Intra- and interobserver concordance in scoring Harris lines: A test on bone sections and radiographs

Little attention has been devoted to assessing the reproducibility of (paleo)pathological observations. Harris lines (HL) are among the markers most used to determine chronology of stresses suffered during growth. Nevertheless, their scoring entails remarkable methodological difficulty. Bone sections (S) and radiographs (R) of 29 adult tibiae of archeological provenance (medieval) were scored for HL by five observers. At regular intervals of time, each observer gave two independent counts on both series. Results show a) a substantial interobserver disagreement of HL estimates for both sectional and radiographic records, and b) a high level of intraobserver error. © 1994 Wiley-Liss, Inc.     

CHROMOSOMAL VERSUS MITOCHONDRIAL DNA EVOLUTION: TRACKING THE EVOLUTIONARY HISTORY OF THE SOUTHWESTERN EUROPEAN POPULATIONS OF THE SOREX ARANEUS GROUP (MAMMALIA,INSECTIVORA)

The shrews of the Sorex araneus group have undergone a spectacular chromosome evolution. The karyotype of Sorex granarius is generally considered ancestral to those of Sorex coronatus and S. araneus. However, a sequence of 777 base pairs of the cytochrome b gene of the mitochondrial DNA (mtDNA) produces a quite different picture: S. granarius is closely related to the populations of S. araneus from the Pyrenees and from the northwestern Alps, whereas S. coronatus and S. araneus from Italy and the southern Alps represent two well-separated lineages. It is suggested that mtDNA and chromosomal evolution are in this case largely independant processes. Whereas mtDNA haplotypes are closely linked to the geographical history of the populations, chromosomal mutations were probably transmitted from one population to another. Available data suggest that the impressive chromosome polymorphism of this group is quite a recent phenomenon.     

A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization,purification and kinetic properties

The enzyme, desacetoxyvindoline 4-hydroxylase, was purified to apparent homogeneity from Catharanthus roseus by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, green 19-agarose, hydroxylapatite, -kg sepharose and Mono Q. The 4-hydroxylase was characterized by its strict specificity for position 4 of desacetoxyvindoline suggesting it to catalyze the second to last step in vindoline biosynthesis. The molecular mass of the native and denatured 4-hydroxylase was 45 kDa and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pIs 4.6, 4.7 and 4.8. The purified 4-hydroxylase exhibited no requirement for divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The enzyme was not inhibited by EDTA or SH-group reagents at concentrations up to 10 mM. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where -kg is the first substrate to bind followed by the binding of O₂ and desacetoxyvindoline. Their K _m values for -kg, O₂ and desacetoxyvindoline are 45 M, 45 M and 0.03 M, respectively. The first product to be released was deacetylvindoline followed by CO₂ and succinate, respectively.Abbreviations -kg -ketoglutarate or 2-oxoglutarate - NMT N-methyltransferase - SAM S-adenosyl-l-methionine - TLC thin layer chromatography - VBL vinblastine - VCR vincristine     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

The subcellular distribution of glucocorticoid-receptor complexes as studied by chemical crosslinking of intact HTC cells

Treatment of intact HTC cells with glutaraldehyde results in redistribution of glucocorticoid binding sites between cytosolic and nuclear fractions. The decrease in cytosolic receptors and their accumulation at the nuclear level were found to be directly related to the glutaraldehyde concentrations employed in our procedure and inversely related to the cell density of samples. When the data from eleven separate experiments were combined, and analyzed by linear regression of cytosolic and nuclear levels of receptor complexes vs the ratios between the DNA and glutaraldehyde concentration of our samples, two lines were obtained whose intercepts on the ordinate yielded values of cytosolic and nuclear receptors corresponding to 37.5 and 62.5% of the total cellular pool, respectively. When we compared the subcellular redistribution of glucocorticoid receptor to that of the cytosolic enzyme lactate dehydrogenase upon HTC cell crosslinking with glutaraldehyde, we found that the cytosolic and nuclear levels of the enzyme were 53.2 and 46.8% of the total content, respectively. If the subcellular distribution of glucocorticoid receptor is corrected for the artefactual redistribution induced by crosslinking, using the values obtained for lactate dehydrogenase, it can be concluded that glucocorticoid receptors in HTC cells are distributed between cytosol and nuclei in a ratio which is about 2:1. Our findings lend further support to theconclusion that only a portion of glucocorticoid receptor is cytosolic in intact cells.     

Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (He) from Carcinus maenas, in the temperature range 300–20 K. The derivatives studied are the mononuclear (Co²⁺)-He with a single cobalt ion in the Cu_A site, the binuclear (Co²⁺)₂-He and the binuclear mixed metal (Co²⁺-Cu¹⁺)-He. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be essentially harmonic in the whole temperature range investigated; moreover the data are consistent with the presence of an exogenous ligand strongly bound to the two metal ions. For the mononuclear derivative an essentially harmonic behavior is evident only up to 200 K where the data are consistent with the presence of an exogenous ligand much less strongly bound, while at higher temperatures the behavior of the spectra indicates the onset of very large anharmonic contributions to motions, that plausibly involve the above exogenous ligand and, quite likely, the entire active site.Abbreviations He Hemocyanin - M₀ zeroth moment - M₁ first moment - M₂ second moment - (Co^2–)₂-He binuclear bicobalt hemocyanin derivative - (Co²⁺)-He mononuclear monocobalt hemocyanin derivative - (Co²⁺-Cu¹⁺)-He binuclear mixed metals hemocyanin derivative - LFT ligand field theory - CT charge transfer - EPR electronic paramagnetic resonance - XANES X-ray absorption near edge structure Correspondence to: L. Cordone     

Artificial nanovesicles for dsRNA delivery in spray-induced gene silencing for crop protection

Spray-induced gene silencing (SIGS) is an innovative and eco-friendly technology where topical application of pathogen gene-targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross-kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNA encapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi-mediated protection against B. cinerea both on pre- and post-harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection.