Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of Sao Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were reisolated in populations ranging from 10(6) to 10(9) CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms.

Influence of light and leaf epicuticular wax layer on Phakopsora pachyrhizi infection in soybean

Influence of light and leaf epicuticular wax layer on Phakopsora pachyrhizi infection in soybean Asian rust, caused by the fungus Phakopsora pachyrhizi, is one of the most serious phytosanitary problems of soybean in Brazil, especially because no cultivars with satisfactory resistance levels as yet exist. The objective of this study was to evaluate the influence of luminosity and of leaf epicuticular wax on the infection of soybean by P. pachyrhizi. The adaxial and abaxial leaflet surfaces of the first trifoliate leaf from cultivar BRS 154, phenological stage V2, were inoculated with a suspension of 105 uredospores/mL. The plants were kept for 24 hours in a humid chamber at temperature of 23 degrees C, in light or dark conditions, using a factorial design. Subsequently, the plants were maintained for 14 days under a 12-hour photoperiod. The disease severity and density were evaluated. For in vitro experiments, in light or dark conditions, the evaluation was done in terms of uredospore germination and appressorium formation. The wax content of adaxial and abaxial leaflets was analyzed quantitatively using chloroform extraction and ultrastructurally using scanning electron microscope. Higher density and severity were observed when the adaxial surface was inoculated, with later incubation of the plants in the dark, with no significant interaction between these factors. Spore germination in the dark (40.7%) was statistically different from spore germination in the light (28.5%). The same effect was observed with appressorium formation, in the dark (24.7%) and in the light (12.8%). The quantity and the ultrastructural aspects of epicuticular wax content did not show differences between the adaxial and abaxial surfaces; nor did they show any effect on infection by Phakopsora pachyrhizi in the soybean cultivar studied.

In vitro safety assessment of papain on human skin: A qualitative Light and Transmission Electron Microscopy (TEM) study

Papain is a thiol proteolytic enzyme widely used in dermatology that found applications in wound treatment. Recently, papain was also used as absorption enhancer which can modify the peptide/ protein material in the bilayer domain. We investigated papain safety using human skin that was exposed to papain in vitro at different times: 4, 24 and 48 hours. The samples were examined using Light and Transmission Electron Microscopy (TEM) to study of the mechanisms involved in enhancer-skin interaction. After 24 hours, changes occurred in corneosomes. However, samples of 48 hours did not show major changes in agreement with the control. These findings indicated that papain could be used safely onto the skin.

Implications of specimen preparation and of surface contamination for the measurement of the grain boundary carbon concentration of steels using x-ray microanalysis in an UHVFESTEM

The purpose of the present investigation was to gain an understanding of the nature of the carbon contamination on the surface of standard steel transmission electron spectroscopy (TEM) specimens, the effect of exposure of a clean specimen to normal laboratory air, and the efficacy of plasma-cleaning treatments. This knowledge is a necessary prerequisite to the development of appropriate specimen preparation and/or specimen cleaning methods. X-ray photoelectron spectroscopy in combination with argon ion beam profiling was used to characterize the specimen surfaces of X65 steel and 316 stainless steel. The only clean carbon-free surface obtained was that during argon etching of the sample in the surface analysis chamber. Any exposure of a previously cleaned sample to laboratory air resulted in a rapid carbon (hydrocarbon) contamination of the sample surface and the development of surface oxidation, Plasma cleaning with subsequent exposure of the specimen to the laboratory air also resulted in a carbon-contaminated surface. This suggests that procedures of preparation of TEM specimens of steels outside an ultrahigh vacuum chamber are unlikely to result in the lowering of contamination rates on specimens to levels where measurements for carbon in the grain boundaries are possible. What is needed is a cleaning system as an integral part of the specimen insertion system into the field-emission scanning transmission electron microscope. This cleaning could be carried out by argon ion etching. Copyright (C) 2000 John Wiley & Sons, Ltd.

Adhesion of microbes using 3-aminopropyl triethoxy silane and specimen stabilisation techniques for analytical transmission electron microscopy

A variety of adhesive support-films were tested for their ability to adhere various biological specimens for transmission electron microscopy. Support films primed with 3-amino-propyl triethoxy silane (APTES), poly-L-lysine, carbon and ultraviolet-B (UV-B)-irradiated carbon were tested for their ability to adhere a variety of biological specimens including axenic cultures of Bacillus subtilis and Escherichia coli and wild-type magnetotactic bacteria. The effects of UV-B irradiation on the support film in the presence of air and electrostatic charge on primer deposition were tested and the stability of adhered specimens on various surfaces was also compared. APTES-primed UV-B-irradiated Pioloform(TM) was consistently the best adhesive, especially for large cells, and when adhered specimens were UV-B irradiated they became remarkably stable under an electron beam. This assisted the acquisition of in situ phase-contrast lattice images from a variety of biominerals in magnetotactic bacteria, in particular metastable greigite magnetosomes. Washing tests indicated that specimens adhering to APTES-primed UV-B-irradiated Pioloform(TM) were covalently coupled. The electron beam stability was hypothesised to be the result of mechanical strengthening of the specimen and support film and the reduced electrical resistance in the specimen and support film due to their polymerization and covalent coupling.

Measuring the interaction forces between protein inclusion bodies and an air bubble using an atomic force microscope

Interaction forces between protein inclusion bodies and an air bubble have been quantified using an atomic force microscope (AFM). The inclusion bodies were attached to the AFM tip by covalent bonds. Interaction forces measured in various buffer concentrations varied from 9.7 nN to 25.3 nN (+/- 4-11%) depending on pH. Hydrophobic forces provide a stronger contribution to overall interaction force than electrostatic double layer forces. It also appears that the ionic strength affects the interaction force in a complex way that cannot be directly predicted by DLVO theory. The effects of pH are significantly stronger for the inclusion body compared to the air bubble. This study provides fundamental information that will subsequently facilitate the rational design of flotation recovery system for inclusion bodies. It has also demonstrated the potential of AFM to facilitate the design of such processes from a practical viewpoint.

Tubular system volume changes in twitch fibres from toad and rat skeletal muscle assessed by confocal microscopy

The volume of the extracellular compartment (tubular system) within intact muscle fibres from cane toad and rat was measured under various conditions using confocal microscopy. Under physiological conditions at rest, the fractional volume of the tubular system (t-sys(Vol)) was 1.38 +/- 0.09% (n = 17),1.41 +/- 0.09% (n = 12) and 0.83 +/- 0.07% (n = 12) of the total fibre volume in the twitch fibres from toad iliofibularis muscle, rat extensor digitorum longus muscle and rat soleus muscle, respectively. In toad muscle fibres, the t-sys(Vol) decreased by 30% when the tubular system was fully depolarized and decreased by 15% when membrane cholesterol was depleted from the tubular system with methyl-beta-cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 mum. There was also an increase by 30% and a decrease by 25% in t-sys(Vol) when toad fibres were equilibrated in solutions that were 2.5-fold hypertonic and 50% hypotonic, respectively. When the changes in total fibre volume were taken into consideration, the t-sys(Vol) expressed as a percentage of the isotonic fibre volume did actually decrease as tonicity increased, revealing that the tubular system in intact fibres cannot be compressed below 0.9% of the isotonic fibre volume. The results can be explained in terms of forces acting at the level of the tubular wall. These observations have important physiological implications showing that the tubular system is a dynamic membrane structure capable of changing its volume in response to the membrane potential, cholesterol depletion and osmotic stress but not when the sarcomere length is changed in resting muscle.

Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta: Liposomes fusion and/or lysis investigated by fluorescence and atomic force microscopy

Three dermaseptins, DS 01, DD K, and DD L, were compared with respect to their structural features and interactions with liposomes. Circular dichroic spectra at alcohols of different chain lengths revealed that DS 01 has the higher helicogenic potential in hydrophobic media. Binding of DS 01, DD K, and DD L to liposomes induced significant blue shifts of the emission spectra of the single tryptophan located at position 3 of all sequences indicating association of the peptides with bilayers. Kinetics evaluation of atomic force microscopy images evidenced the strong fusogenic activity of DS 01 whereas DD K and DD L showed increased lytic activities. (C) 2007 Elsevier Inc. All rights reserved.

