Psychrophilic α-amylase from Aeromonas veronii NS07 isolated from farm soils

Among 120 isolates examined in this study, three isolates were selected for amylase production on starch agar plates following incubation at 10 °C. Identification by 16SrRNA on selected bacterium disclosed the highest similarity for protean regions of this gene as Aeromonas veronii NS07. A 63 kDa psychrophilic amylase enzyme from NS07 strain was purified by two-steps chromatography. The enzyme had the highest specific activity at pH 4 and was active at the range of temperatures from 0 to 50 °C, although the optimum temperature for enzyme activity was found at 10 °C. Analysis of the N-terminal amino acid sequencing disclosed 20 amino acids from purified amylase which had no similarity with other known α-amylases, indicating that the presented enzyme was novel. Amylase activity was enhanced in relation to optimum activity with the presence of sodium sulphate (161%), MnCl₂ (298%), CaCl₂ (175%), FeCl₂ (182%), MgCl₂ (237%), ZnCl₂ (169%), NiCl₂ (139%), NaCl (158%), each at 5 mM, while EDTA, phenylmethane sulphonylfluoride (PMSF) (3 mM), urea (8 M) and SDS (1%) inhibited the enzyme up to 5%, 2%, 80% and 18%, respectively. NS07 strain seems to be suitable as biocatalyst for practical use in liquefaction of starch at low temperatures, detergent and textile industries.     

Tri-nucleotide threading for parallel amplification of minute amounts of genomic DNA

Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.     

Genetic tools for Paracoccus denitrificans

Abstract: Paracoccus denitrificans strain PD1222 (from N. Harms, Vrije Universiteit, Amsterdam, The Netherlands), which is deficient in a host-specific restriction system, could be successfully used for (1) transposon Tn5 mutagenesis, (2) phage P1-mediated generalized transduction and (3) construction of Hfr strains. Experimental protocols and some properties of an ammonium transport-deficient mutant are described.     

A new look at state-space models for neural data

State space methods have proven indispensable in neural data analysis. However, common methods for performing inference in state-space models with non-Gaussian observations rely on certain approximations which are not always accurate. Here we review direct optimization methods that avoid these approximations, but that nonetheless retain the computational efficiency of the approximate methods. We discuss a variety of examples, applying these direct optimization techniques to problems in spike train smoothing, stimulus decoding, parameter estimation, and inference of synaptic properties. Along the way, we point out connections to some related standard statistical methods, including spline smoothing and isotonic regression. Finally, we note that the computational methods reviewed here do not in fact depend on the state-space setting at all; instead, the key property we are exploiting involves the bandedness of certain matrices. We close by discussing some applications of this more general point of view, including Markov chain Monte Carlo methods for neural decoding and efficient estimation of spatially-varying firing rates.     

Morphological Study of the Role of Calcium in the Assembly of the Rotavirus Outer Capsid Protein VP7

Maturation of rotavirus occurs in the endoplasmic reticulum (ER), a site of intracellular calcium storage. It was demonstrated previously that calcium plays an important role in the maturation of bovine rotavirus. We used protein A colloidal gold conjugated to an antibody to localize VP7, the outer capsid protein of the simian rotavius SA11, in permeabilized infected cells in the presence and absence of calcium in the culture medium. In medium containing calcium, VP7 was associated with nonenveloped double-shelled particles and membranous structures of the ER. In calcium-free medium, gold particles were not associated with the ER or with virus particles. Gold particles were distributed through the cytoplasm and were mainly associated with granular structures, but did not assemble onto virus particles. Our data suggest that in calcium-free medium, VP7 is synthesized, but does not remain incorporated, in the ER.