Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Chaperone Hsp27, a novel subunit of AUF1 protein complexes, functions in AU-rich element-mediated mRNA decay

Controlled, transient cytokine production by monocytes depends heavily upon rapid mRNA degradation, conferred by 3' untranslated region-localized AU-rich elements (AREs) that associate with RNA-binding proteins. The ARE-binding protein AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins to attract the mRNA degradation machinery. We refer to this protein assembly as the AUF1- and signal transduction-regulated complex, ASTRC. Rapid degradation of ARE-bearing mRNAs (ARE-mRNAs) requires ubiquitination of AUF1 and its destruction by proteasomes. Activation of monocytes by adhesion to capillary endothelium at sites of tissue damage and subsequent proinflammatory cytokine induction are prominent features of inflammation, and ARE-mRNA stabilization plays a critical role in the induction process. Here, we demonstrate activation-induced subunit rearrangements within ASTRC and identify chaperone Hsp27 as a novel subunit that is itself an ARE-binding protein essential for rapid ARE-mRNA degradation. As Hsp27 has well-characterized roles in protein ubiquitination as well as in adhesion-induced cytoskeletal remodeling and cell motility, its association with ASTRC may provide a sensing mechanism to couple proinflammatory cytokine induction with monocyte adhesion and motility.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Modulation of pro-inflammatory activation of monocytes and dendritic cells by aza-bis-phosphonate dendrimer as an experimental therapeutic agent

Introduction

Our objective was to assess the capacity of dendrimer aza-bis-phosphonate (ABP) to modulate phenotype of monocytes (Mo) and monocytes derived dendritic cells (MoDC) activated in response to toll-like receptor 4 (TLR4) and interferon γ (IFN- γ) stimulation.

Methods

Mo (n = 12) and MoDC (n = 11) from peripheral blood of healthy donors were prepared. Cells were preincubated or not for 1 hour with dendrimer ABP, then incubated with lipopolysaccharide (LPS; as a TLR4 ligand) and (IFN-γ) for 38 hours. Secretion of tumor necrosis factor α (TNFα), interleukin (IL) -1, IL-6, IL-12, IL-10 and IL-23 in the culture medium was measured by enzyme-linked immunosorbent assay (ELISA) and Cytokine Bead Array. Differentiation and subsequent maturation of MoDC from nine donors in the presence of LPS were analyzed by flow cytometry using CD80, CD86, CD83 and CD1a surface expression as markers.

Results

Mo and MoDC were orientated to a pro-inflammatory state. In activated Mo, TNFα, IL-1β and IL-23 levels were significantly lower after prior incubation with dendrimer ABP. In activated MoDC, dendrimer ABP promoted IL-10 secretion while decreasing dramatically the level of IL-12. TNFα and IL-6 secretion were significantly lower in the presence of dendrimer ABP. LPS driven maturation of MoDC was impaired by dendrimer ABP treatment, as attested by the significantly lower expression of CD80 and CD86.

Conclusion

Our data indicate that dendrimer ABP possesses immunomodulatory properties on human Mo and MoDC, in TLR4 + IFN-γ stimulation model, by inducing M2 alternative activation of Mo and promoting tolerogenic MoDC.     

Radioimmunodetection of melanoma: preliminary results of a prospective study

A prospective study to evaluate the clinical usefulness of radioimmunodetection of melanoma in clinical practice is ongoing at the National Cancer Institute of Milan, Italy. Technical conditions for the application of the method were previously reported. In this trial, 99mTc-labelled F(ab')2 fragments of the 225.28S monoclonal antibody were used against a high molecular weight melanoma associated antigen (HMW-MAA). Retrospective studies on radioimmunodetection of melanoma have already been made by our group and by other Centers in about 300 patients. This study concerns the evaluation of the regional extension of primary melanoma. 23 patients with 32 suspected lymphatic involvements of melanoma on the trunk and arms underwent immunoscintigraphy. No false positive results were observed; 3 false negatives, one corresponding to a micrometastasis, were noticed. Specificity corresponds to 100% and sensitivity to 78.6%.     

Multivalent catanionic GalCer analogs derived from first generation dendrimeric phosphonic acids

The synthesis and characterization of a new series of catanionic multivalent analogs of GalCer is described. These systems are based on phosphonic acid terminated dendrimers and N-hexadecylamino lactitol moieties. Despite important structural differences that affect the dendrimers’ outer-shell, these supramolecular assemblies showed a fairly comparable anti-HIV-1 activity. All compounds have submicromolar IC₅₀ in a cell-based HIV-infection model but also a high general cytotoxicity.     

Molecular and cellular immune mediators of neuroprotection

Our view of the immune privileged status of the brain has dramatically changed during the past two decades. Even though systemic immune stimuli have the ability to activate different populations of neurons, cells of monocytic lineage also have access to the neuronal tissue and populate it as microglia. Although such a phenomenon is limited in intact brains, it is greatly increased during neurodegenerative processes associated with innate immunity and the release of pro-inflammatory molecules by either resident microglia or those derived from the bone marrow stem cells. The role of these events is currently a matter of great debate and controversy, especially as it relates to brain protection, repair, or further injury. In recent years, accumulating data have supported the notion that when immune molecules are timely released by microglia, they limit neuronal injury in the presence of pathogens and toxic agents, help clear debris from degenerated cells, and restore the cerebral environment for repair. It has been shown that alteration of the natural innate immune response by microglia has direct consequences in exacerbating the damages following acute injury to neurons. This article presents and discusses these data, supporting a powerful neuroprotective role for microglia and their innate immune reactions in response to pathogens and central nervous system insults.     

Removal of atrial natriuretic peptide by perfused rabbit lungs in situ

Removal of iodinated 28-amino acid atrial natriuretic peptide ([125I]ANP) by rabbit lungs was measured by indicator-dilution methods. After bolus injection of 6.5 pmoles of [125I]ANP, 66.9 +/- 2.9% was removed in a single pass through the lungs. Removal was unaltered by a kininase II inhibitor but was reversibly decreased by unlabelled ANP. Thus the lungs can remove ANP from the pulmonary circulation by a mechanism that does not involve hydrolysis by kininase II. Lungs therefore may be involved in regulating systemic concentrations and hence renal and other actions of ANP.     

Cyclooxygenase 2 (COX-2) inhibition increases the inflammatory response in the brain during systemic immune stimuli

Non-steroidal anti-inflammatory drugs (NSAIDs) and inhibitors of the cyclooxygenase (COX) pathways are currently recommended for the prevention and treatment of several inflammatory diseases, including neurodegenerative disorders. However non-selective blockade of COX was found to have pro-inflammatory properties, because they have the ability to alter the plasma glucocorticoid levels that play a critical role in the control of the innate immune response. The present study investigated the role of non-selective (ketorolac or indomethacin) or specific inhibitors of COX-1 (SC-560) and COX-2 (NS-398) in these effects. Mice challenged systemically with the endotoxin lipopolysaccharide (LPS) exhibited a robust hybridization signal for numerous inflammatory genes in vascular-associated cells of the brain and microglia across the cerebral tissue. Ketorolac, indomethacin and NS-398 significantly increased the ability of LPS to trigger such an innate immune response at time 3 h post challenge, whereas SC-560 failed to change gene expression in the brain of animals treated with the endotoxin. These data together with the crucial role of COX-2-derived prostaglandin E2 (PGE2) in the increase of glucocorticoids during systemic immune stimuli provide evidence that inhibition of this pathway results in an exacerbated early innate immune reaction. This may have a major impact on the use of these drugs in diseases where inflammation is believed to be a contributing and detrimental factor.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).