Publications by authors named "Raffaella Isola"

27 Publications

  • Page 1 of 1

The Role of Lipid Composition in the Sensory Attributes and Acceptability of the Salted and Dried Mullet Roes (Bottarga): A Study in Human and Animal Models.

Nutrients 2020 Nov 11;12(11). Epub 2020 Nov 11.

Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria, SS 554, km 4.5, Monserrato, 09042 Cagliari, Italy.

A taste component is implicated in the oro-sensory detection of dietary lipids and free fatty acids seem to be involved in fatty food recognition. Bottarga, the salted and semi-dried ovary product of mullet ( spp.), is a rich-fat food. A comparative sensory assessment of different commercial bottarga samples was performed in insect and human models in relation to their lipid composition. The bottarga attractant effect to was assessed by behavioral tests. The subjective odor and taste perception of bottarga samples was investigated in human determining the rate of pleasantness, familiarity, and intensity dimensions using the 7-points Likert-type scale. Bottarga samples showed similar lipid profiles, but differences emerged in total and free fatty acid levels. Significant differences were observed in the attractant effect/acceptability of samples to medflies, negatively correlated to their total and free fatty acids. Insect female exhibited the ability to select among bottarga samples based on their visual and olfactory properties. In the human model, a potential contribution of free fatty acid amount in the pleasantness and familiarity dimensions of taste of bottarga samples was evidenced. Women exhibited a greater ability than men to select bottarga samples based on their better olfactory perception. Our results increase the knowledge about this outstanding product with nutritional and nutraceutical properties.
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http://dx.doi.org/10.3390/nu12113454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697620PMC
November 2020

Suitability of the thawed algae for transmission electron microscopy study: Ultrastructural investigation on Coccomyxa melkonianii SCCA 048.

Microsc Res Tech 2020 Oct 23. Epub 2020 Oct 23.

Interdepartmental Center of Environmental Science and Engineering (CINSA), University of Cagliari, Cagliari, Italy.

Morphological and ultrastructural investigations are crucial for the identification and characterization of species such as microalgae, microorganisms that greatly change their morphology and physiology during their life cycle. Transmission electron microscopy (TEM) is an excellent tool for the ultrastructural observation of cells and their components. To date, limited ultrastructural studies have been carried out on microalgae, due to the difficulties in sample preparation. The aim of this work is to establish an appropriate fixation method that allows to better preserve the algal ultrastructure and test the suitability of the thawed algae for TEM observation. Fresh and thawed algae (Coccomyxa melkonianii SCCA 048) were fixed with different TEM fixation methods (a mix of glutaraldehyde and paraformaldehyde for several incubation times, sometimes preceded by a prefixation in cold methanol). The ultrastructural images obtained from fresh algae were compared to those obtained from frozen biomass. The best morphological results were achieved by fixing fresh algae in 1% paraformaldehyde and 1.25% glutaraldehyde for 5 hr. Pretreating with frozen methyl alcohol reduced fixation time to 2 hr. Both fresh and frozen algae ultrastructure were rather well preserved also with 1% paraformaldehyde and 1.25% glutaraldehyde for 2 hr. Ultrastuctural morphological images of the thawed algae demonstrated that also frozen samples can be used in TEM research, widening specimen suitability by means of this technique.
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http://dx.doi.org/10.1002/jemt.23626DOI Listing
October 2020

Neuroprotection by the Immunomodulatory Drug Pomalidomide in the LRRK2 Genetic Model of Parkinson's Disease.

Front Aging Neurosci 2020 13;12:31. Epub 2020 Feb 13.

Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy.

The search for new disease-modifying drugs for Parkinson's disease (PD) is a slow and highly expensive process, and the repurposing of drugs already approved for different medical indications is becoming a compelling alternative option for researchers. Genetic variables represent a predisposing factor to the disease and mutations in leucine-rich repeat kinase 2 (LRRK2) locus have been correlated to late-onset autosomal-dominant PD. The common fruit fly melanogaster carrying the mutation LRRK2 loss-of-function in the WD40 domain (LRRK2), is a simple model of PD and is a valid tool to first evaluate novel therapeutic approaches to the disease. Recent studies have suggested a neuroprotective activity of immunomodulatory agents in PD models. Here the immunomodulatory drug Pomalidomide (POM), a Thalidomide derivative, was examined in the LRRK2 genetic model of PD. Mutant and wild type flies received increasing POM doses (1, 0.5, 0.25 mM) through their diet from day 1 post eclosion, until postnatal day (PN) 7 or 14, when POM's actions were evaluated by quantifying changes in climbing behavior as a measure of motor performance, the number of brain dopaminergic neurons and T-bars, mitochondria integrity. LRRK2 flies displayed a spontaneous age-related impairment of climbing activity, and POM significantly and dose-dependently improved climbing performance both at PN 7 and PN 14. LRRK2 fly motor disability was underpinned by a progressive loss of dopaminergic neurons in posterior clusters of the protocerebrum, which are involved in the control of locomotion, by a low number of T-bars density in the presynaptic bouton active zones. POM treatment fully rescued the cell loss in all posterior clusters at PN 7 and PN 14 and significantly increased the T-bars density. Moreover, several damaged mitochondria with dilated cristae were observed in LRRK2 flies treated with vehicle but not following POM. This study demonstrates the neuroprotective activity of the immunomodulatory agent POM in a genetic model of PD. POM is an FDA-approved clinically available and well-tolerated drug used for the treatment of multiple myeloma. If further validated in mammalian models of PD, POM could rapidly be clinically tested in humans.
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http://dx.doi.org/10.3389/fnagi.2020.00031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031158PMC
February 2020

