Enhanced resolution of specific chromosome and nuclear regions by reflectance laser scanning confocal microscopy

 DNA sequences digested by HaeIII and reconstructed by in situ nick translation employing digoxigenin-labelled nucleotides are usually revealed either by horseradish peroxidase or FITC fluorescence. To obtain a significant improvement in terms of resolution, sensitivity and specificity, colloidal gold has been used instead of FITC (as the reporter molecule) to reveal the labelled DNA. Colloidal gold and propidium iodide were visualised by employing the reflectance mode and the 488-nm laser line of a confocal laser scanning microscope. In chromosomes, the fluorescent reaction pattern showed diffuse areas of labelling in which it was impossible to identify any specific kind of banding along the arms. In some chromosomes and, in particular, 1 and 9, a C-negative banding due to the negativity of the centromeric areas was seen. A more accurate localisation on chromosomes, including telomeric regions, often organised in spot pairs that resembled an R-like banding, was detected using 1-nm colloidal gold. A fine labelling was also demonstrated in nuclei, especially at their peripheral heterochromatin. The non-fading properties of colloidal gold combined with visualisation by reflectance confocal laser scanning microscopy demonstrated the possibility of obtaining a higher spatial resolution than when using conventional fluorophores or higher laser wavelength. This improved way to study the localization of HaeIII digestion sites in single chromosomes and in interphase nuclei made the reaction a valuable tool for the detection of antigens or of specific DNA sequences in biological preparations. Accepted: 5 September 1996     

Rapid leaf development drives the seasonal pattern of volatile organic compound (VOC) fluxes in a ‘coppiced’ bioenergy poplar plantation

Leaves of fast‐growing, woody bioenergy crops often emit volatile organic compounds (VOC). Some reactive VOC (especially isoprene) play a key role in climate forcing and may negatively affect local air quality. We monitored the seasonal exchange of VOC using the eddy covariance technique in a ‘coppiced’ poplar plantation. The complex interactions of VOC fluxes with climatic and physiological variables were also explored by using an artificial neural network (Self Organizing Map). Isoprene and methanol were the most abundant VOC emitted by the plantation. Rapid development of the canopy (and thus of the leaf area index, LAI) was associated with high methanol emissions and high rates of gross primary production (GPP) since the beginning of the growing season, while the onset of isoprene emission was delayed. The highest emissions of isoprene, and of isoprene photo‐oxidation products (Methyl Vinyl Ketone and Methacrolein, i_ox), occurred on the hottest and sunniest days, when GPP and evapotranspiration were highest, and formaldehyde was significantly deposited. Canopy senescence enhanced the exchange of oxygenated VOC. The accuracy of methanol and isoprene emission simulations with the Model of Emissions of Gases and Aerosols from Nature increased by applying a function to modify their basal emission factors, accounting for seasonality of GPP or LAI.     

Ultrastructural organization of vegetative hyphae of Tuber Albidum Pico

The ultrastructural organization of vegetative hyphae ofTuber albidum, an ectomycorrhizal fungus is described. Three zones are recognized: 1) an apical zone with cytoplasmic vesicles; 2) a subapical zone with protoplasmic organelles; 3) a vacuolated zone. This organization corresponds in its main features to the pattern just described for rapidly growing fungi.     

Isoelectric focusing studies of human pancreatic secretion

Pure bile, pancreatic and duodenal human juices have been analyzed by isoelectric focusing, either at rest or upon stimulation with caerulein. In rats, stimulation has also been performed with secretin. Twenty bands have been resolved and quantified in the pancreatic secretion. By developing zymograms, a number of isozymes have been identified: 6 isoamylases [pI's 7.2, 7.1 and 6.6 (major) and pI's 7.4, 6.7 and 5.8 (minor)], 3 lipases [pI's 7.0 and 6.8 (major) and 6.4 (minor)], two major alkaline proteases (pI's 9.8 and 8.4) and one major acidic protease (pI 4.3) and one band of RNAase activity (pI 8.6). The stimulation kinetics follow a mechanism according to Palade, indicating uniform response to secretogogues, parallel intracellular transport and parallel discharge of pancreatic exocrine proteins.     

