Troponin T isoforms alter the tolerance of transgenic mouse cardiac muscle to acidosis

Troponin T (TnT) is an essential protein in the Ca²⁺ regulatory system of striated of muscle. Three fiber type-specific TnT genes have evolved in higher vertebrates to encode cardiac, slow and fast skeletal muscle TnT isoforms. To understand the functional significance of TnT isoforms, we studied the effects of acidosis on the contractility of transgenic mouse cardiac muscle that expresses fast skeletal muscle TnT. Contractility analysis of intact cardiac muscle strips showed that while no differences were detected at physiological pH, the transgenic cardiac muscle had significantly greater decreases in +dF/dt_max at acidic pH than that of the wild-type control. Contractility of skinned cardiac muscles demonstrated that the presence of fast TnT resulted in significantly larger decreases in force and Ca²⁺ sensitivity at acidic pH than that of the wild-type control. The effect of TnT isoforms on the tolerance of muscle to acidosis may explain the higher tolerance of embryonic versus adult cardiac muscles. The results are consistent with the hypothesis that charge differences in TnT isoforms contribute to the contractility of muscle. The data further support a hypothesis that slow TnT is similar to the cardiac, but not fast, and TnT may contribute to the higher tolerance of slow muscles to stress conditions. Therefore, TnT isoform diversity may contribute to the compatibility of muscle thin filaments to cellular environments in different fiber types, during development and functional adaptation.     

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.     

A grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) genetic map integrating the position of 139 expressed genes

Grapevine molecular maps based on microsatellites, AFLP and RAPD markers are now available. SSRs are essential to allow cross-talks between maps, thus upgrading any growing grapevine maps. In this work, single nucleotide polymorphisms (SNPs) were developed from coding sequences and from unique BAC-end sequences, and nested in a SSR framework map of grapevine. Genes participating to flavonoids metabolism and defence, and signal transduction pathways related genes were also considered. Primer pairs for 351 loci were developed from ESTs present on public databases and screened for polymorphism in the “Merzling” (a complex genotype Freiburg 993–60 derived from multiple crosses also involving wild Vitis species) × Vitis vinifera (cv. Teroldego) cross population. In total 138 SNPs, 108 SSR markers and a phenotypic trait (berry colour) were mapped in 19 major linkage groups of the consensus map. In specific cases, ESTs with putatively related functions mapped near QTLs previously identified for resistance and berry ripening. Genes related to anthocyanin metabolism mapped in different linkage groups. A myb gene, which has been correlated with anthocyanin biosynthesis, cosegregated with berry colour on linkage group 2. The possibility of associating candidate genes to known position of QTL is discussed for this plant. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Marzia Salmaso and Giulia Malacarne contributed equally to the present work.     

Dermatophytosis caused by Nannizzia nana in two siblings

Background

Nannizzia nana is a zoophilic dermatophyte that affects animals like pigs, boars and, exceptionally, humans, in whom it causes tinea capitis, as well as tinea corporis and onychomycosis.

Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions

Polystyrene Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of two different enzymatically modified hairy regions (HRs) from pectin containing, for example, rhamnogalacturonan-I and xylogalacturonan structural elements. The two polysaccharide preparations share the same structural elements of apple pectin, but the relative amounts and lengths of the neutral side chains present differ. Surface analysis by X-ray photoelectron spectroscopy, contact angle measurement, and atomic force microscope (AFM) force-separation curves was used to characterize the effects on surface chemistry and interfacial forces of the surface modification process. Cell adhesion experiments using continuous L-929 fibroblasts and primary aortic smooth muscle cells were performed to evaluate the effect of the polysaccharide nature on cell adhesion. Results show that immobilization of the HR affects the interfacial field of forces and the cell behavior: "equilibrium" contact angles, obtained by a recently introduced vibrational approach, decrease after HR immobilization reaching a value close to 20 degrees . AFM force-separation curves show a more extended (or softer) interface in the case of the HR bearing longer side chains. Accordingly, depending on the HR preparation, cells shifted from spread morphology and adhesion behavior quantitatively comparable to that observed on conventional tissue culture polystyrene to rounded morphology and significantly lower adhesion. These data show that engineering of plant pectins can be a valuable tool to prepare novel and finely tuned polysaccharides having different chemico-physical and biological properties, to be used in the surface modification of medical devices and materials.     

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

We studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).     