Tyrosine-hydroxylase, dopamine β-hydroxylase and choline acetyltransferase-like immunoreactive fibres in the human major sublingual gland.

Arch Oral Biol 2020 Jan 23;109:104571. Epub 2019 Sep 23.

Section of Cytomorphology, Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato CA, Italy. Electronic address:

Objective: To study the innervation of the major sublingual gland by means of immunohistochemistry.

Design: Bioptic and autoptic specimens of the major sublingual gland of humans were examined for the presence of immunoreactivity to tyrosine hydroxylase and dopamine-β-hydroxylase, on one hand, and choline acetyltransferase, on the other, to indicate adrenergic and cholinergic nerves, respectively.

Results: Acini and ducts were supplied by both divisions of the autonomic nervous system.

Conclusions: Mucous and seromucous cells of the human major sublingual glands may respond with secretion not only to parasympathetic activity but also to sympathetic activity. The major sublingual gland is therefore a potential contributor to the mucin secretion recently reported in the literature in response to high sympathetic activity during physical exercise.
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http://dx.doi.org/10.1016/j.archoralbio.2019.104571DOI Listing
January 2020

Melatonin release by exocytosis in the rat parotid gland.

J Anat 2019 03 10;234(3):338-345. Epub 2018 Dec 10.

Department of Biomedical Sciences, Division of Cytomorphology, University of Cagliari, Cagliari, Italy.

Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non-protein-bound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the β-adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg ), the right parotid gland was removed; pre-administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty-six per cent of the total granular population (per 100 μm per cell area) displayed melatonin labelling in the matrix; three-quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so-called regulated secretory pathway. During its stay in granules, anti-oxidant melatonin may protect their protein/peptide constituents from damage.
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http://dx.doi.org/10.1111/joa.12921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365479PMC
March 2019

Diabetic Status Influences the Storage of Melatonin in Human Salivary Glands.

Anat Rec (Hoboken) 2018 04 23;301(4):711-716. Epub 2017 Dec 23.

Department of Biomedical Sciences, University of Cagliari, Italy.

Recently we reported on the detailed localization of melatonin (and its receptors) in human salivary glands, revealing that serous cells are able to store and secrete melatonin into saliva. Since we found that type 2 diabetic patients display reduced melatonin content in saliva, our next step was to examine the presence of melatonin in salivary glands removed from type 2 diabetic subjects. The resulting data were compared with those previously obtained by identical procedures in non-diabetics, to establish if the diabetic status may affect melatonin distribution. Bioptic samples of diabetic parotid and submandibular glands were fixed, dehydrated, embedded in Epon Resin and processed to demonstrate melatonin reactivity by the immunogold staining method. The labeling density (expressed as the number of gold particles per μm /granule) and the percentage of melatonin-positive granules were assessed in diabetic samples. These values were compared with those in non-diabetic samples and differences were evaluated. In parotid and submandibular diabetic glands the reactivity for melatonin was specifically associated with secretory granules and small vesicles in serous cells. Melatonin reactivity was higher in parotid than in submandibular glands. Our data were in line with those obtained in our previous study on non-diabetic glands. Diabetic salivary glands showed a higher labeling density and a lower number of melatonin-positive granules compared to non-diabetic glands. Taken together, these data might explain the decreased salivary melatonin content and the associated oral problems observed in diabetics. Anat Rec, 301:711-716, 2018. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ar.23750DOI Listing
April 2018

Marked Differences in the Submandibular Salivary Proteome between Sardinian Alcohol-Preferring and Sardinian Alcohol-Non Preferring Rats Revealed by an Integrated Top-Down-Bottom-Up Proteomic Platform.

