Heat shock induction of apoptosis in promastigotes of the unicellular organism Leishmania (Leishmania) amazonensis

Apoptosis and/or programmed cell death have been described in examples ranging from fungi to man as gene-regulated processes with roles in cell and tissue physiopathology. These processes require the operation of an intercellular communicating network able to deliver alternative signals for cells with different fates and is thus considered a prerogative of multicellular organisms. Promastigotes from Leishmania (Leishmania) amazonensis, when shifted from their optimal in vitro growth temperature (22°C) to the temperature of the mammalian host (37°C), die by a calcium-modulated mechanism. More parasites die in the presence of this ion than in its absence, as detected by a colorimetric assay based on the activity of mitochondrial and cytoplasmic dehydrogenases which measures cell death, independently of the process by which it occurs. A heat shock, unable to induce detectable parasite death (34°C for 1 h), is able to significantly raise the concentration of intracellular free calcium in these cells. Heat-shocked parasites present ultrastructural and molecular features characteristic of cells dying by apoptosis. Morphological changes, observed only in the presence of calcium, are mainly nuclear. Cytoplasmic organelles are preserved. Heat shock is also able to induce DNA cleavage into an oligonucleosomal ladder detected in agarose gels by ethidium bromide staining and autoradiography of [α³²P]ddATP-labeled fragments. These results indicate that death by apoptosis is not exclusive of multicellular organisms. © 1996 Wiley-Liss, Inc.     

A new approach to assess occupational exposure to airborne fungal contamination and mycotoxins of forklift drivers in waste sorting facilities

The waste management industry is an important employer, and exposure of waste-handling workers to microorganisms is considered an occupational health problem. Besides fungal contamination, it is important to consider the co-occurrence of mycotoxins in this setting. Forklifts with closed cabinet and air conditioner are commonly used in waste industry to transport waste and other products within the facilities, possibly increasing the risk of exposure under certain conditions. The aim of this study was to assess the fungal contamination and mycotoxin levels in filters from the air conditioning system of forklift cabinets, as an indicator to assess occupational exposure of the drivers working in a waste sorting facility. Cytotoxicity was also assessed to understand and characterize the toxicity of the complex mixtures as present in the forklift filters. Aqueous extracts of filters from 11 vehicles were streaked onto 2% malt extract agar (MEA) with chloramphenicol (0.05 g/L) media, and in dichloran glycerol (DG18) agar-based media for morphological identification of the mycobiota. Real-time quantitative PCR amplification of genes from Aspergillus sections Fumigati, Flavi, Circumdati, and Versicolores was also performed. Mycotoxins were analyzed using LC-MS/MS system. Cytotoxicity of filter extracts was analyzed by using a MTT cell culture test. Aspergillus species were found most frequently, namely Aspergillus sections Circumdati (MEA 48%; DG18 41%) and Nigri (MEA 32%; DG18 17.3%). By qPCR, only Aspergillus section Fumigati species were found, but positive results were obtained for all assessed filters. No mycotoxins were detected in aqueous filter extracts, but most extracts were highly cytotoxic (n = 6) or medium cytotoxic (n = 4). Although filter service life and cytotoxicity were not clearly correlated, the results suggest that observing air conditioner filter replacement frequency may be a critical aspect to avoid worker’s exposure. Further research is required to check if the environmental conditions as present in the filters could allow the production of mycotoxins and their dissemination in the cabinet during the normal use of the vehicles.     

Interaction of polyamine macrocycles with Zn(II) and ATP in aqueous solution. Binary and ternary systems. A potentiometric,NMR and fluorescence emission study

ATP binding to ligands L1 and L3 and to their Zn(II) complexes has been examined by means of potentiometric and ¹H and ³¹P NMR measurements in aqueous solution. Their coordination features have been compared to those of ligand L2 and its Zn(II) complex. In all the three cases, the Zn(II) complexes proved to be better receptors than free ligands, due to the synergetic action of metal ion and ammonium functions in ATP binding. Among the three complexes, Zn(II) complex with L1 shows the highest equilibrium constant, which can be ascribed to the fact that, being coordinated by the dipyridine nitrogens outside the macrocyclic cavity, it is less saturated by ligand donors. The ³¹P NMR investigation showed that the nucleotide interacts via the P_γ and P_β phosphate groups with both free ligands and complexes, while the ¹H spectra revealed that the binding is reinforced by the presence of π–π interactions. Photophysical studies showed that the fluorescence emission intensity of the Zn(II) complexes is enhanced upon interaction with ATP.     

Simultaneous sleep study and nasoendoscopic investigation in a patient with obstructive sleep apnoea syndrome refractory to continuous positive airway pressure: a case report

Introduction

There seem to be no published data concerning the clinical impact of populations of hepatitis B virus (HBV) in the hepatic and extrahepatic compartments of HIV-infected people with severe acute hepatitis.

Case presentation

A 26-year-old Caucasian man presenting to our hospital with clinical symptoms suggesting acute hepatitis was found to have an acute hepatitis B profile upon admission. He developed fatal fulminant hepatitis and was found to be heavily immunocompromised due to HIV-1 infection. He had a high plasma HBV and HIV load, and analysis of the partial pre-S1/pre-S2 domain showed the presence of mixed infection with D and F genotypes. Analysis of the point mutations within this region revealed the presence of HBV strains with amino acid substitutions at the immunodominant epitopes involved in B or T cell recognition. A homogeneous population of a pre-core mutant strain harbouring the A1896G and A1899G affecting HBeAg expression was invariably found in the liver tissue, plasma and peripheral blood mononuclear cells despite active HBeAg secretion; it was the dominant strain in the liver only, and was characterised by the presence of two point mutations in the direct repeat 1 domain involved in HBV replication activity. Taken together, these mutations are indicative of a highly replicative virus capable of evading immune responses.

Conclusion

This case report provides clinical evidence of a possible association between the rapid spread of highly replicative escape mutants and the development of fulminant hepatitis in a heavily immunocompromised patient. Virological surveillance of severe acute hepatitis B may be important in establishing an early treatment strategy involving antiviral drugs capable of preventing liver failure, especially in individuals for whom liver transplantation is not accepted as a standard indication.     

In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2 % in-house rice flour to encourage sporulation, the addition of 2 % glucose to the culture media (RPMI-1640), and an incubation temperature of 28 °C. In addition, the incubation period was 7 d, the minimum inhibitory concentration (MIC) was defined as 80 % growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC₉₀ (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC₅₀ values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.     

Synthesis, characterization, fluorescence and computational studies of new Cu, Ni and Hg complexes with emissive thienylbenzoxazolyl-alanine ligands

The interaction of four fluorescent compounds containing thiophene and benzoxazole moieties combined with an alanine residue with alkaline, alkaline-earth, transition and post-transition metal ions was explored. The highly fluorescent heterocyclic alanine derivatives are strongly quenched in the solid state after complexation with the paramagnetic metal ions Cu²⁺ and Ni²⁺, and with the diamagnetic Hg²⁺. Absorption and steady-state fluorescence titrations reveal a selective interaction with Cu²⁺, Ni²⁺ and Hg²⁺. In all cases the formation of mononuclear or dinuclear metal complexes in solid state and in solution are postulated. DFT calculations on the mercury(II) complexes confirm the formation of dinuclear species. Our results suggest that one metal ion is coordinated by the chelate group formed by the amine and the protonated carboxylic groups present in the amino acid residue while a second metal ion is directly linked to the chromophore. As parent compound, L4 shows no interaction with Cu²⁺ and Ni²⁺ salts. However, the interaction with Hg²⁺ induces a strong quenching and a red shift of the fluorescence emission.