Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.     

The Escherichia coli lov gene product connects peptidoglycan synthesis, ribosomes and growth rate.

Mecillinam, a beta-lactam antibiotic which binds specifically to penicillin-binding protein 2 (PBP2), blocks lateral cell-wall elongation, induces spherical morphology and ultimately kills bacteria. We describe here a new mecillinam-resistant mutant of Escherichia coli, the lov mutant. It possesses active PBP2, as evidenced by its rod shape in the absence of mecillinam (but not in its presence), its ability to filament when septation is inhibited, and its penicillin-binding ability. The lov mutant grows slowly but seems to regulate its macromolecular parameters properly: cell volume, RNA content (ribosome concentration), and DNA content are appropriate for the growth rate, and the growth yield is identical to that of wild type. The lov mutation is located at 41 min on the E.coli genetic map and is recessive. Certain rpsL (StrR) mutations suppress the lov mutant's mecillinam resistance. The allele specificity of the suppression suggests that the lov gene product may interact directly with the ribosomes. The lov gene product thus seems to define a link between PBP2 (the mecillinam target) and the ribosomes; we propose that this link is involved in transmitting information on the growth rate (ribosome concentration) to the peptidoglycan synthesizing apparatus.     

Isolation ofBacillus schlegelii,a thermophilic,hydrogen oxidizing,aerobic autotroph,from geothermal and nongeothermal environments

Thermophilic hydrogen-oxidizing strains forming round, terminal endospores were isolated from geothermal areas. They were neutrophilic and facultatively autotrophic. They resembledBacillus schlegelii, a thermophilic hydrogen bacterium found so far only in cold environments. Phenotypic similarities, as well as DNA G+C content and DNA:DNA homologies, clearly revealed that the isolated strains belonged to the taxospeciesB. schlegelii. Hence, the strains ofB. schlegelii found in cold environments are probably allochthonous, their origin being geothermal and volcanic areas.     

Determination of monod kinetic coefficients for volatile hydrophobic organic compounds

A new procedure is presented to determine Monod kinetic coefficients and the microbial yield coefficient for volatile hydrophobic compounds such as phenanthrene. Batch experiments were conducted with a mixed culture capable of degrading phenanthrene. The phenanthrene disappearance and carbon dioxide production were monitored with time. A maximum likelihood estimator was formulated to fit the set of equations that describe the system to the measured data. The model takes into account a number of processes such as partition onto the apparatus, volatilization, and partition onto the biomass. The parameters required to describe these processes were obtained by independent experiments. The yield coefficient could be determined within a small range. However, the specific growth rate and the half-saturation constant were found to vary widely, with pairs of them describing the system adequately. It was shown that partition and volatilization processes can significantly affect the determination of the yield and Monod kinetic coefficients and need to be taken into account. (c) 1996 John Wiley & Sons, Inc.     

Tum—mutation P35B generates the MHC-binding site of a new antigenic peptide

Immunogenic tumor cell variant P35 was obtained by mutagen treatment of mouse mastocytoma P815. It express a potent new antigen recognized by syngeneic cytolytic T lymphocytes (CTL). This antigen is the result of a point mutation in a gene that is expressed by most healthy cells. A decapeptide encoded by the region spanning the mutation sensitized P815 cells to the relevant CTL, whereas the homologous decapeptide corresponding to the normal sequence did not. Only the mutant decapeptide was capable of enhancing the expression of the D^d-presenting molecule at the cell surface, indicating that the mutation generates a motif which enables the antigenic peptide to bind to D^d. Correspondence to: T. Boon.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.     

Non-histone protein methylation in Trypanosoma cruzi epimastigotes

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.     

Effects on adenylate cyclase activities of unsaturated fatty acid incorporation into rat liver plasma membrane phospholipids specific modulation by linoleate

The adenylate cyclase activities of the rat liver plasma membrane were measured simultaneously with the incorporation of acyl chains into the membrane phospholipids using oleyl CoA, linoleyl CoA or arachidonyl CoA thioester. The basal, fluoride — and glucagon — stimulated adenylate cyclase activities were increased by the incorporation of linoleate into the plasma membrane phospholipids. Oleyl CoA did not alter the adenylate cyclase activities whereas arachidonyl CoA, at high concentration, decreased the adenylate cyclase activities. These data indicate a specific effect of phospholipid molecular species containing linoleate.     

Biochemical,genetic, psychometric,and neuropsychological studies in heterozygotes of a family with globoid cell leucodystrophy (Krabbe's disease)

Summary Detection of a patient suffering from Krabbe's disease led to carrier screening in his family. Determination of galactosylceramide -galactosidase activity revealed the occurrence of two different alleles among the carriers of the same family. Heterozygotes and their noncarrier relatives were studied using psychometric and neuropsychological tests under blind conditions. It was found that compared to seven adult noncarrier relatives 19 adult carriers differ significantly in their general IQ and some subtests of the Wechsler Intelligence Scale for adults (WISA), including spatial cognition. Reaction times were significantly slower in the carriers with enzyme activity below 25% of the control values. Most of the carriers of this family have had myopia since early childhood.Dedicated to Professor Detlev Ploog on the occasion of his 60th birthday