The assessment of oral cytology utilizing ploidy analysis and virtual microscopy for the early detection of epithelial dysplasia and neoplasia in oral mucosal lesions

Oral squamous cell carcinoma (OSCC) is associated with high morbidity and mortality which is due, at least in part, to late detection. Precancerous and cancerous oral lesions may mimic any number of benign oral lesions, and as such may be left without investigation and treatment until they are well advanced. Over the past several years there has been renewed interest in oral cytology as an adjuvant clinical tool in the investigation of oral mucosal lesions. The purpose of the present study was to compare the usefulness of ploidy analysis after Feulgen stained cytological thin-prep specimens with traditional incisional biopsy and routine histopathological examination for the assessment of the pre-malignant potential of oral mucosal lesions. An analysis of the cytological specimens was undertaken with virtual microscopy which allowed for rapid and thorough analysis of the complete cytological specimen. 100 healthy individuals between 30 and 70 years of age, who were non-smokers, non-drinkers and not taking any medication, had cytological specimens collected from both the buccal mucosa and lateral margin of tongue to establish normal cytology parameters within a control population. Patients with a presumptive clinical diagnosis of lichen planus, leukoplakia or OSCC had lesional cytological samples taken prior to their diagnostic biopsy. Standardised thin preparations were prepared and each specimen stained by both Feuglen and Papanicolau methods. High speed scanning of the complete slide at 40X magnification was undertaken using the Aperio Scanscope TM and the green channel of the resultant image was analysed after threshold segmentation to isolate only nuclei and the integrated optical density of each nucleus taken as a gross measure of the DNA content (ploidy). Preliminary results reveal that ploidy assessment of oral cytology holds great promise as an adjunctive prognostic factor in the analysis of the malignant potential of oral mucosal lesions.

The intercalated disc of monkey myocardial cells and Purkinje fibers as revealed by scanning electron microscopy

The intercalated discs of working myocardium and Purkinje fibers of the monkey heart were examined by scanning and transmission electron microscopy. The NaOH/ultrasonication technique resulted in the digestion of connective tissue and a separation of the intercellular junctions of intercalated discs, such that these could be visualized three-dimensionally. The intercalated discs of ventricular myocytes, atrial myocytes and Purkinje fibers vary considerably in number and configuration, as do the intercalated discs of the three different layers of the ventricular myocardium. Myocytes in the subepicardial, middle and subendocardial layers of the ventricle have 1-3, 4-5 and 5-6 intercalated discs at the end of these cells, respectively, Those in the endocardial layer are characterized by the presence of small laterally-placed intercalated discs. Atrial myocytes and Purkinje fibers usually only have 1-2 intercalated discs, Individual intercalated discs in ventricular myocytes have complicated stairs with 10-30 steps and corresponding risers, while those of atrial myocytes and Purkinje fibers have simple stairs with 1-3 steps and risers, Steps equivalent to the plicate segments are characterized by densely-packed microplicae and finger-like microprojections which greatly increase surface area in vertricular myocytes, Microprojections in atrial myocytes and Purkinje fibers are sparse by comparison, Risers equivalent to the interplicate segments containing large gap junctional areas are most numerous in left ventricular myocytes, followed by right ventricular myocytes, Purkinje fibers and atrial myocytes in decreasing order. The geometric arrangement of the various types of myocytes may be related with impulse propagation. Large intercalated discs of cell trunks and series branches may participate in longitudinal propagation, while small laterally-placed ones may be the site of transverse propagation.

