Properties of p-cresol methylhydroxylase flavoprotein overproduced by Escherichia coli

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.     

Characterization of glucose oxidase immobilization onto mica carrier by atomic force microscopy and kinetic studies

Glucose oxidase (E.C 1.1.3.4) immobilized onto activated surface of mica was analyzed by enzymatic kinetics and visualization with atomic force microscopy (AFM). The activity of the immobilized enzyme decreased with the decrease of concentration of gamma-aminopropyltrimethoxysilane used for the first step of activation of mica, while AFM analysis showed similar homogeneous filling of the surface with the enzyme. The comparison of enzyme activity with its surface filling revealed that there has to be additional vertical structures, which cannot be visualized by the methods of AFM. The simultaneous decrease of the silanizing agent and the concentration of the enzyme led to molecular resolution for the enzyme on the surface of mica. This allows to propose the described method also for analyzing other surfaces of solid materials with coupled biomolecules.     

Group-specific primers for DNA-based detection of springtails (Hexapoda: Collembola) within predator gut contents

Group‐specific, degenerate polymerase chain reaction primers for DNA‐based detection of springtails (Hexapoda: Collembola) within predator gut contents have been developed for the first time. Primers were designed from 18S rDNA and amplified fragments of 272 bp and 177 bp from 17 springtail species collected in agricultural habitats. Specificity tests against 41 nontarget species revealed no cross‐reactivity. Group‐specific polymerase chain reaction is advantageous when working in species‐rich habitats and these primers could facilitate studies of trophic links between springtails and generalist arthropod predators worldwide.     

The Arabidopsis Protein SHI Represses Gibberellin Responses in Arabidopsis and Barley

The current model of gibberellin (GA) signal transduction is based on a derepressible system and a number of candidate negative regulators have been identified in Arabidopsis. We previously have reported the identification of the Arabidopsis gene SHORT INTERNODES (SHI) that causes suppression of GA responses when constitutively activated. In this paper, we show by using reporter gene analysis that the SHI gene is expressed in young organs, e.g. shoot apices and root tips. The model predicts a suppressor of GA responses to be active in these tissues to prevent premature growth or development. To study the effect of SHI on GA signaling, we used a functional assay that measures effects of signaling components on a well-defined GA response; the up-regulation of alpha-amylase in barley (Hordeum vulgare) aleurones in response to GA treatment. We found that SHI was able to specifically block the activity of a high-isoelectric point alpha-amylase promoter following GA(3) treatment, which further supports that SHI is a suppressor of GA responses. We have identified two putative loss-of-function insertion alleles of SHI and lines homozygous for either of the new alleles show no phenotypic deviations from wild type. Because SHI belongs to a gene family consisting of nine members, we suggest that SHI and the SHI-related genes are functionally redundant. We also show that a functional ERECTA allele is able to partly suppress the dwarfing effect of the shi gain-of-function mutation, suggesting that the erecta mutation harbored by the Landsberg erecta ecotype is an enhancer of the shi dwarf phenotype.     

Presence of Leptosphaeria maculans Group A and Group B Isolates in Sweden

Leptosphaeria maculans isolates have been assigned to one of two groups, A or B, on the basis of differences in their characteristics. Group A can further be divided into pathogenicity groups (PG) 2, 3 and 4 and group B into PG1. To determine if isolates belonging to the aggressive canker forming group A are present in Sweden, physiological and genetic characterisation of 120 isolates collected in the year 2000 were performed. Thirty‐seven isolates were classified as belonging to pathogenicity group PG3 and 63 isolates as PG4, based on a cotyledon assay. Twenty isolates did not cause any symptoms at all, and were classified as PG1. When comparing two geographical regions, Skåne and Östergötland, equal numbers of PG3 and PG4 isolates were found. By analysing the isolates by PCR, the collection was further classified into 100 group A isolates and 20 group B isolates. A corresponding classification of the isolates was observed when the ability to produce pigments in Czapek Dox broth was examined. The results showed a clear predominance of group A. This was also the case for the isolate collection from 2001. In a detailed survey of disease development in a L. maculans infected winter oilseed rape field in southern Sweden (Skåne), basal stem canker was not observed until early June Although the disease index value increased from 8.4 in June to 18.0 in July, few severely damaged plants were observed before harvest in mid‐July, despite infection with group A isolates.     

L<Subscript>2</Subscript>-norm multiple kernel learning and its application to biomedical data fusion

Background  

This paper introduces the notion of optimizing different norms in the dual problem of support vector machines with multiple kernels. The selection of norms yields different extensions of multiple kernel learning (MKL) such as L _∞, L ₁, and L ₂ MKL. In particular, L ₂ MKL is a novel method that leads to non-sparse optimal kernel coefficients, which is different from the sparse kernel coefficients optimized by the existing L _∞ MKL method. In real biomedical applications, L ₂ MKL may have more advantages over sparse integration method for thoroughly combining complementary information in heterogeneous data sources.     

Comparative analysis of DNA polymorphisms and phylogenetic relationships among Syzygium cumini Skeels based on phenotypic characters and RAPD technique

The Indian black berry (Syzygium cumini Skeels) has a great nutraceutical and medicinal properties. As in other fruit crops, the fruit characteristics are important attributes for differentiation were also determined for different accessions of S. cumini. The fruit weight, length, breadth, length: breadth ratio, pulp weight, pulp content, seed weight and pulp: seed ratio significantly varied in different accessions. Molecular characterization was carried out using PCR based RAPD technique. Out of 80 RAPD primers, only 18 primers produced stable polymorphisms that were used to examine the phylogenetic relationship. A sum of 207 loci were generated out of which 201 loci found polymorphic. The average genetic dissimilarity was 97 per cent among jamun accessions. The phylogenetic relationship was also determined by principal coordinates analysis (PCoA) that explained 46.95 per cent cumulative variance. The two-dimensional PCoA analysis showed grouping of the different accessions that were plotted into four sub-plots, representing clustering of accessions. The UPGMA (r = 0.967) and NJ (r = 0.987) dendrogram constructed based on the dissimilarity matrix revealed a good degree of fit with the cophenetic correlation value. The dendrogram grouped the accessions into three main clusters according to their eco-geographical regions which given useful insight into their phylogenetic relationships.