Synthesis and crystal structure of the MgCl2(CH3COOC2H5)2· (CH3COOC2H5) adduct

The reaction of α-MgCl₂ with boiling ethyl acetate affords MgCI₂(CH₃COOC₂H₅)₂· (CH₃COOC₂H₅), which is obtained as crystals suitable for X-ray analysis only from the mother liquor. M=315.5, orthorhombic, space group P2₁22₁ (No. 18), a=25.077(3), b=8.616(1), c=7.345(1) Å, V=1587.0(3) ų, Z=4, D_x=1.32 g cm⁻³,λ A(Mo Kα)=0.71069 Å, μ=4.17 cm⁻¹, F(000)=664, T=298 K, observed reflections: 1667, R=0.059 and R_w=0.069. The structure is composed of polymeric chains of MgCl₂(CH₃COOC₂H₅₎₂ and the ethyl acetate molecules occupy a mutually trans position.     

Hypoxia affects dendritic cell survival: role of the hypoxia-inducible factor-1α and lipopolysaccharide

Dendritic cells (DC) are the most potent antigen-presenting cells and during their life cycle they are exposed to different oxygen tensions. Similarly to inflamed and tumor tissues, lymphoid organs are characterized by a hypoxic microenvironment; thus, the modality by which hypoxia may affect DC is important for regulating both the quality and the intensity of the immune response. Here, we show that human monocyte-derived DC, exposed to hypoxia, expressed high levels of the hypoxia-inducible factor (HIF)-1α, associated with upregulation of BNIP3 and BAX expression. This was paralleled with downregulation of the anti-apoptotic molecule Bcl-2, enhanced caspase-3 activity and poly (ADP-ribose) polymerase cleavage, along with cell death. Transfection of HIF-1α siRNA protected DC from the effects of hypoxia. Of interest, when hypoxic DC were maturated with lipopolysaccharide (LPS), we did not observe an increased cell death, while HIF-1α accumulation and BNIP3 expression were still significantly upregulated. In contrast with immature DC, mature DC expressed higher levels of Bcl-2, and, more importantly, of phosphorylated Akt. Transfection of HIF-1α siRNA to mature DC resulted in a significant upregulation of Akt phosphorylation as well. Moreover, inhibition of PI3K/Akt pathway resulted in an increased cell death of hypoxic mature DC. We may conclude that a prolonged exposure to hypoxia induces a cell death program which could be prevented by HIF-1α inhibition and/or LPS maturation. Our results may contribute to further understand the physiology of DC and the molecular mechanisms involved in the survival of DC, with important implications in the regulation of the immune response.     

Mapping the lithic colonization at the boundaries of life in Northern Victoria Land,Antarctica

The endolithic microbial communities of the Antarctic represent a borderline lifestyle in the most hostile ice-free areas of the continent. The extreme adaptation of microbes in these communities renders them very sensitive to environmental changes. To date, the actual distribution of these communities has never been investigated; yet, this information would define the geographic limits for life at present and supply a useful tool for monitoring any possible future variation related to climate change. In this study, most of the outcrops of Northern and of one site in Southern Victoria Land were recorded by altitudinal and sea distance gradients. The presence of endolithic life was determined by in situ observation, by microscopic observation of rock fragments in the laboratory, and, for doubtful samples, by culture experiment. Colonizers were present in more than 87 % of the visited sites. The presence of lithic life in Victoria Land appears to be wider than that reported 14 years earlier. The colonization trend follows climatic variation, with epiliths prevailing in coastal sites and decreasing towards the interior, while chasmoendoliths and cryptoendoliths increase and become predominant from the coast towards the inland sites. Typical cryptoendolithic colonization was exclusive on porous rocks as sandstone, chasmoendolithic colonization occurred even in less porous but translucent rocks as granite and quartz. Multivariate analysis of the combined results clearly indicates the pivotal role of the rock type in the colonization of endolithic micro-organisms; sandstone allows lithobionts to push themselves towards areas characterized by harsher conditions.     

