Internalisation and multiple phosphorylation of γ-Conglutin,the lupin seed glycaemia-lowering protein,in HepG2 cells

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action.     

Alternative splicing of human insulin receptor gene (INSR) in type I and type II skeletal muscle fibers of patients with myotonic dystrophy type 1 and type 2

INSR, one of those genes aberrantly expressed in myotonic dystrophy type 1 (DM1) and type 2 (DM2) due to a toxic RNA effect, encodes for the insulin receptor (IR). Its expression is regulated by alternative splicing generating two isoforms: IR-A, which predominates in embryonic tissue, and IR-B, which is highly expressed in adult, insulin-responsive tissues (skeletal muscle, liver, and adipose tissue). The aberrant INSR expression detected in DM1 and DM2 muscles tissues, characterized by a relative increase of IR-A versus IR-B, was pathogenically related to the insulin resistance occurring in DM patients. To assess if differences in the aberrant splicing of INSR could underlie the distinct fiber type involvement observed in DM1 and DM2 muscle tissues, we have used laser capture microdissection (LCM) and RT-PCR, comparing the alternative splicing of INSR in type I and type II muscle fibers isolated from muscle biopsies of DM1, DM2 patients and controls. In the controls, the relative amounts of IR-A and IR-B showed no obvious differences between type I and type II fibers, as in the whole muscle tissue. In DM1 and DM2 patients, both fiber types showed a similar, relative increase of IR-A versus IR-B, as also evident in the whole muscle tissue. Our data suggest that the distinct fiber type involvement in DM1 and DM2 muscle tissues would not be related to qualitative differences in the expression of INSR. LCM can represent a powerful tool to give a better understanding of the pathogenesis of myotonic dystrophies, as well as other myopathies.     

Effect of tree density on root distribution in Fagus sylvatica stands: a semi-automatic digitising device approach to trench wall method

Knowledge of root profiles is essential for measuring and predicting ecosystem dynamics and function. In the present study, the effects of management practices on root (0.5 ≤ ø < 20 mm) spatial distribution were examined in a 40-year-old coppice stand (CpS 1968) and other two stands converted from coppice to thinned high forest in 1994 (CvS 1994) and 2004 (CvS 2004), respectively. The use of a semi-automatic digitising device approach was compared with a conventional root mapping method in order to estimate the time per person required from fieldwork to the final digital map. In July 2009, six trench walls per stand were established according to tree density, i.e. as equidistant as possible from all surrounding trees. Findings highlighted differences between the stands with CvS 1994 showing a lower number of small roots (2 ≤ ø < 5 mm), a higher mean cross-sectional area (CSA) of coarse roots (5 ≤ ø < 20 mm) and different root depth distribution as compared to CpS 1968 and CvS 2004 whose values were close to each other. The three diameter classes selected in this study showed significant relationships in terms of number of roots, scaling down from coarse- to small- and fine-roots. Forest management practices significantly affected only the number of small roots. The number of fine roots (0.5 ≤ ø < 2 mm) was isometrically related to their root length density (RLD, cm cm^?3). No relationship occurred with RLD of very fine roots (ø < 0.5 mm). In conclusion, forest management practices in terms of conversion thinnings significantly affected belowground biomass distribution of beech forest in space and time. In particular, frequency of coarse roots was related to the stand tree density, frequency of small roots was related to the cutting age. Size of coarse roots was related to tree density but only several years after felling. The allometric relationship occurring between fine- and small-roots highlighted how fine root number and RLD were only indirectly affected by forest management practices. These findings suggest that future investigations on the effect of forest thinning practices on fine-root traits like number, length and biomass several years after felling cannot ignore those on small roots.     

