Mariano Acosta

Folliculostellate cells in pituitary pars distalis of male viscacha: immunohistochemical, morphometric and ultrastructural study

Folliculostellate cells (FSC) have been reported in pituitary of several mammalian species. FSC morphology and secreted substances have been instrumental to the understanding of their function. The purpose of this work was to perform an immunohistochemical, morphometric and ultrastructural study of the pituitary pars distalis FSC in adult male viscacha and to analyze their relation with hormone secreting cells. Immunohistochemistry and image analysis were carried out in different sectors of the gland, from the middle (sector 1) to the glandular periphery (sector 5). Transmission electron microscopy with lanthanum as electrodense tracer was used. FSC formed follicles with PAS-positive colloid inside. They expressed S-100 protein mainly in both nucleus and cytoplasm. FSC were stellate-like in shape and exhibited short cytoplasmic processes that contacted with blood vessels and endocrine cells. In addition, some follicular colloids were immunostained with anti-S-100 protein. A few FSC were immunostained with anti-glial fibrillary acidic protein (GFAP) and anti-vimentin. The morphometric parameters analyzed (percentages of S-100-positive total, cellular and colloidal areas) increased from sector 1 to sector 3 and then decreased to sector 5. Hormone secreting cells, mainly lactotrophs, gonadotrophs and corticotrophs were associated with FSC and follicles. The ultrastructural study demonstrated that FSC developed junctional complexes and desmosomes between their lateral membranes. Lanthanum freely penetrated the spaces between granulated cells and FSC, but did not penetrate into the follicular lumen. In conclusion: 1) the differential expression of S-100 protein, GFAP and vimentin may indicate different physiological stages of FSC; 2) the expression of these proteins suggests a neuroectodermic origin of these cells; 3) FSC spatial distribution, association with endocrine cells, and the generation of an intercellular communication network suggest that FSC are involved in the pituitary pars distalis paracrine regulation of the viscacha.

Sequential determination of lead and cobalt in tap water and foods samples by fluorescence

In this work, a new procedure was developed for the separation and preconcentration of lead(II) and cobalt(II) in several water and foods samples. Complexes of metal ions with 8-hydroxyquinolein (8-HQ) were formed in aqueous solution. The proposed methodology is based on the preconcentration/separation of Pb(II) by solid-phase extraction using paper filter, followed by spectrofluorimetric determination of both metals, on the solid support and the filtered aqueous solution, respectively. The solid surface fluorescence determination was carried out at λem=455 nm (λex=385 nm) for Pb(II)-8-HQ complex and the fluorescence of Co(II)-8-HQ was determined in aqueous solution using λem=355 nm (λex=225 nm). The calibration graphs are linear in the range 0.14-8.03×10(4) μg L(-1) and 7.3×10(-2)-4.12×10(3) μg L(-1), for Pb(II) and Co(II), respectively, with a detection limit of 4.3×10(-2) and 2.19×10(-2) μg L(-1) (S/N=3). The developed methodology showed good sensitivity and adequate selectivity and it was successfully applied to the determination of trace amounts of lead and cobalt in tap waters belonging of different regions of Argentina and foods samples (milk powder, express coffee, cocoa powder) with satisfactory results. The new methodology was validated by electrothermal atomic absorption spectroscopy with adequate agreement. The proposed methodology represents a novel application of fluorescence to Pb(II) and Co(II) quantification with sensitivity and accuracy similar to atomic spectroscopies.

Protective effect of soybeans as protein source in the diet against cadmium-aorta redox and morphological alteration

We investigated the effects of cadmium exposition on thoracic aorta redox status and morphology, and the putative protective effect of soybeans in the diet. Male Wistar rats were separated into 6 groups: 3 fed with a diet containing casein and 3 containing soybeans, as protein source. Within each protein group, one was given tap water (control) and the other two tap water containing 15 and 100 ppm of Cd(2+), respectively, for two months. In rats fed with casein diet, 15 ppm of Cd induced an increase of thiobarbituric acid-reactive substances (TBARS), and of the catalase (CAT) and glutathione peroxidase (GPx) activities, which were even higher with 100 ppm of Cd(2+), in aorta. Also, 100 ppm Cd(2+) exposure increased superoxide dismutase (CuZnSOD) activity; CAT, GPX, SOD, Nrf2 and metallothioneine II mRNA expressions and CAT, GPx and NOX-2 protein levels, compared with control. Aorta endothelial and cytoplasmic alterations were observed. However, with the soybeans diet, 15 and 100 ppm of Cd(2+) did not modify TBARS levels; CAT, GPX and Nrf2 mRNA expressions; CAT, GPx and NOX-2 protein; and the aorta morphology, compared with control. The soybean diet attenuates the redox changes and protects against morphological alterations induced, in a dose-dependent way, by Cd in aorta.