J Proteome Res 2018 01 16;17(1):455-469. Epub 2017 Nov 16.

Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Università Cattolica , Largo Francesco Vito 1 00168, Rome, Italy.

Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at the proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down-bottom-up integrated approach. For this purpose, submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. A total of 200 peptides and proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. The data are available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as those of several unidentified peptides and proteins. sP rats expressed some proteins not detectable in sNP rats such as the glutamine and glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. The submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference and consumption of sP and sNP rats is currently unknown and requires further investigation.
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http://dx.doi.org/10.1021/acs.jproteome.7b00632DOI Listing
January 2018

VGF Peptide Profiles in Type 2 Diabetic Patients' Plasma and in Obese Mice.

PLoS One 2015 12;10(11):e0142333. Epub 2015 Nov 12.

Department of Biomedical Sciences, University of Cagliari, 09042, Monserrato, Italy.

To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142333PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643017PMC
June 2016

Morphometric Study of Diabetes Related Alterations in Human Parotid Gland and Comparison with Submandibular Gland.

Anat Rec (Hoboken) 2015 Nov 31;298(11):1911-8. Epub 2015 Aug 31.

Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy.

Type 2 diabetes mellitus represents one of the principal diseases that afflict the world population and is often associated with malfunction of salivary glands and consequent oral diseases. We recently described significant ultrastructural alterations in the human submandibular gland in diabetic patients without evident oral pathologies. Herein, an analogs morphometrical investigation was focused on the parotid gland in order to evaluate if one of the two glands is more affected by diabetes. Parotid fragments from diabetic and nondiabetic patients were fixed, dehydrated, and processed for light and electron microscopy. Serous cells were randomly photographed and the density and size of several structures involved in the secretory process were examined by morphometry. Scanning electron microscopy images revealed significant changes in the number of apically docked granules and vesicles, suggesting that the last steps in exocytosis are somehow altered in diabetic cells. Other variables analyzed by light and transmission electron microscopy such as the size of acini and secretory granules did not show significant changes, but comparison with previous data obtained with submandibular gland cells demonstrated that the two glands are affected differently.
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http://dx.doi.org/10.1002/ar.23255DOI Listing
November 2015

A reliable model for gamma oscillations in hippocampal tissue.

J Neurosci Res 2015 Jul 24;93(7):1067-78. Epub 2015 Mar 24.

Institute of Physiology and Pathophysiology and Interdisciplinary Center for Neurosciences, University of Heidelberg, Heidelberg, Germany.

Gamma oscillations (30-100 Hz) reflect a fast brain rhythm that provides a fundamental mechanism of complex neuronal information processing in the hippocampus and in the neocortex in vivo. Gamma oscillations have been implicated in higher brain functions, such as sensory perception, motor activity, and memory formation. Experimental studies on synaptic transmission and bioenergetics underlying gamma oscillations have primarily used acute slices of the hippocampus. This study tests whether organotypic hippocampal slice cultures of the rat provide an alternative model for cortical gamma oscillations in vitro. Our findings are that 1) slice cultures feature well-preserved laminated architecture and neuronal morphology; 2) slice cultures of different maturation stages (7-28 days in vitro) reliably express gamma oscillations at about 40 Hz as induced by cholinergic (acetylcholine) or glutamatergic (kainate) receptor agonists; 3) the peak frequency of gamma oscillations depends on the temperature, with an increase of ∼ 3.5 Hz per degree Celsius for the range of 28-36 °C; 4) most slice cultures show persistent gamma oscillations for ∼ 1 hr during electrophysiological local field potential recordings, and later alterations may occur; and 5) in slice cultures, glucose at a concentration of 5 mM in the recording solution is sufficient to power gamma oscillations, and additional energy substrate supply with monocarboxylate metabolite lactate (2 mM) exclusively increases the peak frequency by ∼ 4 Hz. This study shows that organotypic hippocampal slice cultures provide a reliable model to study agonist-induced gamma oscillations at glucose levels near the physiological range.
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http://dx.doi.org/10.1002/jnr.23590DOI Listing
July 2015

Diabetes causes morphological changes in human submandibular gland: a morphometric study.

J Oral Pathol Med 2015 Apr 26;44(4):291-5. Epub 2014 Aug 26.

Department of Biomedical Sciences, University of Cagliari, Monserrato, CA, Italy.

Background: Dataon structural alterations in human diabetic salivary glands are scanty and conflicting. The goal of this study is based on the evaluation of the morphological changes in submandibular glands of subjects with well-controlled diabetes and without evident salivary malfunctions.