Purification and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118

Pseudomonas fluorescens E118 was isolated from soil as an effective eugenol-degrading organism by a screening using eugenol as enrichment substrate. The first enzyme involved in the degradation of eugenol in this organism, eugenol dehydrogenase, was purified after induction by eugenol, and the purity of the enzyme was shown by SDS-PAGE and gel-permeation HLPC. The enzyme is a heterodimer that consists of a 10-kDa cytochrome c and a 58-kDa subunit. The larger subunit presumably contains flavin, suggesting a flavocytochrome c structure and an electron transfer via flavin and cytochrome c during dehydrogenation. The activity of the purified enzyme depended on the addition of a final electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, cytochrome c, or potassium ferricyanide. The enzyme catalyzed the dehydrogenation of three different 4-hydroxybenzylic structures including the conversion of eugenol to coniferyl alcohol, 4-alkylphenols to 1-(4-hydroxyphenyl)alcohols, and 4-hydroxybenzylalcohols to the corresponding aldehydes. The catalytic and structural similarity between this enzyme and a Penicillium vanillyl-alcohol oxidase and 4-alkylphenol methylhydroxylases from several Pseudomonas species is discussed. Received: 17 June 1998 / Accepted: 12 October 1998     

Efficient expression of gene variants that harbour AGA codons next to the initiation codon

In an effort to improve the knowledge about the rules which direct the effect of the early ORF sequences on translation efficiency, we have analyzed the effect of pairs of the six arginine codons at the second and third positions on the expression of lacZ variants. Whereas the pairs of identical AGA or AGG codons were favorable for the gene expression, identical pairs of each of the four CGN codons were very inefficient. This result was unexpected because tandems of AGA or AGG codons located in more internal gene positions provoke deficient expression whilst internally located CGU and CGC are the most abundant and efficiently translated arginine codons. The mixed combinations of AGA and each of the CGN codons usually resulted in efficient rates of lacZ expression independently of the peptidyl-tRNA propensity to dissociate from the ribosome. Thus, the variant harboring the pair of AGA codons was expressed as efficiently as the variant carrying a pair of AAA codons in the same positions, a configuration reported as one of the most common and efficient for gene expression. We explain these results assuming that the presence of adenines in these early positions enhance gene expression. As expected, specific mRNA levels correlated with the intensity of lacZ expression for each variant. However, the induction of lacZ AGA AGA gene in pth cells accumulated peptidyl-tRNA^Arg4 as well as a short 5′-proximal lacZ mRNA fragment suggesting ribosome stalling due to depletion of aminoacylated-tRNA^Arg4.     

Impedimetric immunosensor for the specific label free detection of ciprofloxacin antibiotic

Impedance spectroscopy approaches combined with the immunosensor technology have been used for the determination of trace amounts of ciprofloxacin antibiotic belonging to the fluoroquinolone family. The sensor electrode was based on the immobilization of anti-ciprofloxacin antibodies by chemical binding onto a poly(pyrrole-NHS) film electrogenerated on a solid gold substrate. The electrode surface was modified by electropolymerization of pyrrole-NHS, antibody grafting and ciprofloxacin immunoreaction. The sensitive steps of surface modification, cyclic voltammetry (CV) and atomic force microscopy (AFM) imaging have been used for electrode surface characterization. The immunoreaction of ciprofloxacin on the grafted anti-ciprofloxacin antibody directly triggers a signal via impedance spectroscopy measurements which allows the detection of extremely low concentration of 10 pg/ml ciprofloxacin.     

Repetitive DNA, molecular cytogenetics and genome organization in the King scallop (Pecten maximus)

We studied the structure, organization and relationship of repetitive DNA sequences in the genome of the scallop, Pecten maximus, a bivalve that is important both commercially and in marine ecology. Recombinant DNA libraries were constructed after partial digestion of genomic DNA from scallop with PstI and ApaI restriction enzymes. Clones containing repetitive DNA were selected by hybridisation to labelled DNA from scallop, oyster and mussel; colonies showing strong hybridisation only to scallop were selected for analysis and sequencing. Six non-homologous tandemly repeated sequences were identified in the sequences, and Southern hybridisation with all repeat families to genomic DNA digests showed characteristic ladders of hybridised bands. Three families had monomer lengths around 40 bp while three had repeats characteristic of the length wrapping around one (170 bp), or two (326 bp) nucleosomes. In situ hybridisation to interphase nuclei showed each family had characteristic numbers of clusters indicating contrasting arrangements. Two of the repeats had unusual repetitions of bases within their sequence, which may relate to the nature of microsatellites reported in bivalves. The study of these rapidly evolving sequences is valuable to understand an important source of genomic diversity, has the potential to provide useful markers for population studies and gives a route to identify mechanisms of DNA sequence evolution.