support this view and suggest a key role for the alphaFbeta1 loop in the iron proximal histidine bond breaking process and, therefore, in the sGC activation mechanism.     

Peptidyl-CCA deacylation on the ribosome promoted by induced fit and the O3'-hydroxyl group of A76 of the unacylated A-site tRNA

The last step in ribosome-catalyzed protein synthesis is the hydrolytic release of the newly formed polypeptide from the P-site bound tRNA. Hydrolysis of the ester link of the peptidyl-tRNA is stimulated normally by the binding of release factors (RFs). However, an unacylated tRNA or just CCA binding to the ribosomal A site can also stimulate deacylation under some nonphysiological conditions. Although the sequence of events is well described by biochemical studies, the structural basis of the mechanism underlying this process is not well understood. Two new structures of the large ribosomal subunit of Haloarcula marismortui complexed with a peptidyl-tRNA analog in the P site and two oligonucleotide mimics of unacylated tRNA, CCA and CA, in the A site show that the binding of either CA or CCA induces a very similar conformational change in the peptidyl-transferase center as induced by aminoacyl-CCA. However, only CCA positions a water molecule appropriately to attack the carbonyl carbon of the peptidyl-tRNA and stabilizes the proper orientation of the ester link for hydrolysis. We, thus, conclude that both the ability of the O3′-hydroxyl group of the A-site A76 to position the water and the A-site CCA induced conformational change of the PTC are critical for the catalysis of the deacylation of the peptidyl-tRNA by CCA, and perhaps, an analogous mechanism is used by RFs.     

Electric organ polyamines and their effects on the acetylcholine receptor

1. The electric organ of Torpedo nobiliana contained putrescine (PUT), spermidine (SPD), spermine (SPM), and cadaverine (CAD). Traces of acetylated SPD and SPM were occasionaly seen. 2. Upon fractionation of the tissue by differential centrifugation, the polyamines (PA) were found predominantly in the soluble fraction. The postsynaptic membrane fraction, containing a high concentration of acetylcholine receptor (AChR), was proportionally enriched in SPM. The molar ratio of SPM to AChR was approximately two in these membranes. 3. The effect of exogeneous PA on AChR function was studied by two methods: carbamoylcholine (CCh)-dependent 86Rb+ influx into receptor-rich membrane vesicles and [alpha-125I]bungarotoxin (Bgt) binding to the AChR. 4. SPM inhibited both ion influx and the rate of Bgt binding at concentrations above 1 mM, and therefore it appears to act as a competitive antagonist of the AChR. 5. At submicromolar concentrations, and only after preincubation with the receptor-rich membrane, SPM and PUT increased the ion influx by about 20% over control values. 6. Preincubation with 100 nM SPM did not affect the equilibrium binding of iodinated toxin or the rate of toxin binding, and therefore SPM was not uncovering new receptors. 7. By measuring the initial rate of toxin binding after different periods of preincubation with 1 microM CCh, the rate of the slow phase of receptor desensitization was determined. This rate was not changed by 100 nM SPM. 8. Although these results suggest that at low concentrations SPM is a positive modulator of the AChR, the precise mechanism of action is not determined yet.     

Epigenetic natural variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F₂ families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.     

Loss of plasmids of Borrelia burgdorferi sensu lato during prolonged in vitro cultivation

In the present study we analyzed stability of plasmid content in 34 Borrelia strains of three different species (13 Borrelia afzelii, 10 Borrelia garinii and 11 Borrelia burgodorferi sensu stricto) using pulse field gel electrophoresis (PFGE). During long-term in vitro cultivation consisting of 50 passages, plasmid loss was established in 46% of B. afzelii, 40% of B. garinii and 36% of B. burgdorferi sensu stricto strains. Loss of plasmids occurred as early as between the 5th and 10th passage, affected only plasmids in the range 9-41 kb but not plasmids in the range 50-68 kb and manifested with the loss of one to up to three plasmids.     

Identification of proteins bearing beta1-6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis

The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary beta1-6-N-acetylglucosamine (beta1-6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing beta1-6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35--from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing beta1-6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP-1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing beta1-6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules.     

Effect of indole-3-butyric acid on phytoplasmas in infected Catharanthus roseus shoots grown in vitro

Phytoplasmas are noncultivable bacteria usually maintained in Catharanthus roseus shoots grown in vitro on MS medium with benzylaminopurine. The aim of our research was to examine the influence of indole-3-butyric acid (IBA) on C. roseus shoots infected with three different phytoplasma strains. Supplement of IBA in the medium supported plant growth, photosynthesis and remission of symptoms in all phytoplasma-infected shoots, but had no effect on the presence of EY-C and SA-I phytoplasma strains in tested tissue. However, HYDB phytoplasma was undetectable in approximately half of the tested shoots grown on the medium with IBA. After 1 year of IBA treatment, HYDB-infected periwinkle shoots were retransferred to the medium supplemented with benzylaminopurine. Some of the shoots showing remission of symptoms during the IBA treatment permanently escaped the infection and remained negative when tested for phytoplasma presence. This is the first report on the differential influence of plant growth regulators on phytoplasma-infected C. roseus shoots.     

Eimeria papillata: upregulation of specific miRNA-species in the mouse jejunum

Increasing evidence indicates miRNAs as critical regulators of gene expression, but little information is available for miRNAs in intestinal diseases. Here, we investigated intestinal infections of male Balb/c mice with the coccidian parasite Eimeria papillata. On day 4 after oral infection, mice were shedding 3150 ± 430 oocysts per gram feces. This was associated with a low inflammatory response of the jejunum of mice evidenced by histology, non-response of IL-1β mRNA, even slight downregulation of IL-6 mRNA, only slight increases in iNOS mRNA, nitrate/nitrate, malondialdehyde, and a small decrease in glutathione, respectively. Only IFNγ mRNA was strongly induced. Using miRNA microarray technology, there were significantly upregulated the four miRNA species miR-1959, MCMV-miR-M23-1-5P, miR-203, and miR-21 out of 634 miRNAs, which was also confirmed by quantitative RT-PCR. Our data provide evidence that E. papillata parasites are able to induce specific miRNA species in their host target organ.     

The use of scientometric parameters for the evaluation of scientific contributions

This paper deals with the application of scientometric parameters in the evaluation of scientists, either as individuals or in small formal groups. The parameters are divided into two groups: parameters of scientific productivity and citation parameters. The scientific productivity was further subdivided into three types of parameters: (i) total productivity, (ii) partial productivity, and (iii) productivity in scientific fields and subfields. These citation parameters were considered: (i) impact factors of journals, (ii) impact factors of scientific fields and subfields, (iii) citations of individual papers, (iv) citations of individual authors, (v) expected citation rates and relative citation rates, and (vi) self-citations, independent citations and negative citations. Particular attention was payed to the time-dependence of the scientometric parameters. If available, numeric values of the world parameters were given and compared with the data about the scientific output of Croatian scientists.