Methods: Submandibular gland pieces from diabetic and non-diabetic patients were fixed, dehydrated, and processed to obtain sections for light and electron microscopy. Randomly selected micrographs were statistically analyzed to reveal variations in serous acini.

Results: Morphometrical evaluation allowed us to reveal significant changes such as enlargement of acinar and granule size, reduction of mitochondrial size, increased density of microbuds and protrusions along luminal membranes.

Conclusions: The results indicate that diabetes affects submandibular gland structure even when glandular function appears unaltered and suggest that morphological changes reflect functional changes chiefly regarding the secretory activity.
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http://dx.doi.org/10.1111/jop.12238DOI Listing
April 2015

Animal models are reliably mimicking human diseases? A morphological study that compares animal with human NAFLD.

Microsc Res Tech 2014 Oct 17;77(10):790-6. Epub 2014 Jul 17.

Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, Cagliari, Italy.

Non-alcoholic fatty liver disease (NAFLD) is a clinical-pathological syndrome that includes a wide spectrum of morphological alterations. In research, animal models are crucial in evaluating not only the pathogenesis of NAFLD and its progression, but also the therapeutic effects of various agents. Investigations on the ultrastructural features of NAFLD in humans are not copious, due to the difficulty to obtain human samples and to the long time of NAFLD to evolve. Translational comparative studies on the reliability of animal models in representing the histopathologic picture as seen in humans are missing. To overcome this lack of investigations, we compared the ultrastructural NAFLD features of an animal model versus human. Sprague-Dawley rats were fed with a high fat diet (HFD) for 1-4 weeks, while control rats were fed with a standard diet. Human specimens were collected from patients with diagnosed fatty liver disease, undergoing liver biopsies or surgery. Rat and human samples were examined by light microscopy and by transmission and high resolution scanning electron microscopy. The present work demonstrated that NAFLD in animal model and in human, share overlapping ultrastructural features. In conclusion, animal HFD represent an appropriate tool in studying the pathogenesis of NAFLD.
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http://dx.doi.org/10.1002/jemt.22401DOI Listing
October 2014

Cortical adrenal mitochondrial morphology changes in functional state: new insights.

Cell Tissue Res 2013 Mar 14;351(3):409-17. Epub 2012 Dec 14.

Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, Cagliari, Italy.

We compared changes in the morphology of mitochondrial cristae with those in the blood and adrenal content of steroid hormones after the stimulation or inhibition of steroidogenesis. Rats were treated with adrenocorticotrophic hormone or angiotensin II to elicit steroidogenesis and with dexamethasone to inhibit it. Blood and adrenal glands were collected after several time intervals for measurements of steroids and their main intermediates. In the zona fasciculata, mitochondrial ultrastructure was investigated by high resolution scanning electron microscopy. We found that the morphometric data correlated well with the measurements of hyper- or hypo-activity of steroidogenesis over short periods of time (4 h) but not over longer observation times. A peculiar finding was that, contrary to previous reports, 11-deoxycortisol was present in adult rat adrenal tissue.
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http://dx.doi.org/10.1007/s00441-012-1533-2DOI Listing
March 2013

Morphological evidence that pentagastrin regulates secretion in the human parotid gland.

J Anat 2012 May 14;220(5):447-53. Epub 2012 Mar 14.

Department of Cytomorphology, University of Cagliari, Cagliari, Italy.

Salivary secretion is principally regulated by autonomic nerves. However, recent evidence from in vivo animal experiments suggests that gastrointestinal peptide hormones can also influence saliva production. The aim of the present study was to define the secretagogue activity of the gastrin-analogue pentagastrin in human salivary glands. For this purpose, parotid tissues were exposed to pentagastrin in vitro. Morphological techniques were used to evaluate modifications to serous acinar cells associated with secretion. Using a variant of the osmium maceration method, high resolution scanning electron microscopy allowed assessment of the morphology of the cytoplasmic aspect of the plasmalemma to demonstrate secretory activity. To quantify responses to pentagastrin, we recorded morphometric data on microvilli, microbuds, and protrusions. Dose-dependent morphological changes were observed, whereas protein concentration increased in the incubate. The use of selective receptor antagonists showed pentagastrin to act principally via cholecystokinin-A receptors. The morphological responses observed following exposure to pentagastrin differed from those elicited following exposure to the pan-muscarinic agonist carbachol. This study provides the first demonstration of a direct secretory action of gastrointestinal peptides on salivary glands in humans.
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http://dx.doi.org/10.1111/j.1469-7580.2012.01489.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403275PMC
May 2012

3-D structure of mitochondrial cristae in rat adrenal cortex varies after acute stimulation with ACTH and CRH.

