Raffaella Isola

Homogeneous longitudinal profiles and synchronous fluctuations of mitochondrial transmembrane potential.

This study reports for the first time (a) the longitudinal profile of the transmembrane potential (mΔψ) of single mitochondria using a Nernstian fluorescent probe and (b) the distribution of mΔψ fluctuations of mitochondria undergoing permanent depolarization. Our findings show that (1) mitochondria in different energetic conditions coexist in the same cell, (2) mΔψ is rather homogeneous along the entire length of single mitochondria, (3) mΔψ is not influenced by the surrounding cytoplasmic environment and (4) mΔψ fluctuations occur simultaneously in groups of mitochondria connected in a network. Taken together, these findings provide further evidence for a functional relationship between mitochondrial arrangement and energetic condition.

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

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.

Intra- and Intercellular Distribution of Mitochondrial Probes and Changes after Treatment with MDR Modulators

Fluorescent probes are currently used to evaluate the mitochondrial transmembrane potential in situ. However, in parallel experiments using the probes JC‐1 and TMRM in different cell types (human astrocytes, HEp‐2, Vero, KB, and HeLa cells), we found that the distribution of JC‐1 and TMRM is highly variable not only in different cell types but also in different cells of the same cell type, a condition that has never been documented until our work. This phenomenon depends on a hidden, widespread multidrug resistance (MDR) phenotype that can be recognized only by comparative assays with MDR inhibitors (progesterone, verapamil, and cyclosporin A) and represents a serious risk of error in the evaluation of the mitochondrial potential.

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

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.

Morphological evidence that pentagastrin regulates secretion in the human parotid gland

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.

Diabetes reduces statherin in human parotid: immunogold study and comparison with submandibular gland.

BACKGROUND AND OBJECTIVE Alteration of salivary gland secretion is one of the consequences of diabetes. In a recent study on the submandibular gland of diabetic subjects, we found changed expression of statherin, a salivary protein of fundamental importance in preserving tooth integrity, whose reduction was related with the high incidence of oral diseases in patients with diabetes. The goal of this report is to extend the study to human parotid gland and to compare the effects of diabetes on statherin expression with those previously described in submandibular gland. MATERIALS AND METHODS Fragments of parotid glands obtained from diabetic and non-diabetic patients were fixed, dehydrated, embedded in Epon Resin and processed for the immunogold histochemistry. The staining density was expressed as number of gold particles per μm(2) and statistically evaluated. RESULTS AND CONCLUSIONS In all samples, statherin reactivity was specifically localized in secretory granules of acinar cells. The statistical analysis showed that labelling density was significantly lower in diabetic than in non-diabetic parotid glands and that diabetes affects protein expression at identical extent in parotid and submandibular glands. The results strengthen the hypothesis that a reduced statherin secretion may be responsible for the higher incidence of oral disorders in diabetic subjects.

Diabetes affects statherin expression in human labial glands.

BACKGROUND AND OBJECTIVE Salivary statherin, which plays a special role in the defense of tooth integrity, is secreted by both major and minor salivary glands. A significantly reduced expression of this was recently found in human major salivary glands removed from diabetic subjects and was correlated with the high incidence of dental diseases occurring in patients with diabetes. In this study, we measured the density of gold particles indicating statherin immunoreactivity in labial glands to reveal a significant difference between diabetic and non-diabetic patients. MATERIALS AND METHODS Surgical samples of labial glands obtained from both diabetic and non-diabetic patients were fixed with a glutaraldehyde and paraformaldehyde mixture, embedded in Epon, and treated for immunogold histochemistry using a polyclonal antibody specific for statherin. RESULTS Statherin immunoreactivity was detected onto small vesicles diffused throughout the cytoplasm of serous cells. Statistical analysis revealed that the number of stained particles was significantly lower in the samples from diabetic subjects than from non-diabetic subjects. CONCLUSIONS The results indicate that diabetes affects statherin secretion in labial glands and support the hypothesis that the increased susceptibility to oral diseases associated with diabetes could be related with a reduced statherin secretion.

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

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.

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

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.

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

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.