Topological analysis of a haloacid permease of a Burkholderia sp. bacterium with a PhoA-LacZ reporter

Background  

2-Haloacids can be found in the natural environment as degradative products of natural and synthetic halogenated compounds. They can also be generated by disinfection of water and have been shown to be mutagenic and to inhibit glyceraldehyde-3-phosphate dehydrogenase activity. We have recently identified a novel haloacid permease Deh4p from a bromoacetate-degrading bacterium Burkholderia sp. MBA4. Comparative analyses suggested that Deh4p is a member of the Major Facilitator Superfamily (MFS), which includes thousands of membrane transporter proteins. Members of the MFS usually possess twelve putative transmembrane segments (TMS). Deh4p was predicted to have twelve TMS. In this study we characterized the topology of Deh4p with a PhoA-LacZ dual reporters system.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Divergent and reticulate processes in evolution of Ethiopian Lophuromys flavopunctatus species complex: evidence from mitochondrial and nuclear DNA differentiation patterns

Molecular study of mitochondrial and nuclear genes and cytogenetic analysis were performed to examine possible patterns of speciation in the diverse Lophuromys flavopunctatus species complex of Ethiopia. Phylogenetic analysis of mtDNA data resulted in an unresolved bush of ten deeply diverged haplotype groups corresponding to potential species either well supported by various types of character or 'cryptic'. The cytogenetic analysis showed representatives of five of these mtDNA lineages to share an identical karyotype (2 n  = 70, NFa = 84), that has not been found previously in Ethiopia. One of them, L.  cf.  sikapusi , being a member of the L. flavopunctatus species complex, demonstrates remarkable morphological similarity to representatives of another species complex, L. sikapusi s.l ., which might be considered as a result of convergent evolution in analogous environments. Analysis of RAPD data suggests that at least two mtDNA types might have been subject to interspecific transfer due to hybridization. In the case of two sympatric haplotypes of L. brunneus we may assume that the contemporary pattern of variation between them can be explained by relatively recent hybridization with another distinct species, L. flavopunctatus . The formation of two groups belonging to distinct mitochondrial lineages within northern populations could be associated with more complex processes including ancient hybridization.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 301–316.     

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.     

Synthesis and Antiviral Evaluation of Novel 2,3-Dihydroxypropyl Nucleosides from 2- and 4-Thiouracils

Regioselective alkylation of 2-thiouracils 1a–c and 4-thiouracils 7a,b with 2,3-O-isopropylidene-2,3-dihydroxypropyl chloride (2) afforded 2-{[(2,2-Dimethyl-1,3-dioxolan-4-yl) methyl]thio}pyrimidin-4(1H)-ones 3a–c and 4-{[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl]thio} pyrimidin-2(1H)-ones 8a,b, respectively. Further alkylation with 2 and/or 2,3-O-isopropylidine-1-O-(4-toluenesulfonyl)-glycerol (4) gave the acyclo N-nucleosides 5a–c and 9a,b whose deprotection afforded 6a–c and 10a,b. 2-(Methylthio)pyrimidin-4(1H)-ones 11a–c and 4-(methylthio)pyrimidin-2(1H)-ones 14a,b were treated with 2 and/or 4 to give 12a–c and 15a,b which were deprotected to give 13a–c and 16a,b. Pyrimidine-2,4(1H,3H)-dithiones 17a–c were treated with two equivalents of 2 to give 2,4-bis{[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]thio}pyrimidines 18a–c. Deprotection of compounds 18a–c gave 2,4-bis[(2,3-dihydroxypropyl)thio]pyrimidines 19a-c. The activity of the deprotected nucleosides against Hepatitis B virus was evaluated and showed moderate inhibition activity against HBV with mild cytotoxicity.     

Fermentation of protopanaxadiol type ginsenosides (PD) with probiotic Bifidobacterium lactis and Lactobacillus rhamnosus

Applied Microbiology and Biotechnology - Ginsenosides are believed to be the principal components behind the pharmacological actions of ginseng, and their bioactive properties are closely related...     

Molecular dynamics study of the KcsA potassium channel

The structural, dynamical, and thermodynamic properties of a model potassium channel are studied using molecular dynamics simulations. We use the recently unveiled protein structure for the KcsA potassium channel from Streptomyces lividans. Total and free energy profiles of potassium and sodium ions reveal a considerable preference for the larger potassium ions. The selectivity of the channel arises from its ability to completely solvate the potassium ions, but not the smaller sodium ions. Self-diffusion of water within the narrow selectivity filter is found to be reduced by an order of magnitude from bulk levels, whereas the wider hydrophobic section of the pore maintains near-bulk self-diffusion. Simulations examining multiple ion configurations suggest a two-ion channel. Ion diffusion is found to be reduced to approximately (1)/(3) of bulk diffusion within the selectivity filter. The reduced ion mobility does not hinder the passage of ions, as permeation appears to be driven by Coulomb repulsion within this multiple ion channel.