Ultratrace arsenic determination through hydride trapping on oxidized multiwall carbon nanotubes coupled to electrothermal atomic absorption spectrometry

Arsenic determination in natural waters is an issue of current research. This article reports a novel hydride generation (HG) approach developed for As determination with electrothermal atomic absorption spectrometry (ETAAS) detection. The HG process was interfaced with ETAAS through hydride trapping onto a carbon nanotubes microcolumn. To this end a homemade gas–liquid separator was used, allowing arsine formation and its flow throughout the CNT microcolumn. The retention process involved thus a solid phase extraction from the gas phase to the solid support. Once arsine generation was completed, the elution was carried out with nitric acid directly onto the dosing hole of the graphite furnace. Outstanding sensitivity with detection limit of 1 ng L−1, quantification limit of 5 ng L−1 and the characteristic mass, 5.8 ± 0.4 pg could be achieved. A satisfactory correlation between concentration of As and absorbance (R = 0.9993) from the limit of quantification up to 500 ng L−1, with a relative standard deviation of 6.3% were obtained. A sensitive enhancement factor of 38 was reached when 2 mL of sample were processed and 50 μL of HNO3 were used as eluent. The system was successfully applied to the analysis of a standard reference material, QC LL2 metals in natural waters. In addition tap water analysis provided an As concentration of 0.29 ± 0.03 μg L−1.

Effect of the photoperiod and administration of melatonin on folliculostellate cells of the pituitary pars distalis of adult male viscacha (Lagostomus maximus maximus)

Numerous reports have shown the effect of photoperiod and melatonin administration on the different hormone secreting cell types in the pituitary pars distalis. The viscacha (Lagostomus maximus maximus) is a rodent with photoperiod-dependent seasonal reproduction. The aim of this study was to examine the effect of photoperiod seasonal variations and melatonin administration on the folliculostellate cells in pituitary pars distalis of viscacha. Immunohistochemistry and image analysis were used to measure the percentage of S-100-positive area (total, cellular and colloidal) and the number of folliculostellate cells. The S-100 protein was immunolocalized at intracellular (folliculostellate cells) and extracellular (follicular colloid) levels. The morphometric parameters analyzed exhibited seasonal variations with highest values in the summer (long photoperiod) and lowest values in the winter (short photoperiod). The administration of melatonin caused a significant decrease of immunostaining. Results suggest that the natural photoperiod might be the most important environmental signal causing the decrease in folliculostellate cells immunostaining observed in the winter. These findings agree with seasonal changes previously reported in endocrine cells and suggest that folliculostellate cells may be involved in the paracrine regulation of the secretory activity of pituitary pars distalis through S-100 protein production.

Cellular associations of pituitary gonadotrophs in a rodent (Lagostomus maximus maximus) with photoperiod-dependent reproduction.

The morphological characteristics and percentage of the cellular associations between gonadotrophs (LH- and FSH-secreting cells) and other cellular types were studied in pituitary pars distalis of adult male viscachas (Lagostomus maximus maximus) by double immunohistochemistry using specific antibodies to LH, FSH, PRL, GH, ACTH, TSH and S-100 protein (by folliculostellate cells; FSC), during long and short photoperiods. Bihormonal gonadotrophs were observed in ventro-medial and dorsal regions, interspersed between monohormonal gonadotrophs, and their number increased in short photoperiod. LH- and FSH-gonadotrophs were found around lactotrophs, enclosed by somatotrophs in the dorsal region, and associated with irregular corticotrophs. Gonadotrophs and thyrotrophs were associated along blood vessels and follicular structures. The cytoplasmic prolongations of FSC were in contact with both gonadotrophs. The percentage of LH-FSH, LH-ACTH, LH-FSC, FSH-LH, FSH-PRL, FSH-GH, FSH-ACTH, FSH-TSH and FSH-FSC associations decreased, whereas LH-PRL increased in short as compared to long photoperiod. The most abundant associations were LH-GH and LH-TSH during long photoperiod, but LH-GH and LH-PRL during short photoperiod. FSH-GH and FSH-PRL were the most numerous associations, and LH-FSC and FSH-FSC were the less abundant ones in both photoperiods. These results provide the morphological evidence for specific cellular associations between gonadotrophs and other cellular types of viscacha pituitary.

Caffeine monitoring in biological fluids by solid surface fluorescence using membranes modified with nanotubes.

BACKGROUND In this work, a new methodology based upon enhancement of rhodamine B fluorescent signal is proposed for the quantification of caffeine traces. METHODS Membrane filters treated with multiple wall carbon nanotubes were employed as solid support for determination step by solid surface fluorescence. RESULTS Experimental variables that influence the preconcentration step and fluorimetric sensitivity have been optimized using uni-variation assays, presenting linearity from 1.1 to 9.7×10(3) μg/l, with a correlation coefficient of 0.99. At optimal conditions, a limit of detection of 0.3 μg/l and a limit of quantification of 1.1 μg/l were obtained. The method showed good sensitivity and adequate selectivity and was satisfactorily applied to the determination of trace amounts of caffeine in urine, plasma and serum belonging to subjects with different sex, ages and habit of caffeine intake. CONCLUSIONS Chemofiltration step eliminated the highly fluorescent matrix, thus enabling and allowing CF quantification, in the presence of other methylxanthines. The proposed methodology represents an innovative application of the solid surface fluorescence using membrane filters modified with MWCNTs.

Pituitary pars intermedia of male viscacha (Lagostomus maximus maximus): a morphometric study of seasonal and age-related changes in immunohistochemistry.

The aim of this work was to study the pituitary pars intermedia in seasonally captured adult male and immature viscachas by immunohistochemistry and image analysis. The pituitary pars intermedia exhibited a well-developed parenchyma with scarce connective tissue and vascularization. It was formed by a close association of melanotrophs and folliculostellate cells. The folliculostellate cells were stellate in shape with cytoplasmic processes, and they originated follicles with PAS-positive colloid inside. The morphometric parameters of melanotrophs, follicular colloid and folliculostellate cells (S-100-ir and GFAP-ir) varied seasonally and in relation to age. These parameters showed minimal values in the adult males captured in winter and in immature animals, and they were maximal in summer. The percentage of vimentin-positive area of the folliculostellate cells was maximal in immature animals, decreased in relation to age and did not vary seasonally in the adult animals. The greatest development of pars intermedia in the adult animals in relation to the immature ones is probably related to the adults’ adaptation to the semiarid environment. The expression of the tested proteins suggests a probable neuroectodermic origin for the folliculostellate cells of the viscacha pituitary pars intermedia. In addition, the cytoplasmic processes of folliculostellate cells might originate an intercellular communication network inside the pars intermedia. The decrease in the morphometric parameters melanotrophs, follicular colloid and folliculostellate cells in winter suggests a low endocrine activity of this zone. This fact might be due to the effect of the short photoperiod and high melatonin serum levels.

Sequential determination of nickel and cadmium in tobacco, molasses and refill solutions for e-cigarettes samples by molecular fluorescence

In this work, a new procedure was developed for separation and preconcentration of nickel(II) and cadmium(II) in several and varied tobacco samples. Tobacco samples were selected considering the main products consumed by segments of the population, in particular the age (youth) and lifestyle of the consumer. To guarantee representative samples, a randomized strategy of sampling was used. In the first step, a chemofiltration on nylon membrane is carried out employing eosin (Eo) and carbon nanotubes dispersed in sodium dodecylsulfate (SDS) solution (phosphate buffer pH 7). In this condition, Ni(II) was selectively retained on the solid support. After that, the filtrate liquid with Cd(II) was re-conditioned with acetic acid /acetate buffer solution (pH 5) and followed by detection. A spectrofluorimetric determination of both metals was carried out, on the solid support and the filtered aqueous solution, for Ni(II) and Cd(II), respectively. The solid surface fluorescence (SSF) determination was performed at λem = 545nm (λex = 515nm) for Ni(II)-Eo complex and the fluorescence of Cd(II)-Eo was quantified in aqueous solution using λem = 565nm (λex = 540nm). The calibration graphs resulted linear in a range of 0.058-29.35μgL-1 for Ni(II) and 0.124-56.20μgL-1 for Cd(II), with detection limits of 0.019 and 0.041μgL-1 (S/N = 3). The developed methodology shows good sensitivity and adequate selectivity, and it was successfully applied to the determination of trace amounts of nickel and cadmium present in tobacco samples (refill solutions for e-cigarettes, snuff used in narguille (molasses) and traditional tobacco) with satisfactory results. The new methodology was validated by ICP-MS with adequate agreement. The proposed methodology represents a novel fluorescence application to Ni(II) and Cd(II) quantification with sensitivity and accuracy similar to atomic spectroscopies, introducing for the first time the quenching effect on SSF.

New solid surface fluorescence methodology for lead traces determination using rhodamine B as fluorophore and coacervation scheme: Application to lead quantification in e-cigarette refill liquids.

A new environmental friendly methodology based on fluorescent signal enhancement of rhodamine B dye is proposed for Pb(II) traces quantification using a preconcentration step based on the coacervation phenomenon. A cationic surfactant (cetyltrimethylammonium bromide, CTAB) and potassium iodine were chosen for this aim. The coacervate phase was collected on a filter paper disk and the solid surface fluorescence signal was determined in a spectrofluorometer. Experimental variables that influence on preconcentration step and fluorimetric sensitivity have been optimized using uni-variation assays. The calibration graph using zero th order regression was linear from 7.4×10(-4) to 3.4 μg L(-1) with a correlation coefficient of 0.999. Under the optimal conditions, a limit of detection of 2.2×10(-4) μg L(-1) and a limit of quantification of 7.4×10(-4) μg L(-1) were obtained. The method showed good sensitivity, adequate selectivity with good tolerance to foreign ions, and was applied to the determination of trace amounts of Pb(II) in refill solutions for e-cigarettes with satisfactory results validated by ICP-MS. The proposed method represents an innovative application of coacervation processes and of paper filters to solid surface fluorescence methodology.