Unravelling the response of poplar (Populus nigra) roots to mechanical stress imposed by bending

Abstract

Mechanical stress is a widespread environmental condition that can be caused by several factors (i.e. gravity, touch, wind, soil density, soil compaction and grazing, slope) and that can severely affect plant stability. In response to mechanical stress and to improve their anchorage, plants have developed complex mechanisms to detect mechanical perturbation and to induce a suite of modifications at anatomical, physiological, biochemical, biophysical and molecular level. Although it is well recognized that one of the primary functions of root systems is to anchor the plant to the soil, root response to mechanical stresses have been investigated mainly at morphological and biomechanical level, whereas investigations about the molecular mechanisms underlying these important alterations are still in an initial stage. We have used an experimental system in which the taproot poplar seedlings are bent to simulate mechanical perturbation to begin investigate the mechanisms involved in root response to mechanical stress. The results reported herein show that, in response to bending, the poplar root changes its morphology by emitting new lateral roots, and its biomechanical properties by increasing the root biomass and lignin synthesis. In addition, using a proteomic approach, we found that several proteins involved in the signal transduction pathway, detoxification and metabolism are up-regulated and/or down-regulated in the bent root. These results provide new insight into the obscure field of woody root response to mechanical stress, and can serve as a basis for future investigations aimed at unravelling the complex mechanism involved in the reaction of root biology to environmental stress.     

Membrane- and cell wall-associated heat shock proteins in two genotypes of barley seedlings

ABSTRACT

The heat-shock (HS) response of two genotypes (cv. Onice, winter type, and cv. Georgie, spring type) of barley (Hordeum vulgare L.) was compared. Protein synthesis was markedly reduced by HS in roots and coleoptiles of both genotypes. The reduction in cv. Onice was higher than in cv. Georgie. The pattern of cytosolic, membrane and cell wall HSPs was analysed by SDS-PAGE in coleoptiles and roots of the two genotypes. Differences and similarities in coleoptiles and roots of the same genotype and between the two genotypes were observed. In roots of the genotype Onice, LMW and HMW HSPs isolated from the cytosol, membranes and cell walls were resolved into a diverse array of polypeptides by two-dimensional gel electrophoresis. Present results confirm the de novo synthesis of cytosolic and membrane HSPs, and demonstrate, for the first time, their presence in cell walls.     

Somatic embryogenesis and shoot regeneration from leaf derived callus of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh

Abstract

The development of in vitro regeneration systems for Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh, a medicinal plant used for treating neurological disorders, is described. For the first time in this variety, somatic embryogenesis and shoot regeneration were induced from leaf-derived callus. Well-formed plantlets were obtained through both shoot regeneration and somatic embryogenesis, with separate morphogenetic programmes. Proembryogenic masses were obtained in liquid MS and B5 media supplemented with 5.8 μM 2,4-D, 1.34 μM NAA, and 1.16 μM Kin; after being transferred onto hormone-free medium, they formed whitish and spherical structures that subsequently developed into the heart and torpedo stages.

On MS agarized medium containing thidiazuron (TDZ) at different concentrations (3, 6, 9, 12 μM) combined with 2 μM IBA, only shoot regeneration, and not somatic embryogenesis, was obtained. The mean number of shoots increased significantly when the concentration of TDZ was 3 μM.     

Pedigree Reconstruction of the Italian Grapevine Aglianico (Vitis vinifera L.) from Campania

A total of 41 accessions of Aglianico belonging to three different biotypes (Taburno, Taurasi, and Vulture) and 9 accessions of Sirica grapes were sampled from diverse areas of Campania (Italy). All accessions were first genotyped using 21 microsatellite markers (SSR) to evaluate possible homonymies, synonymies, and the genetic structure of each group. A larger dataset was then constructed adding Italian and International cultivars. On the basis of results obtained analyzing the first dataset, further investigations were carried out enlarging the number of investigated loci (up to 43). The addition of 22 SSRs was useful in the definition of likely genetic relationships linking Aglianico biotypes, Sirica and Syrah. According to their SSR allelic profiles, the monophyletic origin of the three Aglianico biotypes was confirmed. Among Aglianico Taburno accessions, eight samples (called Aglianico like-to-type) performed a different SSR allelic profile from Aglianico true-to-type. Sirica and Syrah proved to be synonyms. This work allowed to determine the genetic relationship between Aglianico and the cultivars supposed to be related. The parentage analysis was investigated. The most likely pedigree has been reconstructed; revealing a second-degree relationship between the worldwide cultivated Syrah from the Rhone Valley and Aglianico. Aglianico like-to-type appeared related to Aglianico in a parent-offspring fashion.     

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide

We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity.