Effect of intratympanic dimethyl sulphoxide (DMSO) in an in vivo model of cisplatin-related ototoxicity.

Although dimethyl sulfoxide (DMSO) is one of the most common solvents employed in otoprotection studies, its effect on the inner ear remains unknown. Only a few in vitro studies have addressed the effect of DMSO in cochlear cells. Up to the date, no in vivo functional studies have been reported. To determine the effect of intratympanic DMSO application in the inner ear, and to evaluate its effect in combination with cisplatin in Wistar rats, twelve Wistar rats were randomly assigned into two groups. Group A received intratympanic 1 % DMSO in both ears. Group B received intraperitoneal cisplatin (10 mg/kg) and intratympanic 0.5 % DMSO in the right ear and saline solution in the left ear. Functional changes were evaluated with Auditory Steady-State Responses before and 5 days after the procedure. Morphological changes were studied by means of confocal laser scanning microscopy following the removal of the temporal bones and cochlear dissection. Hearing threshold levels in group A did not show any statistically significant changes after the treatment. In group B, significant differences between pre- and post-treatment were found, with no statistically significant variations between right (DMSO) and left ear (saline solution). We suggest that DMSO could be safely used to dissolve hydrophobic compounds in otoprotection studies without interfering with the cochlear damage produced by cisplatin.

Nox1-derived oxidative stress as a common pathogenic link between obesity and hyperoxaluria-related kidney injury.

Specific relationships among reactive oxygen species, activation pathways, and inflammatory mechanisms involved in kidney injury were assessed in a combined model of obesity and hyperoxaluria. Male Wistar rats were divided into four groups: Control, HFD (high fat diet), OX (0.75% ethylene glycol), and HFD + OX (combined model) Changes in basal O2- levels were evaluated by chemiluminescence in renal interlobar arteries and renal cortex. Furthermore, the effect of different inhibitors on NADPH-stimulated O2- generation was assessed in renal cortex. Oxidative stress sources, and local inflammatory mediators, were also determined, in parallel, by RT-PCR, and correlated with measures of renal function, urinary biochemistry, and renal structure. Rats from the HFD group developed overweight without lipid profile alteration. Tubular deposits of crystals were seen in OX and severely enhanced in HFD + OX groups along with a significantly higher impairment of renal function. Basal oxidative stress was increased in renal cortex of OX rats and in renal arteries of HFD rats, while animals from the combined HFD + OX group exhibited the highest levels of oxidative stress in renal cortex, derived from xanthine oxidase and COX-2. NADPH oxidase-dependent O2- generation was elevated in renal cortex of the OX group and markedly enhanced in the HFD + OX rats, and associated to an up-regulation of Nox1 and a down-regulation of Nox4 expression. High levels of oxidative stress in the kidney, of OX and HFD + OX groups were also associated to an inflammatory response mediated by an elevation of TNFα, COX-2, NFκB1 MCP-1, and OPN. Oxidative stress is a key pathogenic factor in renal disease associated to hyperoxaluria and a common link underlying the exacerbated inflammatory response and kidney injury found under conditions of both obesity and hyperoxaluria. Nox1 pathway must be considered as a potential therapeutic target.

Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues.

BACKGROUND AND AIMS: The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. METHODS: Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. KEY RESULTS: Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. CONCLUSIONS: The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies.

NETs detection and quantification in paraffin embedded samples using confocal microscopy.

Detection and quantification of Neutrophil Extracellular Traps (NETs) in tissue samples has become a topic of great interest to understand their pathological role in various diseases. We describe a semi-automatic method of visualization and quantification of NETs in paraffin-embedded intracoronary thrombus aspirate samples. This study is based on colocalization of myeloperoxidase (MPO) and citrullinated histone 3 (H3Cit) as hallmark of the presence of NETs. For the analysis we used the confocal immunofluorescence microscopy technology to quantify the number of fields and the total area (in µm2) containing NETs in each thrombus sample. This observer-independent quantification method could be a useful tool to standardize the study of NETs in paraffin-embedded tissues, enabling comparison of results among different laboratories.

