Growth-inhibitory effects of Galla Rhois-derived tannins on intestinal bacteria

The growth-inhibitory activity of Galla Rhois-derived materials towards 17 intestinal bacteria was evaluated using an impregnated paper disc method. The biologically active components of Galla Rhois were characterized as the tannins methyl gallate (MG) and gallic acid (GA) by spectral analysis. The growth responses varied with bacterial strain tested. In the test using 10 mg disc⁻¹, MG and GA produced a clear inhibitory effect on harmful bacteria such as Clostridium perfringens , Cl. paraputrificum , Eubacterium limosum , Bacteroides fragilis , Staphylococcus aureus and Escherichia coli . Methyl gallate showed no growth-inhibitory activity towards Bifidobacterium adolescentis or B. longum whereas the growth of B. bifidum , B. breve , B. infantis , B. animalis , B. thermophilum , Lactobacillus acidophilus , Lact. plantarum and Streptococcus faecalis was slightly affected. However, GA did not adversely affect the growth of the bifidobacteria and lactobacilli. At 5 mg disc⁻¹, MG significantly inhibited the growth of Cl. perfringens and Cl. paraputrificum but did not affect the growth of the bifidobacteria and lactobacilli. At 1 mg disc⁻¹, MG greatly inhibited the growth of Cl. perfringens alone. These results may be an indication of at least one of the pharmacological actions of Galla Rhois.     

Combined effects of chemical and visual information in eliciting antipredator behaviour in juvenile Atlantic salmon Salmo salar

Under natural conditions, both young-of-the-year (YOY; 0+ year) and parr (1+ year) Atlantic salmon Salmo salar exhibited strong antipredator behaviour ( e.g. increase in latency to resume foraging) following the exposure to damage-released chemical alarm cues relative to a stream water control. Subsequent exposure to a novel visual stimulus had contrasting results. Parr increased their reactive distance to the visual stimulus if they had been previously exposed to a chemical alarm cue, whereas YOY did not. On the other hand, both YOY and parr took significantly longer to resume foraging when exposed to a visual stimulus if they had been previously exposed to a chemical alarm cue than control groups. While YOY and parr differed in the type and intensity of antipredator responses to both chemical and visual stimuli, perhaps due to differential costs and benefits associated with age, both used the chemical and the visual information in a combined manner.     

The 2DX robot: A membrane protein 2D crystallization Swiss Army knife

Among the state-of-the-art techniques that provide experimental information at atomic scale for membrane proteins, electron crystallography, atomic force microscopy and solid state NMR make use of two-dimensional crystals. We present a cyclodextrin-driven method for detergent removal implemented in a fully automated robot. The kinetics of the reconstitution processes is precisely controlled, because the detergent complexation by cyclodextrin is of stoichiometric nature. The method requires smaller volumes and lower protein concentrations than established 2D crystallization methods, making it possible to explore more conditions with the same amount of protein. The method yielded highly ordered 2D crystals diffracting to high resolution from the pore-forming toxin Aeromonas hydrophila aerolysin (2.9 Å), the plant aquaporin SoPIP2;1 (3.1 Å) and the human aquaporin-8 (hAQP8; 3.3 Å). This new method outperforms traditional 2D crystallization approaches in terms of accuracy, flexibility, throughput, and allows the usage of detergents having low critical micelle concentration (CMC), which stabilize the structure of membrane proteins in solution.     

Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial species were belonging to the genera Acidithiobacillus, Leptospirillum, Sulfobacillus, and Sphingomonas, accounting for 6.3, 66.7, 18.8, and 8.3%, respectively; the sole archaeal species was Ferroplasma sp. (100%). Quantitative RT-PCR revealed that the 16S rRNA gene copy numbers (per gram of concentrates) of bacteria and archaea were 4.59 × 10⁹ and 6.68 × 10⁵, respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor _Fx, sor _SA, and sor _SB were identified from metagenomic DNAs of the bioreactors. The sor _Fx is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor _SA and sor _SB showed no significant identity to any genes in GenBank databases. The sor _SB was cloned and expressed in Escherichia coli, and SOR activity was determined. Quantitative RT-PCR determination of the gene densities of sor _SA and sor _SB were 1,000 times higher than archaeal 16S rRNA gene copy numbers, indicating that these genes were mostly impossible from archaea. Furthermore, with primers specific to the sor _SB gene, this gene was PCR-amplified from the newly isolated Acidithiobacillus sp. strain SM-1. So far as we know, this is the first time to determine SOR activity originating from bacteria and to document SOR gene in bioleaching reactors and Acidithiobacillus species.     

Molecular characterization of the <Emphasis Type="Italic">GmAMS1</Emphasis> gene encoding β-amyrin synthase in soybean plants

Suppression subtractive hybridization was used to isolate wound-induced cDNAs from soybean plants. One of wound-induced cDNA, gmwi33 designated GmAMS1, showed a high homology with genes encoding β-amyrin synthase. GmAMS1 protein showed 89% identity with licorice GgbAS1. In five-day-old dark-grown seedlings, RNA expression of GmAMS1 was strongly induced by light, weakly induced by methyl jasmonate and low temperature, and was not induced by elicitor or UV-B treatment. Genomic blot and FISH analyses confirmed that the soybean genome has two copies of the GmAMS1 gene. To our knowledge, this is the first report on β-amyrin synthase from soybean. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 584–589. The text was submitted by the authors in English.     

