Assessment of bacteria and archaea in metalworking fluids using massive parallel 16S rRNA gene tag sequencing.

Determination of the bacterial diversity in industry-based liquid in-use water-miscible metalworking fluid (MWF) samples was targeted by massive parallel multiplex DNA sequencing, either directly or upon pretreatment with propidium monoazide (PMA) that allows differentiation between intact and physically damaged cells. As MWFs provide a suitable basis of life for micro-organisms, the majority is preserved by biocides. 'Bio-concept' fluids on the other hand are bactericide free, which intentionally leads to substantial bacterial populations. Samples from both fluid types were chosen: A median of 51 operational taxonomic units at genera level (OTUs) were detected per sample, but only 13 were present at or above 1·0% of the total population in any PMA-treated sample analysed. As both fluid types were mainly dominated by Pseudomonas spp., we resolved this genus on the species level and found the Pseudomonas oleovorans/pseudoalcaligenes group to predominate. We also looked for archaea and detected Methanobrevibacter spp., albeit in <3% of all samples analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Water-miscible metalworking fluids provide a suitable base of life for micro-organisms, mainly bacteria and fungi. Earlier publications suggested that the diversity is rather low, but these studies were largely based on heterotrophic plate counts. This might have resulted in underestimation of population density and microbial diversity as some organisms might just refuse to grow. This study used high-throughput sequencing in the absence and presence of propidium monoazide to explore bacterial and archaeal presence in metalworking fluids. We established that diversity is low and bacterial populations are dominated by the genus Pseudomonas spp.

(E,Z)-3-(3',5'-Dimethoxy-4'-hydroxy-benzylidene)-2-indolinone blocks mast cell degranulation.

(E,Z)-3-(3',5'-Dimethoxy-4'-hydroxy-benzylidene)-2-indolinone (indolinone) is an alkaloid that has been identified as a pharmacologically active compound in extracts of the traditional anti-inflammatory herb Isatis tinctoria. Indolinone has been shown to inhibit compound 48/80-induced mast cell degranulation in vitro. Application of indolinone to bone marrow derived mast cells showed that it was uniformly distributed in the cytoplasm and that cellular uptake was terminated within minutes. Pre-treatment of IgE-sensitized mast cells with 100nM indolinone rendered them insensitive against FcvarepsilonRI-receptor dependent degranulation. However, upstream signalling induced by antigen such as activation of PI3-K and MAPK remained unaffected. We conclude that indolinone blocks mast cell degranulation at the level of granule exocitosis with an IC(50) of 54nm.

Inhibition of apoptosis by intracellular protozoan parasites.

Protozoan parasites which reside inside a host cell avoid direct destruction by the immune system of the host. The infected cell, however, still has the capacity to counteract the invasive pathogen by initiating its own death, a process which is called programmed cell death or apoptosis. Apoptotic cells are recognised and phagocytosed by macrophages and the parasite is potentially eliminated together with the infected cell. This potent defence mechanism of the host cell puts strong selective pressure on the parasites which have, in turn, evolved strategies to modulate the apoptotic program of the host cell to their favour. Within the last decade, the existence of cellular signalling pathways which inhibit the apoptotic machinery has been demonstrated. It is not surprising that intracellular pathogens subvert these pathways to ensure their own survival in the infected cell. Molecular mechanisms which interfere with apoptotic pathways have been studied extensively for viruses and parasitic bacteria, but protozoan parasites have come into focus only recently. Intracellular protozoan parasites which have been reported to inhibit the apoptotic program of the host cell, are Toxoplasma gondii, Trypanosoma cruzi, Leishmania sp., Theileria sp., Cryptosporidium parvum, and the microsporidian Nosema algerae. Although these parasites differ in their mechanism of host cell entry and in their final intracellular localisation, they might activate similar pathways in their host cells to inhibit apoptosis. In this respect, two families of molecules, which are known for their capacity to interrupt the apoptotic program, are currently discussed in the literature. First, the expression of heat shock proteins is often induced upon parasite infection and can directly interfere with molecules of the cellular death machinery. Secondly, a more indirect effect is attributed to the parasite-dependent activation of NF-kappaB, a transcription factor that regulates the transcription of anti-apoptotic molecules.

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation.

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3-K-generated phosphoinositides, in Theileria-transformed leucocytes. Akt/PKB is constitutively activated in a PI3-K- and parasite-dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF-kappaB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3-K-Akt/PKB pathway and NF-kappaB activation, resulting in protection against apoptosis. In Theileria-transformed leucocytes, activation of the NF-kappaB and the PI3-K-Akt/PKB pathways are not directly linked. The PI3-K-Akt/PKB pathway does not contribute to the persistent induction of IkappaBalpha phosphorylation, NF-kappaB DNA-binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF-kappaB-dependent protection against apoptosis is not dependent on a functional PI3-K-Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria-transformed T cells.