Mitochondrion 2010 Aug 27;10(5):472-8. Epub 2010 May 27.

Department of Cytomorphology, University of Cagliari, Cittadella Universitaria di Monserrato, Cagliari, Italy.

We attempted to determine whether acute treatment with adrenocorticotropin hormone (ACTH) and corticotropin releasing hormone (CRH) affects mitochondrial morphology, as evaluated by the HRSEM and osmium maceration methods. We quantified CRH and ACTH effects on HRSEM images in rat glomerulosa and fasciculata. After ACTH or CRH treatment, mitochondrial cristae increased the number of globular expansions, whereas mitochondrial volume decreased in glomerulosa. As the morphological variations reported may be linked to increased hormonal production, further studies using parallel measurements of circulating and tissue hormones are now in progress, and may aid in clarifying their functional significance.
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http://dx.doi.org/10.1016/j.mito.2010.05.007DOI Listing
August 2010

Suggestive evidence of a vesicle-mediated mode of cell degranulation in chromaffin cells. A high-resolution scanning electron microscopy investigation.

J Anat 2010 Apr 28;216(4):518-24. Epub 2010 Jan 28.

Department of Medical and Morphological Research, Section of Anatomy, University of Udine, Udine, Italy.

In this study we used a modified osmium maceration method for high-resolution scanning electron microscopy to study some ultrastructural details fitting the schema of piecemeal degranulation in chromaffin cells. Piecemeal degranulation refers to a particulate pattern of cell secretion that is accomplished by vesicle-mediated extracellular transport of granule-stored material. We investigated adrenal samples from control and angiotensin II-treated rats, and identified a variable proportion of smooth, 30-60-nm-diameter vesicles in the cytoplasm of chromaffin cells. A percentage of these vesicles were interspersed in the cytosol among chromaffin granules but the majority appeared to be attached to granules. Remarkably, the number of unattached cytoplasmic vesicles was greatly increased in chromaffin cells from angiotensin II-treated animals. Vesicles of the same structure and dimension were detected close to or attached to the cytoplasmic face of the plasma membrane; these, too, were increased in number in chromaffin cells from rats stimulated with angiotensin II. In specimens shaken with a rotating agitator during maceration, the cytoplasmic organelles could be partially removed and the fine structure of the vesicular interaction with the inner side of the plasma membrane emerged most clearly. A proportion of chromaffin granules showed protrusions that we interpreted as vesicular structures budding from the granular envelope. In some instances, the transection plane intersected granules with putative vesicles emerging from the surfaces. In these cases, the protrusions of budding vesicles could be observed from the internal side. This study provides high-resolution scanning electron microscopy images compatible with a vesicle-mediated degranulation mode of cell secretion in adrenal chromaffin cells. The data indicating an increase in the number of vesicles observed in chromaffin cells after stimulation with the chromaffin cell secretagogue angiotensin II suggests that this secretory process may be susceptible to fine regulation.
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http://dx.doi.org/10.1111/j.1469-7580.2009.01198.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849529PMC
April 2010

Mitochondria of human Leydig cells as seen by high resolution scanning electron microscopy.

Arch Histol Cytol 2010 ;73(1):37-44

Department of Cytomorphology, School of Medicine, University of Cagliari, Italy.

The three-dimensional ultrastructure of over 1000 mitochondria in human Leydig cells (from twelve sexually mature patients) was examined by high resolution scanning electron microscopy (HRSEM) of osmium-macerated specimens, as well as by transmission electron microscopy of conventional ultrathin sections. The stereo-pair imaging of the osmium-macerated specimens by HRSEM is also very useful for investigating the three-dimensional structure of cytoplasmic membranous organelles with great clarity. The mitochondria, which mainly are elongated (although some are ovate), possess cristae that are almost exclusively tubular and that occasionally display constrictions and terminal bulbules. Lamelliform cristae are quite rare. Occasionally, the tubular cristae are joined together to form a simple network. Classic crista junctions could not be identified with certainty, although the base of the tubular cristae might correspond functionally to such junctions. As a whole, in line with the identical and common embryological origin of adrenal cortex and gonads, mitochondria of human Leydig cell closely resemble those of steroidogenic cells of human suprarenal cortex treated by the same maceration method.
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http://dx.doi.org/10.1679/aohc.73.37DOI Listing
August 2011

Oxytocin immunoreactivity in the human urethral (Littrè's) glands.

J Reprod Dev 2010 Feb 5;56(1):94-7. Epub 2009 Nov 5.

Department of Cytomorphology, University of Cagliari.

