Decomposition of woody debris in Mediterranean ecosystems: the role of wood chemical and anatomical traits

Plant and Soil - Data about woody debris (WD) decomposition are very scarce for the Mediterranean basin. The specific aim of this work is to explore the relationships between WD traits with the...     

Novel RasGRF1-derived Tat-fused peptides inhibiting Ras-dependent proliferation and migration in mouse and human cancer cells

Mutations of RAS genes are critical events in the pathogenesis of different human tumors and Ras proteins represent a major clinical target for the development of specific inhibitors to use as anticancer agents. Here we present RasGRF1-derived peptides displaying both in vitro and in vivo Ras inhibitory properties. These peptides were designed on the basis of the down-sizing of dominant negative full-length RasGRF1 mutants. The over-expression of these peptides can revert the phenotype of K-RAS transformed mouse fibroblasts to wild type, as monitored by several independent biological readouts, including Ras-GTP intracellular levels, ERK activity, morphology, proliferative potential and anchorage independent growth. Fusion of the RasGRF1-derived peptides with the Tat protein transduction domain allows their uptake into mammalian cells. Chemically synthesized Tat-fused peptides, reduced to as small as 30 residues on the basis of structural constraints, retain Ras inhibitory activity. These small peptides interfere in vitro with the GEF catalyzed nucleotide dissociation and exchange on Ras, reduce cell proliferation of K-RAS transformed mouse fibroblasts, and strongly reduce Ras-dependent IGF-I-induced migration and invasion of human bladder cancer cells. These results support the use of RasGRF1-derived peptides as model compounds for the development of Ras inhibitory anticancer agents.     

The effects of l-DOPA on root growth, lignification and enzyme activity in soybean seedlings

In the present study, we investigated the effects of l-DOPA (l-3,4-dihydroxyphenylalanine), an allelochemical exuded from the velvetbean (Mucuna pruriens L DC. var. utilis), on the growth and cell viability of soybean (Glycine max L. Merrill) roots. We analyzed the effects of l-DOPA on phenylalanine ammonia lyase (PAL), cinnamyl-alcohol dehydrogenase (CAD) and cell wall-bound peroxidase (POD) activities as well as its effects on phenylalanine, tyrosine and lignin contents in the roots. 3-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), with or without 0.5?mM l-DOPA, in a growth chamber at 25?°C for 6, 12, 18 or 24?h with a day/night regime of 1:1, and a photon flux density of 280???mol?m^?2 s^?1. In general, the length, fresh weight and dry weight of the roots decreased followed by a significant loss of cell viability. Phenylalanine, tyrosine and lignin contents as well as PAL, CAD and cell wall-bound POD activities increased after l-DOPA treatment. These results reinforce the susceptibility of soybean to l-DOPA, which increases the enzyme activity in the phenylpropanoid pathway and, therefore, provides precursors for the polymerization of lignin. In brief, these findings suggest that the inhibition of soybean root growth induced by exogenously applied l-DOPA may be due to excessive production of lignin in the cell wall.     

Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.     

USER friendly DNA engineering and cloning method by uracil excision

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.     

Malnutrition enhances cardiovascular responses to chemoreflex activation in awake rats

Several studies in the literature suggest that low-protein intake is associated with increases in sympathetic efferent activity and cardiovascular disease. Among the possible mechanisms, changes in the neurotransmission of cardiovascular reflexes have been implicated. Therefore, the present study comprised the evaluation of chemoreflex responsiveness in rats subjected to a low-protein diet during the 35 days after weaning. As a result, we observed that malnourished rats presented higher levels of baseline mean arterial pressure and heart rate and exhibited a mild increase in the pressor response to chemoreflex activation. They also exhibited a massive bradycardic response to chemoreflex activation. Interestingly, bilateral ligature of the carotid body arteries further increased baseline mean arterial pressure and heart rate in malnourished animals. The data suggest severe autonomic imbalance and/or change in the central interplay between neural and cardiovascular mechanisms.     

A series of novel, potent, and selective histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors offer a promising strategy for cancer therapy and the first generation HDAC inhibitors are currently in clinical trials. A structurally novel series of HDAC inhibitors based on the natural cyclic tetrapeptide Apicidin is described. Selected screening of the sample collection looking for L-2-amino-8-oxodecanoic acid (L-Aoda) derivatives identified a small acyclic lead molecule 1 with the unusual ketone zinc binding group. SAR studies around this lead resulted in optimization to potent, low molecular weight, selective, non-hydroxamic acid HDAC inhibitors, equipotent to current clinical candidates.     

Fungal antagonists of the plant pathogen Rhizoctonia solani: selection, control efficacy and influence on the indigenous microbial community

A broad spectrum of fungal antagonists was evaluated as potential biocontrol agents (BCAs) against the soil-borne pathogen Rhizoctonia solani using a new combination of in vitro and in vivo assays. The in vitro characterisation of diverse parameters including the ability to parasitise mycelium and to inhibit the germination of Rhizoctonia sclerotia at different temperatures resulted in the selection of six potential fungal antagonists. These were genotypically characterised by their BOX-PCR fingerprints, and identified as Trichoderma reesei and T. viride by partial 18S rDNA sequencing. When potato sprouts were treated with Trichoderma, all isolates significantly reduced the incidence of Rhizoctonia symptoms. Evaluated under growth chamber conditions, the selected Trichoderma isolates either partly or completely controlled the dry mass loss of lettuce caused by R. solani. Furthermore, the antagonistic Trichoderma strains were active under field conditions. To analyse the effect of Trichoderma treatment on indigenous root-associated microbial communities, we performed a DNA-dependent SSCP (Single-Strand Conformation Polymorphism) analysis of 16S rDNA/ITS sequences. In this first assessment study for Trichoderma it was shown that the pathogen and the vegetation time had much more influence on the composition of the microbiota than the BCA treatment. After evaluation of all results, three Trichoderma strains originally isolated from Rhizoctonia sclerotia were selected as promising BCAs.     

Presence of fimH,mrkD, and irp2 Virulence Genes in KPC-2-Producing Klebsiella pneumoniae Isolates in Recife-PE,Brazil

Klebsiella pneumoniae strains can produce different virulence factors, such as fimbrial adhesins and siderophores, which are important in the colonization and development of the infection. The aims of this study were to determine the occurrence of fimH, mrkD, and irp2 virulence genes in 22 KPC-2-producing K. pneumoniae isolates as well as 22 not producing-KPC isolates, from patients from different hospitals in Recife-PE, Brazil, and also to analyze the clonal relationship of the isolates by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The genes were detected by PCR and DNA sequencing. The bla _KPC-2 gene was identified in 22 KPC-positive isolates. On analyzing the antimicrobial susceptibility profile of the isolates, it was detected that polymyxin and amikacin were the antimicrobials of best activity against K. pneumoniae. On the other hand, five isolates exhibited resistance to polymyxin. In the KPC-positive group, was observed a high rate of resistance to cephalosporins, followed by carbapenems. Molecular typing by ERIC-PCR detected 38 genetic profiles, demonstrating a multiclonal spread of the isolates analyzed. It was observed that the virulence genes irp2, mrkD, and fimH were seen to have together a higher frequency in the KPC-positive group. The accumulation of virulence genes of KPC-positive K. pneumoniae isolates, observed in this study, along with the multi-resistance impose significant therapeutic limitations on the treatment of infections caused by K. pneumoniae.