Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.     

Recombinant artificial forisomes provide ample quantities of smart biomaterials for use in technical devices

Forisomes are mechanoproteins that undergo ATP-independent contraction–expansion cycles triggered by divalent cations, pH changes, and electrical stimuli. Although native forisomes from Medicago truncatula comprise a number of subunits encoded by separate genes, here we show that at least two of those subunits (MtSEO1 and MtSEO4) can assemble into homomeric forisome bodies that are functionally similar to their native, multimeric counterparts. We expressed these subunits in plants and yeast, resulting in the purification of large quantities of artificial forisomes with unique characteristics depending on the expression platform. These artificial forisomes were able to contract and expand in vitro like native forisomes and could respond to electrical stimulation when immobilized between interdigital transducer electrodes. These results indicate that recombinant artificial forisomes with specific characteristics can be prepared in large amounts and used as components of microscale and nanoscale devices.     

Characteristics of artificial forisomes from plants and yeast

Forisomes are protein bodies found exclusively in the phloem of the Fabaceae (legumes). In response to wounding, the influx of Ca ( 2+) induces a conformational change from a condensed to a dispersed state which plugs the sieve tubes and prevents the loss of photoassimilates. This reversible, ATP-independent reaction can be replicated with purified forisomes in vitro by adding divalent cations or electrically inducing changes in pH, making forisomes ideal components of technical devices. Although native forisomes comprise several subunits, we recently showed that functional homomeric forisomes with distinct properties can be expressed in plants and yeast, providing an abundant supply of forisomes with tailored properties. Forisome subunits MtSEO-F1 and MtSEO-F4 can each assemble into homomeric artificial forisomes, which indicates functional redundancy. However, we provide further evidence that both proteins are subunits of the native heteromeric forisome body in planta. We also show that the properties of artificial forisomes can be modified by immobilization, which is a prerequisite for their incorporation into technical devices.     

Pre-haustorial resistance to broomrape (Orobanche cumana) in sunflower (Helianthus annuus): cytochemical studies

Sunflower broomrape (Orobanche cumana Wallr.) is a root holoparasitic angiosperm considered as one of the major constraints for sunflower production in Mediterranean areas. Breeding for resistance is regarded as the most effective, feasible, and environmentally friendly solution to control this parasite. However, the existing sources of genetic resistance are defeated by the continuous emergence of new more virulent races of the parasite. In this work, the interaction between sunflower and O. cumana has been analysed in order to gain insights into the mechanisms involved in resistance. Two sunflower genotypes were selected showing different behaviour against the new race F of O. cumana, HE-39998 (susceptible) and HE-39999 (resistant), and both compatible and incompatible interactions were compared. Pot and Petri dish bioassays revealed that only HE-39998 plants were severely affected, supporting a high number of successfully established broomrapes to mature flowering, whereas in HE-39999 root tubercles were never observed, resistance being associated with browning symptoms of both parasite and host tissues. Histological aspects of the resistance were further investigated. Suberization and protein cross-linking at the cell wall were seen in the resistant sunflower cells in contact with the parasite, preventing parasite penetration and connection to the host vascular system. In addition, fluorescence and confocal laser microscopy (CLM) observations revealed accumulation of phenolic compounds during the incompatible reaction, which is in agreement with these metabolites playing a defensive role during H. annuus-O. cumana interaction.     

Simple,rapid and reliable methods to obtain high quality RNA and genomic DNA from <Emphasis Type="Italic">Quercus ilex</Emphasis> L. leaves suitable for molecular biology studies

Isolation of high-quality RNA and genomic DNA (gDNA) from many samples is a necessary step before accomplishing molecular biology studies. The particular composition of Quercus ilex leaves, specially hard and rich in cell wall material, polyphenolics and secondary metabolites, usually results in preparations contaminated with non-nucleic acid compounds. Although many methods have been developed, each case of study demands a protocol adapted to the specific plant sample and the pursued research objectives. We have evaluated several protocols to establish the methodology that best suited to our current genetic and molecular studies on Q. ilex. Our priority was to select the simplest methods reducing the plant starting material and the time employed, without compromising yield, quality and integrity of the isolated nucleic acids. Our results point to two protocols based on silica-membrane purification, as the most convenient for Q. ilex leaf tissue, and both procedures are greatly improved by adding insoluble polyvinyl polypyrrolidone during the isolation process. The protocols optimized here can be completed at the microfuge scale and allow a researcher to process 48 samples in 1 h, producing high quality preparations suitable for the routinely molecular biology applications with higher efficiency than other more labour and time-consuming protocols.     

Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis

This work was performed to compare three precipitation protocols of protein extraction for 2-DE proteomic analysis using Arabidopsis leaf tissue: TCA-acetone, phenol, and TCA-acetone-phenol. There were no statistically significant differences in protein yield between the three methods. Samples were subjected to 2-DE in the 5 to 8 pH and 14-80 kDa ranges. The TCA-acetone-phenol protocol provided the best results in terms of spot focusing, resolved spots, spot intensity, unique spots detected, and reproducibility. In all, 93 qualitative or quantitative statistically significant differential spots were found between the three protocols. The 2-DE map of TCA-acetone-phenol extracts presented more resolved spots above 40 kDa, with no pI-dependent differences observed between the three protocols. 54 spots were selected for trypsin digestion, and the peptides were analyzed by MALDI-TOF-TOF MS. After database search using peptide mass fingerprinting, and MS/MS combined search, 30 proteins were identified, the proteins from chloroplastic photosynthetic and carbohydrate metabolism being those most highly represented. From these data, we were able to conclude that each extraction protocol had its main features. Considering this, the workflow of any standard comparative proteomic experiment should include the optimization and adaptation of the protein extraction protocol to the plant tissue and to the particular objective pursued.     

17β-Estradiol Directly Lowers Mitochondrial Membrane Microviscosity and Improves Bioenergetic Function in Skeletal Muscle

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Estrogenic regulation of skeletal muscle proteome: a study of premenopausal women and postmenopausal MZ cotwins discordant for hormonal therapy

Female middle age is characterized by a decline in skeletal muscle mass and performance, predisposing women to sarcopenia, functional limitations, and metabolic dysfunction as they age. Menopausal loss of ovarian function leading to low circulating level of 17β‐estradiol has been suggested as a contributing factor to aging‐related muscle deterioration. However, the underlying molecular mechanisms remain largely unknown and thus far androgens have been considered as a major anabolic hormone for skeletal muscle. We utilized muscle samples from 24 pre‐ and postmenopausal women to establish proteome‐wide profiles, associated with the difference in age (30–34 years old vs. 54–62 years old), menopausal status (premenopausal vs. postmenopausal), and use of hormone replacement therapy (HRT; user vs. nonuser). None of the premenopausal women used hormonal medication while the postmenopausal women were monozygotic (MZ) cotwin pairs of whom the other sister was current HRT user or the other had never used HRT. Label‐free proteomic analyses resulted in the quantification of 797 muscle proteins of which 145 proteins were for the first time associated with female aging using proteomics. Furthermore, we identified 17β‐estradiol as a potential upstream regulator of the observed differences in muscle energy pathways. These findings pinpoint the underlying molecular mechanisms of the metabolic dysfunction accruing upon menopause, thus having implications for understanding the complex functional interactions between female reproductive hormones and health.     

Proteomic analysis of Arabidopsis protein S-nitrosylation in response to inoculation with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis>

Nitric oxide (NO) is a key signaling molecule in plants, being its biological effects mainly mediated through S-nitrosylation of cysteine thiols. Using the biotin switch method combined with mass spectrometry analysis we have identified 127 targets of S-nitrosylation in Arabidopsis cell suspension cultures and leaves challenged with virulent and avirulent isolates of Pseudomonas syringae pv. tomato. The NO targets are proteins associated with carbon, nitrogen, and sulpfur metabolism, photosynthesis, the cytoskeleton, stress-, pathogen- and redox-related and signaling proteins. Some proteins were previously identified in plants and mammals, while others (63%) represent novel targets of S-nitrosylation. Our data suggest that NO might be orchestrating the whole plant physiology, presumably through covalent modification of proteins.