Oxytocin is a cyclic nonapeptide whose best known effects are stimulation of uterine smooth muscle cells during labor and of milk ejection during lactation. Circulating oxytocin originates from the hypothalamus, but its production has also been documented in peripheral tissues. Furthermore, seminal plasma also contains oxytocin, but its functional role is still unknown, although its secretion is generally ascribed to the prostate. In this study, we investigated the possibility that seminal oxytocin is also secreted by other exocrine glands of the human male genital tract. Intramural (Littrè's) glands isolated from bioptic specimens of normal urethrae were processed for immunogold localization of oxytocin. Immunostaining was detected in principal cells, with gold particles specifically found on secretory granules. Basal and endocrine cells were unstained. The present findings suggest that urethral glands not only produce the mucinous layer that protects and lubricates the urethral wall, but also are potential sources of other seminal components, such as oxytocin, which probably play still unclear roles in reproductive physiology.
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http://dx.doi.org/10.1262/jrd.09-063eDOI Listing
February 2010

The three-dimensional morphology of Candida albicans as seen by high-resolution scanning electron microscopy.

J Microbiol 2009 Jun 26;47(3):260-4. Epub 2009 Jun 26.

Department of Cytomorphology, Cittadella Universitaria di Monserrato, University of Cagliari, Monserrato 09042, Italy.

The fine structure of Candida albicans has been repeatedly described by transmission electron microscopy, whereas studies by high-resolution scanning electron microscopy (HRSEM) are rare and devoted solely to the study of its external morphology. This report describes the results of an HRSEM study on C. albicans carried out by an osmium maceration protocol modified to better retain the structural characteristics of this yeast. Thus, we visualized various intracellular structures including invaginations of cell membrane (plasmalemmasomes), nuclear envelope, mitochondria, the vacuolar system, and two additional structures that might represent a form of endoplasmic reticulum and the Golgi apparatus. The present investigation, which for the first time shows the organelles of C. albicans at the 3D level, may lead to a better understanding of its cell physiology.
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http://dx.doi.org/10.1007/s12275-008-0212-1DOI Listing
June 2009

Acute nicotine changes dynorphin and prodynorphin mRNA in the striatum.

Psychopharmacology (Berl) 2009 Jan 21;201(4):507-16. Epub 2008 Sep 21.

Department of Psychiatry, College of Medicine, Ohio State University, Columbus, OH 43210, USA.

Rationale: Nicotine displays rewarding and aversive effects, and while dopamine has been linked with nicotine's reward, the neurotransmitter(s) involved with aversion remains speculative. The kappa-dynorphinergic system has been associated with negative motivational and affective states, and whether dynorphin (Dyn) contributes to the behavioral pharmacology of nicotine is a pertinent question.

Objective: We determined whether administration of a single dose of nicotine alters the biosynthesis of Dyn in the striatum of mice.

Results: Nicotine free base, 1 mg/kg, sc, induced a biphasic, protracted increase of striatal Dyn, an initial rise by 1 h, which declined to control levels by 2 h, and a subsequent increase, between 6 and 12 h, lasting over 24 h. At 1 h, the nicotine effect was dose dependent, with doses>or=0.5 mg/kg inducing a response. Prodynorphin mRNA increased by 30 min for over 24 h, and in situ hybridization demonstrated elevated signal in caudate/putamen and nucleus accumbens. The nicotinic antagonist mecamylamine prevented the Dyn response, and a similar effect was observed with D1- and D2-like dopamine receptor antagonists, SCH 23390, sulpiride, and haloperidol. The glutamate NMDA receptor antagonist MK-801 reversed the nicotine-induced increase of Dyn, while the AMPA antagonist NBQX had a marginal effect.

Conclusions: We interpret our findings to indicate that acute nicotine enhances the synthesis and release of striatal Dyn. We propose that nicotine influences Dyn primarily through dopamine release and that glutamate plays a modulatory role. A heightened dynorphinergic tone may contribute to the aversive effects of nicotine in naive animals and first-time tobacco smokers.
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http://dx.doi.org/10.1007/s00213-008-1315-4DOI Listing
January 2009

Dynorphin and prodynorphin mRNA changes in the striatum during nicotine withdrawal.

Synapse 2008 Jun;62(6):448-55

Department of Psychiatry, College of Medicine, Ohio State University, Columbus, Ohio 43210, USA.