Isolation and Screening of Black Fungi as Degraders of Volatile Aromatic Hydrocarbons

Black fungi reported as degraders of volatile aromatic compounds were isolated from hydrocarbon-polluted sites and indoor environments. Several of the species encountered are known opportunistic pathogens or are closely related to pathogenic species causing severe mycoses, among which are neurological infections in immunocompetent individuals. Given the scale of the problem of environmental pollution and the phylogenetic relation of aromate-degrading black fungi with pathogenic siblings, it is of great interest to select strains able to mineralize these substrates efficiently without any risk for public health. Fifty-six black strains were obtained from human-made environments rich in hydrocarbons (gasoline car tanks, washing machine soap dispensers) after enrichment with some phenolic intermediates of toluene and styrene fungal metabolism. Based on ITS sequencing identification, the majority of the obtained isolates were members of the genus Exophiala. Exophiala xenobiotica was found to be the dominant black yeast present in the car gasoline tanks. A higher biodiversity, with three Exophiala species, was found in soap dispensers of washing machines. Strains obtained were screened using a 2,6-dichlorophenol-indophenol (DCPIP) assay, optimized for black fungi, to assess their potential ability to degrade toluene. Seven out of twenty strains tested were able to use toluene as carbon source.     

Comparison of exhaled breath condensate pH using two commercially available devices in healthy controls,asthma and COPD patients

Background

Analysis of exhaled breath condensate (EBC) is a non-invasive method for studying the acidity (pH) of airway secretions in patients with inflammatory lung diseases.

Aim

To assess the reproducibility of EBC pH for two commercially available devices (portable RTube and non-portable ECoScreen) in healthy controls, patients with asthma or COPD, and subjects suffering from an acute cold with lower-airway symptoms. In addition, we assessed the repeatability in healthy controls.

Methods

EBC was collected from 40 subjects (n = 10 in each of the above groups) using RTube and ECoScreen. EBC was collected from controls on two separate occasions within 5 days. pH in EBC was assessed after degasification with argon for 20 min.

Results

In controls, pH-measurements in EBC collected by RTube or ECoScreen showed no significant difference between devices (p = 0.754) or between days (repeatability coefficient RTube: 0.47; ECoScreen: 0.42) of collection. A comparison between EBC pH collected by the two devices in asthma, COPD and cold patients also showed good reproducibility. No differences in pH values were observed between controls (mean pH 8.27; RTube) and patients with COPD (pH 7.97) or asthma (pH 8.20), but lower values were found using both devices in patients with a cold (pH 7.56; RTube, p < 0.01; ECoScreen, p < 0.05).

Conclusion

We conclude that pH measurements in EBC collected by RTube and ECoScreen are repeatable and reproducible in healthy controls, and are reproducible and comparable in healthy controls, COPD and asthma patients, and subjects with a common cold.     

Involvement of TRAIL/TRAIL-receptors in human intestinal cell differentiation

Despite the fact that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptors (TRAIL-Rs) are expressed in intestinal mucosa, little is known about the biological role of this system in intestinal cell physiology. The expression of surface TRAIL and TRAIL-R1, -R2, -R3, -R4 were examined by flow cytometry in the immortalized human cell line tsFHI under culture conditions promoting growth or growth arrest and expression of differentiated traits. A progressive increase of surface TRAIL expression paralleled tsFHI differentiation, consistently with immunohistochemistry analysis showing an increase of TRAIL immunostaining along the crypt-villus axis in normal jejuneal mucosa. In spite of the presence of TRAIL-R1 and TRAIL-R2 "death receptors," recombinant TRAIL was not cytotoxic for tsFHI cells. Exposure of tsFHI to recombinant TRAIL rather increased/anticipated the expression levels of the cyclin-dependent kinase inhibitors p21 and p27, which mediate the induction of growth arrest and the stabilization of differentiated traits, respectively, as well as of the canonical differentiation marker DPPIV. The differentiation inducing activity of TRAIL was abolished by pre-incubation with a Fc-TRAIL-R2 chimera. On the other hand, TRAIL did not significantly modulate the levels of osteoprotegerin (OPG), CXCL8/IL-8, CXCL9/MIG, and CXCL10/IP10 spontaneously released or induced by inflammatory cytokines. Taken together, these data suggest that TRAIL might act as a paracrine trophic cytokine on intestinal epithelium, promoting intestinal cell differentiation.