Modulation of the biological activity of a tobacco LTP1 by lipid complexation

Plant lipid transfer proteins (LTPs) are small, cysteine-rich proteins secreted into the extracellular space. They belong to the pathogenesis-related proteins (PR-14) family and are believed to be involved in several physiological processes including plant disease resistance, although their precise biological function is still unknown. Here, we show that a recombinant tobacco LTP1 is able to load fatty acids and jasmonic acid. This LTP1 binds to specific plasma membrane sites, previously characterized as elicitin receptors, and is shown to be involved in the activation of plant defense. The biological properties of this LTP1 were compared with those of LTP1-linolenic and LTP1-jasmonic acid complexes. The binding curve of the LTP1-linolenic acid complex to purified tobacco plasma membranes is comparable to the curve obtained with LTP1. In contrast, the LTP1-jasmonic acid complex shows a strongly increased interaction with the plasma membrane receptors. Treatment of tobacco plants with LTP1-jasmonic acid resulted in an enhancement of resistance toward Phytophthora parasitica. These effects were absent upon treatment with LTP1 or jasmonic acid alone. This work presents the first evidence for a biological activity of a LTP1 and points out the crucial role of protein-specific lipophilic ligand interaction in the modulation of the protein activity.     

Cathepsin-dependent apoptosis triggered by supraoptimal activation of T lymphocytes: a possible mechanism of high dose tolerance

High doses of Ag can paradoxically suppress immune responses in vivo. This Ag-specific unresponsiveness (termed high dose tolerance) involves extrathymic mechanisms in mature T lymphocytes. To investigate these mechanisms, we used the in vitro model of PBL activated with anti-CD3 or PHA. In these conditions, increasing mitogen concentrations resulted in a reduction of the proliferative response, associated with an increased percentage of apoptotic cells. Apoptosis did not require prior exposure to IL-2, it was not the consequence of CD178/CD95 or TNF/TNFR interactions, and was therefore clearly distinct from activation-induced cell death. Although the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) decreased DNA fragmentation, cytochrome c release and caspase-9 and caspase-3 activation were not implicated, suggesting that this apoptosis did not primarily involve the intrinsic mitochondrial pathway. E64d, a cysteine protease inhibitor, as well as specific inhibitors of cathepsin B and cathepsin L conferred protection. We further demonstrated that cathepsin B and cathepsin L were released from the lysosomes and catalytically active in the cytosol. Release of cathepsin B and cathepsin L was the consequence of lysosomal membrane permeabilization without complete disruption of the cytosol-lysosome pH gradient. These results demonstrate a role for cathepsins in supraoptimal activation-induced apoptosis in vitro and suggest their possible participation in high dose tolerance in vivo.     

First record of the microsporidian parasite Steinhausia mytilovum in Mytilus sp. (Bivalvia: Mytilidae) from France

Steinhausia mytilovum is a globally distributed microsporidian parasite which infects the oocytes of the blue mussels Mytilus edulis and M. galloprovincialis. Despite the intensive monitoring effort made on mussel populations, the parasite has not previously been reported in France. We report herein on the occurrence of S. mytilovum in Mytilus sp. from 1 cultured and 2 natural populations on the northern coast of France, thus extending the parasite's known distribution northwards. We also report on the observation in 1989 of S. mytilovum in M. galloprovincialis from the Golfe de Fos area in the Mediterranean Sea (South of France). S. mytilovum was observed in the European hybrid zone between M. edulis and M. galloprovincialis, which therefore renders the exact taxonomic status of the infected hosts unknown. The prevalence of the parasite was low, which suggests that its effect on mussel populations was probably limited.     