Metabolic syndrome contributes to renal injury mediated by hyperoxaluria in a murine model of nephrolithiasis.

Metabolic syndrome (MS) individuals have a higher risk of developing chronic kidney disease through unclear pathogenic mechanisms. MS has been also related with higher nephrolithiasis prevalence. To establish the influence of MS on renal function, we designed a murine model of combined metabolic syndrome and hyperoxaluria. Four groups of male Sprague-Dawley rats were established: (1) control group (n = 10) fed with standard chow; (2) stone former group (SF) (n = 10) fed with standard chow plus 0.75% ethylene glycol administered in the drinking water; (3) metabolic syndrome group (MS) (n = 10), fed with 60% fructose diet; (4) metabolic syndrome + stone former group (MS + SF) (n = 10), 60% fructose diet and 0.75% EG in the drinking water. MS group showed a significant injury to renal function when hyperoxaluria was induced. It was demonstrated by a significant decrease of creatinine clearance (p < 0.001), with higher tubular damage (34.3%, CI 95% 23.9-44.7, p < 0.001), produced by deposition of crystals, and increased tubular synthesis of osteopontin as a response to tubular damage. Induction of hyperoxaluria in rats with MS causes severe morphological alterations with a significant impairment of renal function. This impairment is not produced in rats without MS. Therefore, this model can be useful for the study of the influence of MS in stone formation.

Differentiating plant cells switched to proliferation remodel the functional organization of nuclear domains.

The immature pollen grain, the microspore, under stress conditions can switch its developmental program towards proliferation and embryogenesis. The comparison between the gametophytic and sporophytic pathways followed by the microspore permitted us to analyse the nuclear changes in plant differentiating cells when switched to proliferation. The nucleus is highly dynamic, the architecture of its well organised functional domains--condensed chromatin, interchromatin region, nuclear bodies and nucleolus--changing in response to DNA replication, RNA transcription, processing and transport. In the present work, the rearrangements of the nuclear domains during the switch to proliferation have been determined by in situ molecular identification methods for the subcellular localization of chromatin at different functional states, rDNA, elements of the nuclear machinery (PCNA, splicing factors), signalling and stress proteins. The study of the changes in the nuclear domains was determined by a correlative approach at confocal and electron microscopy levels. The results showed that the switch of the developmental program and the activation of the proliferative activity affected the functional organization of the nuclear domains, which accordingly changed their architecture and functional state. A redistribution of components, among them various signalling molecules which targeted structures within the interchromatin region upon translocation from the cytoplasm, was also observed.

Chemical characterization and spectroscopic analysis of the solubilization products from wheat straw produced by Streptomyces strains grown in solid-state fermentation.

The effects of two extraction procedures on the yield and properties of APPL (acid-preciµltable polymeric lignin, or solubilized lignocellulose) produced by four streptomycetes during growth in solid-state fermentation were examined. When APPL was extracted with NaOH (0.1 M) rather than distilled water, yields increased threefold, with Streptomyces chattanoogensis exhibiting maximum solubilization levels [163 mg product (g straw)-1]. Alterations in the characteristics of APPL obtained during extraction with NaOH were detected using cross-polarization and magic-angle sµlnning (CPMAS) 13C NMR and IR spectroscopy and by GC-MS analysis after CuO oxidation, with the most significant changes detected in the cinnamic acid and lignin moieties. When APPL was extracted with NaOH, ester links between hemicellulose and lignin and between hemicellulose and cinnamic acid were cleaved, resulting in a decrease in the alkyl and carbonyl groups attached to lignin, enabling greater solubilization. Yields of APPL extracted with water were lower, but spectral characterization of this APPL suggested a possible role for actinomycete peroxidases and phenolic acid esterases in lignin solubilization. For industrial solubilization of lignocellulose, a possible role for the application of streptomycetes, or their enzymes, in alkali extraction is suggested as a means of increasing solubilization levels.

Analysis of alkali-lignin in a paper mill effluent decolourised with two Streptomyces strains by gas chromatography-mass spectrometry after cupric oxide degradation.