Nicotine withdrawal causes somatic and negative affective symptoms that contribute to relapse and continued tobacco smoking. So far, the neuronal substrates involved are not fully understood, and an opioid role has been suggested. In this regard, the opioid dynorphin (Dyn) is of interest as it produces aversive states and has been speculated to play a role in the nicotine behavioral syndrome. These studies explore whether Dyn metabolism is altered during withdrawal following chronic administration of nicotine. Mice were administered nicotine, 2 mg/kg, s.c., four times daily for 14 days, and Dyn and prodynorphin (PD) mRNA estimated in selective brain regions at various times (30 min to 96 h) following drug discontinuation. The content of Dyn, estimated by RIA, was decreased in the striatum for a protracted time, from 30 min to over 72 h. In contrast, the mRNA for PD, evaluated by Northern blot, was elevated, appearing by 8 h and lasting over 96 h. Dyn was decreased in both ventral and dorsal striatum, and PD mRNA was differentially increased in the two striatal compartments as demonstrated by in situ hybridization. PD message was predominantly augmented in the nucleus accumbens, rostral pole, core, and shell, and the medial aspects of caudate/putamen. We interpret these data to indicate increased activity of striatal, particularly accumbal, dynorphinergic neurons during nicotine withdrawal resulting in enhanced peptide release and compensatory synthesis. Heightened dynorphinergic tone might be responsible, in part, for the emergence of the negative affective states observed during nicotine withdrawal.
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http://dx.doi.org/10.1002/syn.20515DOI Listing
June 2008

Intracellular distribution of fluorescent probes delivered by vesicles of different lipidic composition.

Colloids Surf B Biointerfaces 2007 Jun 6;57(2):143-51. Epub 2007 Feb 6.

Dipartimento Farmaco Chimico Tecnologico, Università di Cagliari, Via Ospedale 72, Cagliari, Italy.

In order to study mechanisms involved in liposome-cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle-cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker.
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http://dx.doi.org/10.1016/j.colsurfb.2007.01.016DOI Listing
June 2007

Histatin-induced alterations in Candida albicans: a microscopic and submicroscopic comparison.

Microsc Res Tech 2007 Jul;70(7):607-16

Department of Cytomorphology, University of Cagliari, Cittadella Universitaria di Monserrato, Monserrato (CA), Italy.

Despite the numerous studies performed in an attempt to clarify the issue, the mechanism of action of salivary histatins remains unclear. The aim of the present study was to correlate histatin-induced morphological changes in Candida albicans by fluorescence microscopy (FM), transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM). Each of the fluorescent dyes used by FM (i.e., tetramethylrhodamine methyl ester perchlorate for mitochondrial potential, Lysotracker for lysosome acidic compartment, and 4',6-diamino-2-phenylindole dihydrochloride for DNA) exhibited a specific staining in control cells. Following histatin treatment, we observed a recurring staining pattern, corresponding to fluorescence concentration along the cell periphery, suggesting a loss of dye specificity. To assess histatin-induced cytoplasmic modifications, ultrastructural analysis was then carried out. After treatments with histatins, TEM revealed characteristic intracellular modifications including: vacuole overgrowth, nuclear disappearance, loss of organelle identity, as well as the appearance of electron-dense membranes, likely of mitochondrial origin. Additionally, structures resembling autophagosomes were occasionally observed. By HRSEM, mitochondrial swelling was invariably the first sign of a histatin-induced effect. Other modifications included intracellular membrane disarrangement, organelles in disarray, and a large central cavity with deformed bodies displaced to the cell periphery, similar to what was detected by TEM. In summary, our study illustrates the occurrence of ultrastructural modifications following administration of histatins. Observations made with FM, TEM, and HRSEM provided different views of the same signs, demonstrating a definite action of histatins on C. albicans morphology. The possible functional meanings of these morphological results is discussed in light of the most recent biochemical data on histatin fungicidal activity.
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http://dx.doi.org/10.1002/jemt.20441DOI Listing
July 2007

Mitochondrial alterations and autofluorescent conversion of Candida albicans induced by histatins.

Microsc Res Tech 2005 Apr;66(5):219-28

Dipartimenti di Citomorfologia, Università di Cagliari, 09042 Monserrato (Cagliari), Italy.

