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.     

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.     

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.     

Reproductive cycle of gilt sardine,Sardinella aurita,Valenciennes 1847, in the eastern middle Adriatic Sea

The annual reproduction cycle of gilt sardine, Sardinella aurita, based on gonad maturity stages, gonad weight and gonadosomatic index was the subject of this study. A total of 2033 gilt sardines (983 males, 1021 females and 29 undetermined) were analysed. Fish were collected monthly from commercial purse seiners between November 2007 and January 2009 in the eastern middle Adriatic Sea (mesh size 8 mm/bar length/; sampling: five boats per month). Based on the monthly evolution of gonad maturation stages, gonad weights and gonadosomatic index, the peak spawning season was determined to be from June to August. Variations in sea surface temperature (SST) coincided with monthly variations of the gonadosomatic index. Highest monthly average values for both analysed parameters were recorded in July (GSI = 3.38; T = 26.5°C). Fifty per cent (L₅₀) of males and females reached sexual maturity at TL 15.8 cm and at 16.6 cm, respectively. Absolute fecundity ranged from 8458 to 48 032 (mean 34 565 ± 10 310), whereas relative fecundity was from 171 to 722 (mean 385 ± 104.35). Mean value of the oocyte size was 0.53 ± 0.10 mm.     

Influence of Mannosylation on Immunostimulating Activity of Adamant‐1‐yl Tripeptide

The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH₂CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H₂O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH₂)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.     

Functional tyrosine residue in the active center of human dipeptidyl peptidase III

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.     

Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin

Background and Aims

In the genus Anemone two small groups of taxa occur with the highest ploidy levels 2n = 6x = 48, belonging to the closely related clades: the montane/alpine Baldensis clade and the more temperate Multifida clade. To understand the formation of polyploids within these groups, the evolution of allohexaploid A. baldensis (AABBDD, 2n = 6x = 48) from Europe and allotetraploid Anemone multifida (BBDD, 2n = 4x = 32) from America was analysed.

Methods

Internal transcribed spacer and non-transcribed spacer sequences were used as molecular markers for phylogenetic analyses. Cytogenetic studies, including genomic in situ hybridization with genomic DNA of potential parental species as probe, fluorescence in situ hybridization with 5S and 18S rDNA as probes and 18S rDNA restriction analyses, were used to identify the parental origin of chromosomes and to study genomic changes following polyploidization.

Key Results

This study shows that A. multifida (BBDD, 2n= 4x = 32) and A. baldensis (AABBDD, 2n = 6x = 48) are allopolyploids originating from the crosses of diploid members of the Multifida (donor of the A and B subgenomes) and Baldensis groups (donor of the D subgenome). The A and B subgenomes are closely related to the genomes of A. sylvestris, A. virginiana and A. cylindrica, indicating that these species or their progeny might be the ancestral donors of the B subgenome of A. multifida and A and B subgenomes of A. baldensis. Both polyploids have undergone genomic changes such as interchromosomal translocation affecting B and D subgenomes and changes at rDNA sites. Anemone multifida has lost the 35S rDNA loci characteristic of the maternal donor (B subgenome) and maintained only the rDNA loci of the paternal donor (D subgenome).

Conclusions

It is proposed that A. multifida and A. baldensis probably had a common ancestor and their evolution was facilitated by vegetation changes during the Quaternary, resulting in their present disjunctive distribution.