Alkali-lignin samples obtained from an untreated paper mill effluent and from the effluent decolourised by the strains Streptomyces avermitilis CECT 3339 and Streptomyces scabies UAH 51 were analysed by gas chromatography-mass spectrometry (GC-MS) after cupric oxide degradation. The analysis of the depolymerisation products of the alkali-lignin from the decolourised effluents showed a strain specific modification of the aromatic moiety of the alkali-lignin. Moreover, both strains were able to breakdown the aryl-alkyl ether linkages between the cinnamic acids and the lignin. Finally, GC-MS analysis showed that both strains oxidised the alkali-lignin regardless of its initial degree of oxidation.

Influence of salt concentration on the susceptibility of moderately halophilic bacteria to antimicrobials and its potential use for genetic transfer studies.

The influence of salinity on the susceptibility of 13 moderately halophilic collection strains belonging to the genera Chromohalobacter, Deleya, Halomonas, Vibrio, and Volcaniella to 10 common antimicrobials has been studied. Three different patterns of tolerance were found when salinity was varied from 10 to 1% (wt/vol) total salts in the testing media. The first one included the responses to ampicillin and rifampicin, where only minimal effects on the susceptibility were found. All moderate halophiles showed a high sensitivity to rifampicin regardless of the salt concentration. In the second group, including the responses to the aminoglycosides gentamycin, kanamycin, neomycin, and streptomycin, a remarkable and gradual increase of the toxicity was detected at lower salinities. Thirdly, the highest heterogeneity was found for the rest of antimicrobials assayed (trimethoprim, nalidixic acid, spectinomycin, and tetracycline), where the effect of salinity was moderate and dependent on both the individual strain and the antimicrobial tested. The data presented here should facilitate genetic studies on moderate halophiles. Thus, they simplify the design of selection media for genetic exchange experiments. Besides, by using low-salinity media, genes encoding resistance to a number of antimicrobials, especially to aminoglycosides, can be used as genetic markers for plasmids or transposons to be transferred to these extremophiles.

Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis.

A multidisciplinary study was carried out to analyse the chromosome doubling process during the early stages of in vitro maize microspore embryogenesis. The main stages (microspore derivatives) that were formed in the course of the culture were analysed. Chromosome number was determined from squashed cells, and DNA content was measured by cytometry. In parallel, an ultrastructural analysis of the microspore derivatives demonstrated the occurrence of a nuclear fusion process. It seems likely that nuclear fusion ensures chromosome doubling at early stages of induced microspore embryogenesis. It occurs precisely at the 5/7 day stage in the embryonic domain and probably leads to polyploidy in the endosperm domain of the microspore derivatives. As a conclusion a scheme summarises the results and proposes an interpretation of the sequence of chromosome doubling events during early maize microspore embryogenesis. Understanding of this process will be important for future efforts to increase the percentage of homozygous plants for crop improvement.

MAPKs entry into the nucleus at specific interchromatin domains in plant differentiation and proliferation processes.

Mitogen-activated protein kinases (MAPKs) are involved in the signaling of extracellular stimuli in eukaryotes, including plants. Different MAPKs have recently been shown to be expressed during plant cell proliferation and developmental processes such as pollen development and embryogenesis, but the structural subdomain where these MAPKs are targeted in the nucleus has not yet been characterized. We have determined the changes in the expression and subcellular localization of ERK homologues, proteins belonging to the MAPK family, and MAPK-active forms in two plant developmental processes which involved differentiation (pollen maturation) and proliferation (the initials of pollen embryogenesis). Immunofluorescence and immunogold labeling in the species studied showed that the progression of differentiation and proliferation was accompanied by an increase in the expression of ERKs and MAPK activation together with a translocation to the nucleus. Combining ultrastructural cytochemistry and immunogold for RNA and phosphorylated proteins we have identified the nuclear sites housing these MAPKs in areas of the interchromatin region enriched in RNA and phosphoproteins that include clusters of interchromatin granules. This could suggest a role of these MAPKs in the early events of activation of the transcription and processing machinery, via phosphorylation, which subsequently would be recruited to the transcription sites. The association of the nuclear localization of MAPKs with the progression through the cell cycle and the commitment toward differentiation in the two plant developmental processes can be correlated.