Prediction of protein subcellular localization by support vector machines using multi-scale energy and pseudo amino acid composition

As more and more genomes have been discovered in recent years, there is an urgent need to develop a reliable method to predict the subcellular localization for the explosion of newly found proteins. However, many well-known prediction methods based on amino acid composition have problems utilizing the sequence-order information. Here, based on the concept of Chou's pseudo amino acid composition (PseAA), a new feature extraction method, the multi-scale energy (MSE) approach, is introduced to incorporate the sequence-order information. First, a protein sequence was mapped to a digital signal using the amino acid index. Then, by wavelet transform, the mapped signal was broken down into several scales in which the energy factors were calculated and further formed into an MSE feature vector. Following this, combining this MSE feature vector with amino acid composition (AA), we constructed a series of MSEPseAA feature vectors to represent the protein subcellular localization sequences. Finally, according to a new kind of normalization approach, the MSEPseAA feature vectors were normalized to form the improved MSEPseAA vectors, named as IEPseAA. Using the technique of IEPseAA, C-support vector machine (C-SVM) and three multi-class SVMs strategies, quite promising results were obtained, indicating that MSE is quite effective in reflecting the sequence-order effects and might become a useful tool for predicting the other attributes of proteins as well.     

Specific reversal of multidrug resistance to colchicine in CEM/VLB100 cells by Gynostemma pentaphyllum extract

P-glycoprotein (P-gp)-mediated multiple drug resistance (MDR) is perhaps the most thoroughly studied cellular mechanism of cytotoxic drug resistance. Its efflux function can be circumvented by a wide range of pharmacological agents in vitro and in vivo. Most of these agents are pharmaceuticals used clinically for conditions other than cancer. However, their use in alleviating MDR is limited because the concentrations required for inhibition of the pump surpass their dose-limiting toxicity. The aim of this research is to study the role of gypenosides, isolated from Gynostemma pentaphyllum, as modulators of P-gp-mediated MDR in tumor cells, at both cellular and plasma membrane level. In the presence of total gypenoside preparation (0.1 mg/ml), an approximately 15-fold reversal of colchicine (COL) resistance was observed in P-gp-overexpressed CEM/VLB₁₀₀ cells. However, the gypenoside sample showed no reversal effect in cells treated with vinblastine and taxol. A purified gypenoside sample (gypenoside fraction 100) exhibited even more significant reversal of COL resistance (42-fold) in the CEM/VLB₁₀₀ cells. Further examination of the reversal effect of fraction 100 in membrane vesicles derived from CEM/VLB₁₀₀ cells using the continuous fluorescence method found that gypenoside fraction 100 at 0.1 mg/ml completely abolished the transport of fluorescein–COL.     

Probing Structure–Function Relationships of Serine Hydrolases and Proteases with Carbamate and Thiocarbamate Inhibitors

Benzene-1,3-di-N-n-octylcarbamate (1), benzene-1-hydroxyl-3-N-n-octylcarbamate (2), benzene-1,3-di-N-n-ocztylthiocarbamate (3), and benzene-1-hydroxyl-3-N-n-octylthiocarbamate (4) are synthesized from 1,3-benzene-diol and are characterized as the pseudo-substrate inhibitors of acetylcholinesterase, butyrylcholinesterase, cholesterol esterase, lipase, trypsin, and chymotrypsin. For these six enzyme inhibitions by 1-4, the pKi values are linearly correlated with their log ki values - Br?nsted plots. Therefore, 1-4 inhibit these enzymes through a common mechanism. Moreover, both pKi and log ki values for the inhibitions by 1,3, and 4 are linearly correlated with both pKi and log ki values for the inhibitions by 2, respectively. Thus, the pKi values for the inhibitions by 2 are defined as the nucleophilicity constants of these enzymes (nenzyme). The log k2 values for the inhibitions by 1-4 are also linearly correlated with the nenzyme values. Therefore, the nucleophilicity for serine hydrolases and proteases toward 1-4 also applies the Swain-Scott correlations.     

Frameshifting in Alphaviruses: A Diversity of 3′ Stimulatory Structures

Programmed ribosomal frameshifting allows the synthesis of alternative, N-terminally coincident, C-terminally distinct proteins from the same RNA. Many viruses utilize frameshifting to optimize the coding potential of compact genomes, to circumvent the host cell's canonical rule of one functional protein per mRNA, or to express alternative proteins in a fixed ratio. Programmed frameshifting is also used in the decoding of a small number of cellular genes. Recently, specific ribosomal − 1 frameshifting was discovered at a conserved U_UUU_UUA motif within the sequence encoding the alphavirus 6K protein. In this case, frameshifting results in the synthesis of an additional protein, termed TF (TransFrame). This new case of frameshifting is unusual in that the − 1 frame ORF is very short and completely embedded within the sequence encoding the overlapping polyprotein.The present work shows that there is remarkable diversity in the 3′ sequences that are functionally important for efficient frameshifting at the U_UUU_UUA motif. While many alphavirus species utilize a 3′ RNA structure such as a hairpin or pseudoknot, some species (such as Semliki Forest virus) apparently lack any intra-mRNA stimulatory structure, yet just 20 nt 3′-adjacent to the shift site stimulates up to 10% frameshifting. The analysis, both experimental and bioinformatic, significantly expands the known repertoire of − 1 frameshifting stimulators in mammalian and insect systems.