The leishmania ARL-1 and Golgi traffic

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.     

A rat model reproducing key pathological responses of alcoholic chronic pancreatitis

Although alcohol abuse is the major cause of chronic pancreatitis, the pathogenesis of alcoholic chronic pancreatitis (ACP) remains obscure. A critical obstacle to understanding the mechanism of ACP is lack of animal models. Our objective was to develop one such model. Rats were pair-fed for 8 wk ethanol or control Lieber-DeCarli liquid diet. For the last 2 wk, they received cyclosporin A (CsA; 20 mg/kg once daily) or vehicle. After 1 wk on CsA, one episode of acute pancreatitis was induced by four 20 microg/kg injections of cerulein (Cer); controls received saline. Pancreas was analyzed 1 wk after the acute pancreatitis. CsA or Cer treatments alone did not result in pancreatic injury in either control (C)- or ethanol (E)-fed rats. We found, however, that alcohol dramatically aggravated pathological effect of the combined CsA+Cer treatment on pancreas, resulting in massive loss of acinar cells, persistent inflammatory infiltration, and fibrosis. Macrophages were prominent in the inflammatory infiltrate. Compared with control-fed C+CsA+Cer rats, their ethanol-fed E+CsA+Cer counterparts showed marked increases in pancreatic NF-kappaB activation and cytokine/chemokine mRNA expression, collagen and fibronectin, the expression and activities of matrix metalloproteinase-2 and -9, and activation of pancreatic stellate cells. Thus we have developed a model of alcohol-mediated postacute pancreatitis that reproduces three key responses of human ACP: loss of parenchyma, sustained inflammation, and fibrosis. The results indicate that alcohol impairs recovery from acute pancreatitis, suggesting a mechanism by which alcohol sensitizes pancreas to chronic injury.     

Synthesis and anti-inflammatory activity of natural and semisynthetic geranyloxycoumarins

Nine new 7-geranyloxycoumarin derivatives differently substituted at position 8 were semi-synthesised. Their topical anti-inflammatory activity was evaluated using the Croton oil ear test in mice as a model of acute inflammation. Auraptene (7-geranyloxycoumarin), its 8-methoxy (collinin, 1) and 8-acetoxy derivatives (5) (1 micromol/cm(2)) provoked 50% oedema reduction, similarly to 0.25 micromol/cm(2) of the reference drug indomethacin, a nonsteroidal anti-inflammatory drug.     

Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids

Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools. In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M. pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 x 10(-6) transformants/CFU/ micro g of plasmid DNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region. Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.     

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis

BackgroundDuring pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence.MethodsWe examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis.ResultsPancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin.ConclusionOur findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.     

Thymosin beta4 and thymosin beta4-derived peptides induce mast cell exocytosis

The peptide thymosin beta4 (Tbeta4) promotes angiogenesis and wound healing. Mast cells are involved in these processes as well and therefore we investigated the effect of Tbeta4 on mast cells. Exposure to 0.2-2000nM Tbeta4 induced mediator release (up to 23%) in murine peritoneal and human HMC-1 mast cells in a concentration-dependent manner. While the peptide AcSDKP, matching the 4 N-terminal amino acid residues of Tbeta4, mediated low but detectable mediator release, peptides corresponding to the Tbeta4 amino acid sequences 16-38 and 17-23 stimulated mast cells mediator release on a level equal to or higher than that observed with native Tbeta4. These observations and certain characteristics of Tbeta4-mediated mast cell activation suggest that the actin-binding motif LKKTET present in Tbeta4 (amino acid 17-22) might be implicated in this process. Thus, Tbeta4 activates mediator release in mast cells by a process that possibly involves an actin-binding motif and this could be important for understanding the mechanisms of Tbeta4-mediated effects in vivo.     

The penetrating properties of the tumor homing peptide LyP‐1 in model lipid membranes

Cell‐penetrating peptides (CPPs) have the property to cross the plasma membrane and enhance its permeability. CPPs were successfully used to deliver numerous cargoes such as drugs, proteins, nucleic acids, imaging and radiotherapeutic agents, gold and magnetic nanoparticles, or liposomes inside cells. Although CPPs were intensively investigated over the past 20 years, the exact molecular mechanisms of translocation across membranes are still controversial and vary from passive to active mechanisms. LyP‐1 is a cyclic 9‐amino‐acids homing peptide that specifically binds to p32 receptors overexpressed in tumor cells. tLyP‐1 peptide is the linear truncated form of LyP‐1 and recognizes neuropilin (NRP) receptors expressed in glioma tumor tissue. Here, we investigate the interaction of the cyclic LyP‐1 peptide and linear truncated tLyP‐1 peptide with model plasma membrane in order to understand their passive, energy‐independent mechanism of uptake. The experiments reveal that internalization of tLyP‐1 peptides depends on membrane lipid composition. Inclusion of negatively charged phosphatidylserine (PS) or cone‐shaped phosphatidylethanolamine (PE) lipids in the composition of giant unilamellar vesicles facilitates the membrane adsorption and direct penetration but without inducing pore formation in membranes. In contrast, cyclic LyP‐1 peptide mostly accumulates on the membrane, with very low internalization, regardless of the lipid composition. Thus, the linear tLyP‐1 peptide has enhanced penetrating properties compared with the cyclic LyP‐1 peptide. Development of a mutant peptide containing higher number of arginine amino acids and preserving the homing properties of tLyP‐1 may be a solution for new permeable peptides that facilitate the internalization in cells and further the endosomal escape as well.     

Reconstituted NALP1 inflammasome reveals two-step mechanism of caspase-1 activation

Interleukin (IL)-1beta maturation is accomplished by caspase-1-mediated proteolysis, an essential element of innate immunity. NLRs constitute a recently recognized family of caspase-1-activating proteins, which contain a nucleotide-binding oligomerization domain and leucine-rich repeat (LRR) domains and which assemble into multiprotein complexes to create caspase-1-activating platforms called "inflammasomes." Using purified recombinant proteins, we have reconstituted the NALP1 inflammasome and have characterized the requirements for inflammasome assembly and caspase-1 activation. Oligomerization of NALP1 and activation of caspase-1 occur via a two-step mechanism, requiring microbial product, muramyl-dipeptide, a component of peptidoglycan, followed by ribonucleoside triphosphates. Caspase-1 activation by NALP1 does not require but is enhanced by adaptor protein ASC. The findings provide the biochemical basis for understanding how inflammasome assembly and function are regulated, and shed light on NALP1 as a direct sensor of bacterial components in host defense against pathogens.