Defined nuclear changes accompany the reprogramming of the microspore to embryogenesis.

The switch of the gametophytic developmental program toward pollen embryogenesis to form a haploid plant represents an important alternative for plant breeding. In the present study, the switch of the gametophytic developmental program toward a sporophytic pathway, "embryogenesis," has been studied in three different plant species, Brassica, tobacco, and pepper. The switch has been induced by stress (heat shock) at the very responsive stage of the microspore, which is the vacuolate period. As a result, the cell nucleus undergoes striking structural changes with regard to late gametophytic development, including alterations of biosynthetic activities and proliferative activity. An enrichment in HSP70 heat-shock protein and in the presence of Ntf6-MAP kinase was observed after inductive treatment in the nuclei during early embryogenesis. This apparently reflected the possible roles of these proteins, specifically the protective role of HSP70 for the nuclear machinery, and signal transduction of Ntf6-MAPK for the entry of cells into proliferation. Importantly, the observed nuclear changes were similar in the three species investigated and represented convenient markers for early monitoring of embryogenesis and selection purposes for obtaining double-haploid plants in plant breeding.

Ultrastructural distribution of a MAP kinase and transcripts in quiescent and cycling plant cells and pollen grains.

Mitogen-activated protein kinases (MAPKs) are components of a kinase module that plays a central role in the transduction of diverse extracellular stimuli, including mitogens, specific differentiation and developmental signals and stress treatments. This shows that reversible protein phosphorylation cascades play a pivotal role in signal transduction in animal cells and yeast, particularly the entry into mitosis of arrested cells. Homologues of MAPKs have been found and cloned in various plant species, but there have been no data about their in situ localization at the subcellular level and their expression in plant cells so far. In the present paper we report the first data on the ultrastructural in situ localization of MAPK and their mRNAs in various plant cells. Proliferating and quiescent meristematic plant cells were studied to evaluate whether changes in MAPK presence, distribution and expression accompany the entry into proliferation of dormant cells. Moreover, MAPK localization was analyzed in vacuolate microspores. Polyclonal antibodies against the deduced MAPK from the tobacco Ntf6 clone were able to recognize homologue epitopes by immunocytochemical techniques in the cell types studied. The pattern of protein distribution is similar in all the cases studied: it is localized in the cytoplasm and in the nucleus, mainly in the interchromatin region. The quantitative study of the density showed that MAPK labelling is more abundant in cycling than in quiescent cells, also suggesting that, in plants, MAPK pathways might play a role in cell proliferation. RNA probes for conserved regions of the catalytic domain of plant MAPK homologue genes were used to study MAPK expression in those plant cells. In situ hybridization (ISH) showed the presence of MAPK transcripts in the three plant cell types studied, but levels were very low in quiescent cells compared to those in cycling cells. The quantification of labelling density of ISH signals strongly suggests a higher level of MAPK expression in proliferating cells, but also some basal messenger presence and/or expression in the quiescent ones. Immunogold and ISH results show the presence and distribution of MAPK proteins and mRNAs in vacuolate microspores. This represents a very dynamic stage during pollen development in which the cell nucleus is being prepared for an asymmetrical mitotic division, giving rise to both the generative and the vegetative nuclei of the bicellular pollen grain. Taken together, the data indicate a role played by MAPK in the re-entry into proliferation in plant cells.

Degradation of alkali-lignin residues from solid-state fermentation of wheat straw by streptomycetes.

The ability of three Streptomyces strains to degrade alkali-lignin, produced from the treatment of wheat straw by the same organisms, was examined. Decolourisation and loss of alkali-lignin was only detected in cultures supplemented with ammonium as an inorganic N source. The pH of cultures supplemented with inorganic N reached lower pH than in those supplemented with yeast extract. From FT-IR spectra corresponding to the alkalilignin obtained from the same cultures, a degradation of carbohydrate component concomitant with a modification in the aromatic moiety of lignin could be inferred. The results indicate that streptomycetes are suitable for use in the treatment of alkali-lignin effluents from the biological treatment of wheat straw by the same organisms and therefore support the role for these organisms in the development of clean technologies in pulp and paper industry.