Determination of albumin concentration by MIP-QCM sensor

An MIP-QCM sensor able to detect micro-determine albumin concentrations was prepared by imprinting albumin with 3-dimethylaminopropyl methacrylamide-acrylate. The albumin MIP was coated on a QCM Au electrode. The adsorption characteristics of different electrodes, such as Au-OH, Au-COOH, Au-NH2 and Au electrodes, with albumin or mixture was examined. In the tetraethyleneglycol dimethacrylate crosslinking agent system, the adsorption capacity of different Au electrodes is in the order Au-OH > Au-COOH > Au-NH2 > Au. Additionally, the time taken to receive a steady-state frequency is in the order Au-NH2 < Au-OH < Au-COOH < Au. However, in the trimethylolpropane trimethacrylate crosslinking agent system, the adsorption capacity is in the order Au > Au-NH2 > Au-OH > Au-COOH electrode. Hence, the crosslinking agent had a significant effect on the MIP-QCM. On the adsorption selectivity, the albumin MIP-QCM exhibited higher response to albumin, the adsorption mass ratio of cytochrome c:lysozyme:albumin:myoglobin was 160:1:1942:30. On the other hand, in the non-MIP-QCM, the adsorption mass ratio of cytochrome c:lysozyme:albumin:myoglobin was 13:1:249:86. Additionally, a linear fitting was established and a clinical real sample was tested. This novel potential application of molecular imprinting to the recognition element of an MIP-QCM sensor appears to be promising.     

Thoracic vein ablation terminates chronic atrial fibrillation in dogs

The thoracic vein hypothesis of chronic atrial fibrillation (AF) posits that rapid, repetitive activations from muscle sleeves within thoracic veins underlie the mechanism of sustained AF. If this is so, thoracic vein ablation should terminate sustained AF and prevent its reinduction. Six female mongrel dogs underwent chronic pulmonary vein (PV) pacing at 20 Hz to induce sustained (>48 h) AF. Bipolar electrodes were used to record from the atria and thoracic veins, including the vein of Marshall, four PVs, and the superior vena cava. Radio frequency (RF) application was applied around the PVs and superior vena cava and along the vein of Marshall until electrical activity was eliminated. Computerized mapping (1,792 electrodes, 1 mm resolution) was also performed. Sustained AF was induced in 30.6 +/- 6.5 days, and ablation was done 17.3 +/- 8.5 days afterward. Before ablation, the PVs had shorter activation cycle lengths than the atria, and rapid, repetitive activations were observed in the PVs. All dogs converted to sinus rhythm during (n = 4 dogs) or within 90 min of completion of RF ablation. Rapid atrial pacing afterward induced only nonsustained (<60 s) AF in all dogs. Average AF cycle lengths after reinduction were significantly (P = 0.01) longer (183 +/- 31.5 ms) than baseline (106 +/- 16.2 ms). There were no activation cycle length gradients after RF application. We conclude that thoracic vein ablation converts canine sustained AF into sinus rhythm and prevents the reinduction of sustained AF. These findings suggest that thoracic veins are important in the maintenance of AF in dogs.     

Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.     

Fus3-regulated Tec1 degradation through SCFCdc4 determines MAPK signaling specificity during mating in yeast

Signaling specificity is fundamental for parallel mitogen-activated protein kinase (MAPK) cascades that control growth and differentiation in response to different stimuli. In Saccharomyces cerevisiae, components of the pheromone-responsive MAPK cascade activate Fus3 and Kss1 MAPKs to induce mating and Kss1 to promote filamentation. Active Fus3 is required to prevent the activation of the filamentation program during pheromone response. How Fus3 prevents the crossactivation is not clear. Here we show that Tec1, a cofactor of Ste12 for the expression of filamentation genes, is rapidly degraded during pheromone response. Fus3 but not Kss1 induces Tec1 ubiquination and degradation through the SCFCdc4 ubiquitin ligase. T273 in a predicted high-affinity Cdc4 binding motif is phosphorylated by Fus3 both in vitro and in vivo. Tec1T273V blocks Tec1 ubiquitination and degradation and allows the induction of filamentation genes in response to pheromone. Thus, Fus3 inhibits filamentous growth during mating by degrading Tec1.     

Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the <Emphasis Type="Italic">gpd</Emphasis> promoter for transformation in <Emphasis Type="Italic">Flammulina velutipes</Emphasis>

The glyceraldehyde-3-phosphate dehydrogenase gene of Flammulina velutipes was isolated. The complete gpd sequence (from ATG to TAA) was 1,489 bp in length and contained nine introns. The locations of these nine introns were similar to those of other basidiomycetes, which might reflect the evolutionary divergence of these mushrooms. The F. velutipes gpd gene was found to encode a protein of 339 amino acids and its putative amino acid sequence revealed a high similarity to an analogous protein deriving from other basidiomycetes. Results of Southern blot analysis suggested that there existed only one copy of the gpd gene in the genome of F. velutipes and that there was one typical TATA box and two CAAT boxes located in the 5 flanking region. The F. velutipes gpd promoter was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selection marker. Using the resulting construction, hph was efficiently transformed into F. velutipes by basidiospore electroporation. No false-positive antibiotic-resistant cultures were detected by PCR amplification and the hygromycin resistance trait was maintained stably during mitotic cell division for 3 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. This rapid and convenient electroporation procedure offers new prospects for the genetic manipulation and a tool for tagging genes of this important edible mushroom species. Sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AF515622.     

Three new compounds from the plant Lippia alva as inhibitors of chemokine receptor 5 (CCR5)

The 70% aqueous methanol extract of the Peruvian plant Lippia alva (Verbenaceae) was found to contain three novel compounds, 1, 2, and 3, which were identified as inhibitors of the chemokine receptor CCR5. The structures of 1-3 were established based on extensive NMR studies. Compounds 1-3 inhibited CCR5 receptor signaling as measured by a calcium mobilization assay with IC(50) values of 5.5, 6.0, and 7.2 microg/mL, respectively.     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

Molecular therapeutic target for type-2 diabetes

Many lines of evidences indicate that increased flux of glucose through the pathway, in which glutamine:fructose-6-phosphate amidotransferase (GFPT or GFAT) is a key catalyst while uridine-5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) functions as an energy sensor, can lead to the insulin resistance that is characteristic of Type-2 diabetes. In view of this, GFAT and its interaction mechanism with UDP-GlcNAc may become a novel therapeutic target for the treatment of type 2 diabetes. To stimulate the structure-based drug design, the three-dimensional structures of human GFAT1 monomer and dimer have been developed. It has been found by docking UDP-GlcNAc to the dimer (the smallest unit for catalyzing the substrate) that UDP-GlcNAc is bound to the interface of the dimer by 12 hydrogen bonds. On the basis of the docking results, a binding pocket of human GFAT1 dimer for UDP-GlcNAc is defined. All of these findings can serve as a reference or footing in developing new therapeutic strategy for the treatment of type-2 diabetes.     

Insights from modeling three-dimensional structures of the human potassium and sodium channels

Ion channels allow the movement of ions across cell membranes. Nearly all cells have membranes spanned by ion channels, without which human nerves simply would not work. Ion channels are formed by the aggregation of subunits into a cylindrical configuration that allows a pore, thus forming a kind of tube for ion trafficking. In the present study, the subunits of the human potassium channel are formed by four identical protein chains, whereas for the case of the human sodium channel, the corresponding subunits are actually four hetero-domains formed by the folding of a very large but single protein chain. Since both of the two ion channels are important targets for drug discovery, the 3D (dimensional) structures of their pore regions were developed. On the basis of the 3D models, some important molecular biological mechanisms were discussed that may stimulate novel strategies for therapeutic treatment of the diseases related to ion channel disorders, such as long QT syndrome and chronic pain.