Segmental distribution and morphometric features of primary sensory neurons projecting to the tibial periosteum in the rat

Previous reports have demonstrated very rich innervation pattern in the periosteum. Most of the periosteal fibers were found to be sensory in nature. The aim of this study was to identify the primary sensory neurons that innervate the tibial periosteum in the adult rat and to describe the morphometric features of their perikarya. To this end, an axonal fluorescent carbocyanine tracer, DiI, was injected into the periosteum on the medial surface of the tibia. The perikarya of the sensory fibers were traced back in the dorsal root ganglia (DRG) L1-L6 by means of fluorescent microscopy on cryosections. DiI-containing neurons were counted in each section and their segmental distribution was determined. Using PC-assisted image analysis system, the size and shape of the traced perikarya were analyzed. DiI-labeled sensory neurons innervating the periosteum of the tibia were located in the DRG ipsilateral to the injection site, with the highest distribution in L3 and L4 (57% and 23%, respectively). The majority of the traced neurons were of small size (area < 850 microm2), which is consistent with the size distribution of CGRP- and SP-containing cells, regarded as primary sensory neurons responsible for perception of pain and temperature. A small proportion of labeled cells had large perikarya and probably supplied corpuscular sense receptors observed in the periosteum. No differences were found in the shape distribution of neurons belonging to different size classes.     

An overview of the KIN1/PAR-1/MARK kinase family

Members of the KIN1/PAR-1/MARK kinase family are conserved from yeast to humans and share a similar primary structural organization. Several kinases of this family appear to be at the crossroads of various biological functions including cell polarity, cell cycle control, intracellular signalisation, microtubules stability and protein stability. Here we present an overview of known roles of KIN1/PAR-1/MARK kinases including pEg3 a newly identified member which is regulated during the cell cycle and is a potential regulator of the cell cycle progression. Some common modes of action can be deciphered for this protein kinase family.     

Release of DNA into the medium by competent Streptococcus pneumoniae: kinetics, mechanism and stability of the liberated DNA

The release of chromosomal DNA into culture media has been reported for several naturally transformable bacterial species, but a direct link between competence development and the liberation of DNA is generally lacking. Based on the analysis of strains with mutations in competence-regulatory genes and the use of conditions favouring or preventing competence, we provide evidence that DNA release is triggered by the induction of competence in Streptococcus pneumoniae. Kinetic analyses revealed that whereas competence was maximal 20 min after addition of competence-stimulating peptide, and then decreased, the amount of liberated DNA continued to increase and reached a maximum in stationary phase, when cells are no longer competent for DNA uptake. These data are not consistent with the proposal that release of DNA by a fraction of the population is coordinated with uptake by the remainder. Moreover, we observed that an unidentified DNase was specifically induced or released in competent cultures, and that together with the major pneumococcal endonuclease, EndA, it could degrade released DNA. Nearby complete abolition of release in a mutant lacking both the major autolysin, LytA, and the autolytic lysozyme, LytC, indicated that DNA liberation occurs by LytA-LytC-dependent cell lysis. These observations suggest that competence-dependent DNA release is one facet of a more general phenomenon of sensitization to autolysis that reaches its maximum in stationary phase.     

Rational design of potent and selective NH-linked aryl/heteroaryl cathepsin K inhibitors

Prior reports from our laboratories have identified the nonpeptidic inhibitor 2 as a potent and selective Cathepsin K (Cat K) inhibitor. Modelling studies suggested that the introduction of a NH linker between the P3 aryl and P2 leucinamide moieties would allow the formation of a H-bond with the Gly66 residue of Cat K, hopefully increasing potency. Aniline 4 was thus synthesized and showed improved potency over its predecessor 2. Further modelling concluded that a 2-substituted five membered ring could more adequately place the P3 moiety of 4 into the S3 pocket of Cat K. The synthesis of the 2-substituted thiophene 5 confirmed this hypothesis by displaying a slight increase in potency against Cat K (>10-fold increase in potency vs 2) and a good selectivity profile against Cathepsins B, L, and S. This rationally designed inhibitor 5 also displayed increased potency in a functional bone resorption assay (10nM) versus 2 (95 nM).