The mechanism of the candidacidal activity of histatins 3 and 5 (Hst) is still a matter of debate. Previous studies have indicated that Hst induce cell permeabilization, generation of reactive oxygen species (ROS) by mitochondria, inhibition of the respiratory chain, and energy-dependent cytotoxic release of ATP. On the other hand, the multiplicity of effects and the apparent contrast between experimental data continue to render the mechanism of Hst-induced killing of C. albicans unclear. In this investigation, using fluorescent probes (the potential-sensitive mitochondrial probe tetramethylrhodamine methyl ester perchlorate, TMRM; the ROS-sensitive probe dihydrofluorescein diacetate, DHF; the membrane-impermeant probe, calcein) and autofluorescence data we observed that Hst induce ROS generation by mitochondria undergoing a high energy swelling condition, accompanied by oxidation of cytosolic NAD(P)H and mitochondrial flavoproteins. ROS generation and swelling, attributable to an inhibition of the respiratory chain and to impairment of the K/H-exchanger, were followed by mitochondrial depolarization. Mitochondrial changes were accompanied by massive calcein influx, indicative of cell permeabilization, and prominent alterations of the cell size, shape, and optical density. The loss of proliferative activity was correlated, on a single cell basis, to the acquisition of a lipofuscin-like autofluorescence.
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http://dx.doi.org/10.1002/jemt.20161DOI Listing
April 2005

Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivation.

FEBS J 2005 Apr;272(7):1649-59

Department of Cytomorphology, University of Cagliari, Monserrato, Italy.

Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were analyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochondrial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase). Succinic acid (mitochondrial complex II substrate, given in the permeable form of dimethyl ester) abolished the rotenone-induced inhibition of R phases. Taken together, these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions, resulting in a progressive depolarization of mitochondria accompanied by NAD(P)H oxidation and Ca2+ influx. D phases were not blocked by cyclosporin A (inhibitor of the permeability transition pore) or o-phenyl-EGTA (a Ca2+ chelator), suggesting that the permeability transition pore was not involved in mitochondrial potential fluctuations.
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http://dx.doi.org/10.1111/j.1742-4658.2005.04586.xDOI Listing
April 2005

Mitochondrial localization of reactive oxygen species by dihydrofluorescein probes.

Histochem Cell Biol 2003 Oct 20;120(4):319-25. Epub 2003 Sep 20.

Department of Cytomorphology, University of Cagliari, Cittadella Universitaria Monserrato, 09042, Monserrato (CA), Italy.

Mitochondria are the main source of reactive oxygen species (ROS). The aim of this work was to verify the ROS generation in situ in HeLa cells exposed to prooxidants and antioxidants (menadione, tert-butyl hydroperoxide, antimycin A, vitamin E, N-acetyl-L-cysteine, and butylated hydroxytoluene) using the ROS-sensitive probes 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate di-acetomethyl ester (DCDHF) and dihydrofluorescein diacetate (DHF). Mitochondria were counterstained with the potential-sensitive probe tetramethylrhodamine methyl ester perchlorate (TMRM). Both DCDHF and DHF were able to detect the presence of ROS in mitochondria, though with distinct morphological features. DCDHF fluorescence was invariably blurred, smudged, and spread over the cytoplasm surrounding the major mitochondrial clusters. On the contrary, DHF fluorescence was sharp and delineated thin filaments which corresponded in all details to TMRM-stained mitochondria. These data suggest that DCDHF does not reach the mitochondrial matrix but is oxidized by ROS released by mitochondria in the cytosol. On the other hand, DHF enters mitochondria and reacts with ROS released in the matrix. Cytosolic (DCDHF+) ROS but not matrix (DHF+) ROS, were significantly decreased by vitamin E. N-acetyl-L-cysteine was effective in reducing DCDHF and DHF photooxidation in the medium, but was unable to reduce intracellular ROS. ROS generation was accompanied by partial mitochondrial depolarization.
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http://dx.doi.org/10.1007/s00418-003-0566-8DOI Listing
October 2003

Met-enkephalin and preproenkephalin mRNA changes in the striatum of the nicotine abstinence mouse.

Neurosci Lett 2002 May;325(1):67-71

Department of Psychiatry, The Ohio State University School of Medicine and Public Health, 5034 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210, USA.

We studied the changes of met-enkephalin (Met-Enk) content and preproenkephalin (PPE) mRNA in the striatum in a mouse model of nicotine abstinence. Nicotine, 2 mg/kg, s.c., was administered four times daily for 14 days and Met-Enk and PPE mRNA evaluated at various times (4-96 h) following drug discontinuation. Met-Enk, assayed by radioimmunoassay, was increased in the ventral (nucleus accumbens) but not dorsal (putamen/caudate) striatum, while PPE mRNA, assayed in whole striatum by Northern blotting was elevated. Both changes were seen early during withdrawal and lasted over 72 h. In situ hybridization revealed enhanced signal in the dorsal striatum, mostly laterally, and smaller increases in the rostral pole, core and shell of the nucleus accumbens. These observations indicate that during nicotine withdrawal, striatal enkephalinergic neurons undergo adaptative responses, which might contribute to the abstinence behavioral syndrome.
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http://dx.doi.org/10.1016/s0304-3940(02)00240-9DOI Listing
May 2002