Cyclic mismatch binding ligands interact with disease-associated CGG trinucleotide repeats in RNA and suppress their translation

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.     

Halogenated imidazole derivatives block RNA polymerase II elongation along mitogen inducible genes

Background  

Aberrant activation of protein kinases is one of the essential oncogenic driving forces inherent to the process of tumorigenesis. The protein kinase CK2 plays an important role in diverse biological processes, including cell growth and proliferation as well as in the governing and transduction of prosurvival signals. Increased expression of CK2 is a hallmark of some cancers, hence its antiapoptotic properties may be relevant to cancer onset. Thus, the designing and synthesis of the CK2 inhibitors has become an important pursuit in the search for cancer therapies.     

Dietary Macrogard reduces <Emphasis Type=Italic>Aeromonas hydrophila</Emphasis> mortality in tench (<Emphasis Type=Italic>Tinca tinca</Emphasis>) through the activation of cellular and humoral defence mechanisms

Influence of beta-1.3/1.6-glucan (Macrogard) on the innate immunity and protection against Aeromonas hydrophila in tench (Tinca tinca (L.)) was assessed. Macrogard was fed at doses of 0, 0.5, 1 and 2 g kg⁻¹ of pellets for 1 month. The blood, spleen and head kidney from 10 fish of each group were separated and analysed for immunity parameters. Twenty tench from each group were infected with A. hydrophila. Macrogard at doses 1 and 2 g kg⁻¹ of feed significantly (P < 0.05) increased the phagocytic activity of macrophages and proliferative response of mitogens stimulated lymphocytes. The same doses significantly (P < 0.05) increased lysozyme activity and Ig level in serum, compared to the control and dose 0.5 g kg⁻¹ of feed. The challenge test showed that Macrogard reduced mortality of tench after experimental infection (5–35%).     

Evaluation of fresh and frozen-thawed fowl semen by flow cytometry

The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.     

Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis

Background  

Recently, HEN1 protein from Arabidopsis thaliana was discovered as an essential enzyme in plant microRNA (miRNA) biogenesis. HEN1 transfers a methyl group from S-adenosylmethionine to the 2'-OH or 3'-OH group of the last nucleotide of miRNA/miRNA* duplexes produced by the nuclease Dicer. Previously it was found that HEN1 possesses a Rossmann-fold methyltransferase (RFM) domain and a long N-terminal extension including a putative double-stranded RNA-binding motif (DSRM). However, little is known about the details of the structure and the mechanism of action of this enzyme, and about its phylogenetic origin.     

New derivative of 1alpha,25-dihydroxy-19-norvitamin D3 with 3'-alkoxypropylidene moiety at C-2: synthesis, biological activity and conformational analysis

In pursuit of novel biologically active Vitamin D compounds of potential therapeutic value, 1alpha,25-dihydroxy-2-[3'-(methoxymethoxy)propylidene]-19-norvitamin D(3) (7) was efficiently prepared in a convergent synthesis, starting with (-)-quinic acid and the protected 25-hydroxy Grundmann ketone 16. The key synthetic step involved Lythgoe type Wittig-Horner coupling of 16, with the phosphine oxide 15. Molecular modeling was employed to establish the A-ring conformation of the synthesized Vitamin 7. Also, preliminary modeling of its complex with the rVDR was performed and interactions between ligand and the binding domain analyzed. Analog 7 was found to be only six times less potent than 1alpha,25-(OH)(2)D(3) (1) in binding to the rat recombinant Vitamin D receptor (VDR). In comparison with hormone 1, it also showed slightly lower cellular HL-60-differentiation activity. Preliminary in vivo tests indicated unusually high calcemic activity of 7.     

Molecular basis of cellulose biosynthesis disappearance in submerged culture of Acetobacter xylinum

Acetobacter xylinum strains are known as very efficient producers of bacterial cellulose which, due to its unique properties, has great application potential. One of the most important problems faced during cellulose synthesis by these bacteria is generation of cellulose non-producing cells, which can appear under submerged culture conditions. The reasons of this remain unknown. These studies have been undertaken to compare at the molecular level wild-type, cellulose producing (Cel(+)) A. xylinum strains with Cel(-) forms of cellulose-negative phenotype. Comparison of protein profiles of both forms of A. xylinum by 2D electrophoresis allowed for the isolation of proteins which were produced exclusively by either Cel+ or Cel- cells. Sequences of peptides derived from these proteins were aligned with those of proteins deposited in databases. This analysis revealed that Cel(-) cells lacked two enzymes: phosphoglucomutase and glucose-1-phosphate uridylyltransferase, which generates UDP-glucose being the substrate for cellulose synthase. DNA was analyzed by ligation-mediated PCR carried out at low denaturation temperature (PCR-MP). Two DNA fragments of different thermal stability (218 and 217 bp) were obtained from the DNA of Cel(+) and Cel(-) forms, respectively. The only difference between these Cel(-) and Cel(+) DNA fragments is deletion of one T residue. Alignment of those two sequences with those deposited in the GenBank database revealed that similar fragments are present in the genomes of some bacterial cellulose producers and are located downstream from open reading frames (ORF) encoding phosphoglucomutase. The meaning of this observation is discussed.     

Comparison of the localization and post-translational modification of Campylobacter coli CjaC and its homolog from Campylobacter jejuni, Cj0734c/HisJ

Campylobacter is an asaccharolytic microorganism which uses amino acids as a source of carbon and energy. CjaC/HisJ is a ligand-binding protein, a component of the ABC transport system. Campylobacter CjaC/HisJ is post-translationally modified by glycosylation. The number of glycosylation motifs present in the CjaC protein is species-specific. C. coli CjaC has two and C. jejuni one motif (E/DXNYS/T) which serves as a glycan acceptor. Although the two C. coli CjaC motifs have identical amino-acid sequences they are not glycosylated with the same efficiency. The efficacy of CjaC glycosylation in Escherichia coli containing the Campylobacter pgl locus is also rather low compared to that observed in the native host. The CjaC localization is host-dependent. Despite being a lipoprotein, CjaC is recovered in E. coli from the periplasmic space whereas in Campylobacter it is anchored to the inner membrane.     

Structure of the O-specific polysaccharide of Proteus vulgaris O15 containing a novel regioisomer of N-acetylmuramic acid, 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O15 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, and H-detected 1H,(13)C HMQC experiments. The polysaccharide was found to contain an ether of GlcNAc with lactic acid, and the following structure of the repeating unit was established:-->3)-alpha-D-GlcpNAc4(R-Lac)6Ac-(1-->2)-beta-D-GlcpA-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where L-6dTal and D-GlcNAc4(R-Lac) are 6-deoxy-L-talose and 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose, respectively. The latter sugar, which to our knowledge has not been hitherto found in nature, was isolated from the polysaccharide by solvolysis with anhydrous triflic acid and identified by comparison with the authentic synthetic compound. Serological studies with the Smith-degraded polysaccharide showed an importance of 2-substituted GlcA for manifesting of the immunospecificity of P. vulgaris O15.