Effect of balloon pre-dilation on performance of self-expandable nitinol stent in femoropopliteal artery

Balloon pre-dilation is usually performed before implantation of a nitinol stent in a femoropopliteal artery in a case of severe blockage or calcified plaque. However, its effect on performance of the nitinol stent in a diseased femoropopliteal artery has not been studied yet. This study compares the outcomes of stenting with pre-dilation and without it by modelling the entire processes of stent deployment. Fatigue deformation of the implanted stent is also modelled under diastolic–systolic blood pressure, repetitive bending, torsion, axial compression and their combination. Reduced level of stress in the stent occurs after stenting with pre-dilation, but causing the increased damage in the media layer, i.e. the middle layer of the arterial wall. Generally, pre-dilation increases the risk of nitinol stent’s fatigue failure. Additionally, the development of in-stent restenosis is predicted based on the stenting-induced tissue damage in the media layer, and no severe mechanical irritation is induced to the media layer by pre-dilation, stent deployment or fatigue loading.

     

A new strategy for cellulases application in high temperature industrial scenarios with syringic acid assisting

In the process of bioethanol production, more stable and active cellulase in high temperature condition is required. In this study, syringic acid was applied in cellulase hydrolysis system. At 70°C, TvEG3 activity increased 201.36%, CtBglA activity decreased 72.79% by syringic acid. With syringic acid assisting, TvEG3 thermostability was improved, CtBglA thermostability was reduced. Syringic acid scarcely affected CtCBH. In hydrolysis system with the cellulases containing TvEG3, CtCBH, and CtBglA, the reducing sugar yield improved by 28.37% with syringic acid assisting. With the molecular dynamic simulation in syringic acid system, the backbone root-mean-square deviation (RMSD) and the residue root-mean-square fluctuation (RMSF) of TvEG3, CtCBH reduced, while the RMSD and RMSF of CtBglA increased. The reduction in the number of secondary structures, especially α-helix, caused the structure of CtBglA in the presence of syringic acid to collapse at high temperature. More secondary structures in TvEG3 and more α-helix in CtCBH in the presence of syringic acid make them more stable at high temperatures. These means syringic acid can stabilize TvEG3 and CtCBH structure, destabilize CtBglA structure at high temperature. In summary, this study not only provides insight into cellulase hydrolysis at high temperature with syringic acid assisting but also demonstrates the promoting mechanism of syringic acid.     

Screening of Korean herbal medicines used to improve cognitive function for anti-cholinesterase activity.

Methanolic extracts of seven herbs (Acorus calamus, Acorus gramineus, Bupleurm facaltum, Dioscorea batatas, Epimedium koreanum, Poria cocos and Zizyphi jujuba) used in traditional Korean medicine for improvement of memory and cognition in old age were tested for cholinesterase inhibitory properties using the Ellman colorimetric method. Significant inhibition of the enzyme at 200 microg/ml was observed for extracts from A. calamus and E. koreanum. The possible bases for the reputation of these and the other herbs tested are discussed in the light of previous investigations into their chemistry and biological activity.     

Cell separation from high cell density broths of Alcaligenes eutrophus by using a coagulant

Summary Alcaligenes eutrophus was successfully recovered from high cell density broths by pre-treatment with polyaluminium hydroxide chloride silicate as a coagulant at 36–90 mg Al/l. The optimum pH range for cell coagulation was 10–12. Subsequent centrifugation (45×g) and filtration (pore size 0.5 mm) gave a cell recovery of higher than 90%. The energy demand for cell recovery with the coagulant was only 3–11% of that without it.     

Impaired interleukin-3 (IL-3) response of the A/J mouse is caused by a branch point deletion in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) alone does not support hematopoietic colony formation of bone marrow cells from the A/J mouse. To elucidate the molecular lesion in A/J mice, we examined expression of the alpha and beta subunits of the IL-3 receptor (IL-3R). While IL-3R beta was normally expressed, IL-3R alpha was not detectable on the surface of A/J-derived cells by antibody staining. Genetic linkage analysis using recombinant inbred mouse strains between A/J and IL-3-responsive C57BL/6 indicated that the IL-3R alpha gene locus was responsible for the impaired IL-3 response in A/J mice. Molecular cloning and characterization of A/J-derived IL-3R alpha cDNA revealed that it lacked the sequence corresponding to exon 8, which encodes 10 amino acid residues in the extracellular domain. The aberrant splicing was due to a 5 base pair deletion at the branch point in intron 7 and was reproduced in heterologous cells by transfecting with an IL-3R alpha minigene carrying the deleterious intron. The A/J-specific abnormal form of IL-3R alpha was localized inside the cells, but not on the cell surface, providing the molecular basis for the impaired IL-3 response in the A/J mouse.     

Mismatch Repair by Efficient Nick-Directed, and Less Efficient Mismatch-Specific, Mechanisms in Homologous Recombination Intermediates in Chinese Hamster Ovary Cells

Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in ~10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A -> G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was >80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803.

Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 micromol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30 degrees C and 7.0, respectively. The enzyme was inactivated very rapidly at 70 degrees C. The enzyme required Mg2+ and thiamine pyrophosphate for maximal activity. Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor. Erythrose 4-phosphate, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The Kms for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 microM, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.