Study of direct immunofluorescence, immunofluorescence mapping and light microscopy in porphyria cutanea tarda

BACKGROUND: Even though porphyria cutanea tarda is the most frequent type of porphyria, there are few studies about its cutaneous physiopathology. OBJECTIVE: To evaluate skin changes in porphyria cutanea tarda using light microscopy and direct immunofluorescence before and after treatment with chloroquine. To perform antigen immunomapping of bullae to study their level of cleavage. METHODS: Light microscopy and direct immunofluorescence of 28 patients are reported in three different phases: 23 patients with active porphyria before treatment (Phase A), 7 patients with clinical remission during treatment (Phase B), and 8 patients with biochemical remission (Phase C). Immunomapping was performed on 7 patients. RESULTS: In active porphyria, direct immunofluorescence showed homogenous and intense fluorescence on the inside and on the walls of blood vessels as well as in the dermal-epidermal junction. In clinical remission (Phase B) and biochemical remission (Phase C), the deposit of immunoglobulins was maintained, but the deposit of complement was reduced in most cases. Immunomapping revealed no standard cleavage plane. CONCLUSION: No correlation was observed between clinical response and immunoglobulin deposits. The reduction of complement favors the hypothesis that activation of the complement cascade represents an additional pathway that leads to endothelial damage.

The ""single-section"" Golgi method adapted for formalin-fixed human brain and light microscopy

The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The ""single-section"" Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, ""sandwiched"" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the ""single-section"" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results. (C) 2010 Elsevier B.V. All rights reserved.

Plasma hormonal profiles and dendritic spine density and morphology in the hippocampal CA1 stratum radiatum, evidenced by light microscopy, of virgin and postpartum female rats

Successful reproduction requires that changes in plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), oxytocin (OT), estrogen (E-2) and progesterone (P-4) occur together with the display of maternal behaviors. Ovarian steroids and environmental stimuli can affect the dendritic spines in the rat hippocampus. Here, studying Wistar rats, it is described: (a) the sequential and concomitant changes in the hormonal profile of females at postpartum days (PP) 4, 8, 12, 16, 20 and 24, comparing to estrous cycle referential values; (b) the dendritic spine density in the stratum radiatum of CA1 (CA1-SR) Golgi-impregnated neurons in virgin females across the estrous cycle and in multiparous age-matched ones; and (c) the proportion of different types of spines in the CAI-SR of virgin and postpartum females, both in diestrus. Plasma levels of gonadotrophins and ovarian hormones remained low along PP while LH increased and PRL decreased near the end of the lactating period. The lowest dendritic spine density was found in virgin females in estrus when compared to diestrus and proestrus phases or to postpartum females in diestrus (p < 0.03). Other comparisons among groups were not statistically significant (p > 0.4). There were no differences in the proportions of the different spine types in nulliparous and postpartum females (p > 0.2). Results suggest that medium layer CA1-SR spines undergo rapid modifications in Wistar females across the estrous cycle (not quite comparable to Sprague-Dawley data or to hormonal substitutive therapy following ovariectomy), but persistent effects of motherhood on dendritic spine density and morphology were not found in this area. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Dorsal cutaneous branch of the ulnar nerve: a light and electron microscopy histometric study

This study describes the normal morphology and morphometry of the dorsal cutaneous branch of the ulnar nerve (DCBU) in humans. Fourteen nerves of eight donors were prepared by conventional techniques for paraffin and epoxy resin embedding. Semiautomatic morphometric analysis was performed by means of specific computer software. Histograms of the myelinated and unmyelinated fiber population and the G-ratio distribution of fibers were plotted. Myelinated fiber density per nerve varied from 5,910 to 10,166 fibers/mm(2), with an average of 8,170 +/- 393 fibers/mm(2). The distribution was bimodal with peaks at 4.0 and 9.5 mu m. Unmyelinated fiber density per nerve varied from 50,985 to 127,108, with an average of 78,474 +/- 6, 610 fibers/mm(2), with a unimodal distribution displaying a peak at 0.8 mu m. This study thus adds information about the fascicles and myelinated and unmyelinated fibers of DCBU nerves in normal people, which may be useful in further studies concerning ulnar nerve neuropathies, mainly leprosy neuropathy.