Energetics of hydrogen bond networks in RNA: hydrogen bonds surrounding G+1 and U42 are the major determinants for the tertiary structure stability of the hairpin ribozyme

The hairpin ribozyme, a small catalytic RNA consisting of two helix-loop-helix motifs, serves as a paradigm for RNA folding. In the active conformer, the ribozyme is docked into a compact structure via loop-loop interactions. The crystal structure of the docked hairpin ribozyme shows an intricate network of hydrogen bonding interactions at the docking interface, mediated by the base, sugar, and phosphate groups of U42 and G+1 [Rupert, P. B., and Ferre-D'Amare, A. R. (2001) Nature 410, 780-786]. To elucidate the determinants for tertiary structure stability in the hairpin ribozyme, we evaluated the energetic contributions of hydrogen bonds surrounding U42 and G+1 by time-resolved fluorescence resonance energy transfer using modified ribozymes that lack one or more of the individual interactions. Elimination of a single tertiary hydrogen bond consistently resulted in a net destabilization of approximately 2 kJ/mol. The results of double- and triple-mutant cycles suggest that individual hydrogen bonds surrounding G+1 or U42 act cooperatively and form extended hydrogen bond networks that stabilize the docked ribozyme. These results demonstrate that RNAs, similar to proteins, can exploit coupled hydrogen bond networks to stabilize the docking of distant structural domains.     

Evaluation of Probiotic <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> CCM 7421 Administration with Alginite in Dogs

There are growing efforts to find applications for various naturally occurring organo-mineral rocks. They have so far been preferentially used in agriculture and forestry; however, medicine and nutrition may also be interesting areas for their application. This study investigates the effects of dietary supplementation with canine-derived probiotic strain Lactobacillus fermentum CCM 7421 in combination with alginite in dogs. Alginite is a loam-like material of volcanic origin composed of clay minerals and fossilised unicellular algae. The effects of these additives on faecal microbiota, faecal characteristics, short-chain fatty acid profile, haematology, serum biochemistry and cellular immunity parameters were monitored. Forty dogs were randomly divided into four treatment groups: control group (C), alginite-supplemented group (A; 1% diet), probiotic group (LF; L. fermentum CCM 7421 at a dose of 10⁹ cfu/day/dog) and combined group (A + LF group); 10 dogs in each group. The experiment lasted for 35 days with a 14-day treatment period (sample collection at days 0, 7, 14 and 35). The results of this straightforward experiment showed beneficial effects in the combined A + LF group. In detail, a decrease in faecal coliforms and clostridia and an increase in lactic acid bacteria, haemoglobin and serum magnesium levels compared to baseline were observed in the A + LF group (P?<?0.05). In contrast, sole application of alginite (A group) led to several unexpected effects such as an increase in clostridial population and serum alanine aminotrasferase and a decrease in haemoglobin concentration (P?<?0.05). The addition of alginite prevented a decrease in faecal pH and serum mineral content observed in the LF group. This indicates the possibility of applying alginite also in the nutrition of dogs as a combinative additive with probiotic bacteria for restoring optimal acid-alkali balance without affecting positive probiotic effects.     

Differences between ecological niches in juvenileSalix cinerea andS. pentandra

Comparison of growth rates ofSalix cinerea andS. pentandra seedlings in terrestric, limosal, and littoral ecophases showed different life strategies of juvenile stages of these willows. Several characters determining ecological niches of both species are summarized.     

An operator binding-negative mutation of Xyl repressor from Bacillus subtilis is trans dominant in Bacillus megaterium

Abstract We have selected a Bacillus subtilis 168-borne xylR Ser to Leu mutation at position 41 of the encoded amino acid sequence showing a constitutive expression phenotype for the xyl operon. When cloned on a multi-copy plasmid in a B. megaterium strain harbouring a single-copy xylA-lacZ fusion it leads to derepressor of β-galactosidase expression. Thus, it is trans dominant over the endogenous xylR , indicating that Xyl repressor functions as a multimer. This result also supports the assumption that the mutation is in a putative α-helix-turn-α-helix operator binding motif of Xyl repressor.     

The utilization of interphase cytogenetic analysis for the detection of mosaicism

This study describes a method for defining mosaic aneuploidy by interphase cytogenetics based on statistical limits established from control specimens. Fluorescence in situ hybridization (FISH) has been used to detect the number of copies of specific chromosomes in interphase nuclei from placental tissues of diploid controls and mosaic placentas. FISH was performed using probes D7Z1/D7Z2, D9Z1, D10Z1, and D18Z1, all purchased from Oncor, Inc. Statistical analysis of data obtained from diploid controls was used to determine the one-sided upper reference limit and corresponding 95% confidence interval for the proportion of cells with one and three signals for each of the probes used. The one-sided upper reference limits established the lower levels of monosomy and trisomy detectable using each of the four probes. These statistical parameters were then used to interpret the results obtained by FISH applied to the study of term placentas for the confirmation of prenatally diagnosed chromosomal mosaicism.     

Consequences of Cis-amide Bond Simulation in Opioid Peptides

Summary Six analogs of leucine-enkephalin were synthesized in which a 1,5-disubstituted tetrazole ring was incorporated in order to lock selected peptide bonds in cis geometry. The obtained compounds were examined based on their biological effects in vivo and in vitro. Only one analog was completely inactive in binding assays being very weakly active in the antinociceptive test. The remaining five compounds displayed at least weak receptor affinity and in vivo activity.     

Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3′ end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34⁺ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1⁺ lin⁻ primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.