Low Voltage Transmission Electron Microscopy in Cell Biology.

Low voltage transmission electron microscopy (LVTEM) was employed to examine biological tissues with accelerating voltages as low as 5kV. Tissue preparation was modified to take advantage of the low-voltage techniques. Treatments with heavy metals, such as post-fixation with osmium tetroxide, on block and counterstaining were omitted.

Perspectives on low voltage transmission electron microscopy as applied to cell biology.

Low voltage transmission electron microscopy (LVTEM) with accelerating voltages as low as 5 kV was applied to cell biology. To take advantage of the increased contrast given by LVTEM, tissue preparation was modified omitting all heavy metals such as osmium, uranium, and lead from the fixation, on block staining and counterstaining. Nonstained ultra-thin tissue sections (40 nm thick) generated highly contrasted images.

Role of the apical and basolateral domains of the enterocyte in the regulation of cholesterol transport by a high glucose concentration.

We have recently shown that a high glucose (HG) concentration raised intestinal cholesterol (CHOL) transport and metabolism in intestinal epithelial cells. The objective of the present work is to determine whether the stimulus for increased CHOL absorption by glucose originates from the apical site (corresponding to the intestinal lumen) or from the basolateral site (related to blood circulation). We tackled this issue by using differentiated Caco-2/15 cells.

Caspase activation regulates the extracellular export of autophagic vacuoles.

The endothelium plays a central role in the regulation of vascular wall cellularity and tone by secreting an array of mediators of importance in intercellular communication. Nutrient deprivation of human endothelial cells (EC) evokes unconventional forms of secretion leading to the release of nanovesicles distinct from apoptotic bodies and bearing markers of multivesicular bodies (MVB). Nutrient deficiency is also a potent inducer of autophagy and vesicular transport pathways can be assisted by autophagy.

A review on gastric leptin: the exocrine secretion of a gastric hormone.

A major advance in the understanding of the regulation of food intake has been the discovery of the adipokine leptin a hormone secreted by the adipose tissue. After crossing the blood-brain barrier, leptin reaches its main site of action at the level of the hypothalamic cells where it plays fundamental roles in the control of appetite and in the regulation of energy expenditure. At first considered as a hormone specific to the white adipose tissue, it was rapidly found to be expressed by other tissues.

Regulation of leptin receptor expression in human polarized Caco-2/15 cells.

Background: Leptin receptors (LEPR) are expressed in intestinal epithelial cells from the duodenum to the colon. Since their role is fundamental for the proper control of nutrient absorption, mucus secretion and mucosa renewal, the regulation of LEPR expression is for the first time investigated as a function of various potential effectors.

Methodology/principal Findings: Fully differentiated Caco-2/15 cells were incubated for 24 hours with nutrients [carbohydrates, fatty acids (FA), amino acids and sterols], hormones (leptin, insulin, hydrocortisone and epithelial growth factor), inflammatory agents (Interferon-γ, LPS, TNF-α), and PPAR agonists (rosiglitazone and WY14643).

Psammomys obesus, a particularly important animal model for the study of the human diabetic nephropathy.

The Psammomys obesus lives in natural desert habitat on low energy (LE) diet, however when maintained in laboratory conditions with high energy (HE) diet it exhibits pathological metabolic changes resembling those of type 2 diabetes. We have evaluated and correlated the histopathology, metabolic and functional renal alterations occurring in the diabetic Psammomys. Renal function determined by measuring glomerular filtration rate (GFR), protein excretion, protein/creatinine ratio and morpho-immunocytochemical evaluations were performed on HE diet diabetic animals and compared to LE diet control animals.

Compartmentalization of pancreatic secretory zymogen granules as revealed by low-voltage transmission electron microscopy.

Low-voltage (5-kV) transmission electron microscopy revealed a novel aspect of the pancreatic acinar cell secretory granules not previously detected by conventional (80-kV) transmission electron microscopy. Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV.

Cross-talk between adipose and gastric leptins for the control of food intake and energy metabolism.

The understanding of the regulation of food intake has become increasingly complex. More than 20 hormones, both orexigenic and anorexigenic, have been identified. After crossing the blood-brain barrier, they reach their main site of action located in several hypothalamic areas and interact to balance satiety and hunger.

Receptor-Mediated Transcytosis of Leptin through Human Intestinal Cells In Vitro.

Gastric Leptin is absorbed by duodenal enterocytes and released on the basolateral side towards the bloodstream. We investigated in vitro some of the mechanisms of this transport. Caco-2/15 cells internalize leptin from the apical medium and release it through transcytosis in the basal medium in a time- temperature-dependent and saturable fashion.

Nestin action during insulin-secreting cell differentiation.

Nestin, which was initially identified as a marker of neural stem cells, has been reported in regenerating pancreas as well as in early embryonic stem (ES) cell derivatives. However, little is known about its specific roles in stem cells as a functional regulator. We investigated the source of the action of nestin in ES and adult pancreatic ductal stem (PDS) cells in regard to the neogenesis of insulin-secreting beta-cells.

Association of AMP-activated protein kinase subunits with glycogen particles as revealed in situ by immunoelectron microscopy.

Immunogold cytochemistry was applied to reveal the intracellular location of AMP-activated protein kinase (AMPK) subunits in liver tissue of normal rats fed ad libitum. AMPK alpha and beta subunits were located both in the cytosol and in close association with rosettes of glycogen particles (alpha particles). To reveal their true in situ association with glycogen, particular tissue processing conditions that retain glycogen in the cells were required.

Localization, function and regulation of the two intestinal fatty acid-binding protein types.

Although intestinal (I) and liver (L) fatty acid binding proteins (FABP) have been widely studied, the physiological significance of the presence of the two FABP forms (I- and L-FABP) in absorptive cells remains unknown as do the differences related to their distribution along the crypt-villus axis, regional expression, ontogeny and regulation in the human intestine. Our morphological experiments supported the expression of I- and L-FABP as early as 13 weeks of gestation. Whereas cytoplasmic immunofluorescence staining of L-FABP was barely detectable in the lower half of the villus and in the crypt epithelial cells, I-FABP was visualized in epithelial cells of the crypt-villus axis in all intestinal segments until the adult period in which the staining was maximized in the upper part of the villus.

Adiponectin stimulates phosphorylation of AMP-activated protein kinase alpha in renal glomeruli.

Adiponectin receptor ADIPOR1 activates the intracellular second messenger AMP-activated protein kinase (AMPK) that participates in the control of the oxidative stress and apoptosis. This study reveals the presence of a functional ADIPOR1 receptor in all the cells of the renal glomeruli. Isolated glomeruli were incubated in vitro with adiponectin and proteins analysed by western blot.

Up-regulation of P-glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line.

A major concern regarding the chronic administration of antiretroviral drugs is the potential for induction of drug efflux transporter expression (i.e., P-glycoprotein, P-gp) at tissue sites that can significantly affect drug distribution and treatment efficacy.

Low-voltage transmission electron microscopy reveals SV40 viral particles within secretory granules in pancreatic cells.

Novel approach in low voltage transmission electron microscopy (TEM) has revealed the presence of SV40 viral like particles in the secretory zymogen granules and in spherical membrane-bound dense bodies of SV40 infected pancreatic cells. The presence of SV40 antigen in these cellular compartments was confirmed by immunocytochemistry of the VP1 antigen. Visualization of the viral particles was only possible by examining ultrathin tissue sections with low-voltage TEM that significantly enhances imaging contrast.

Pancreatic acinar and islet cell infection by low-dose SV40 administration.

Objectives: Viral vector uptake into the pancreas is rare. The few viral vectors reported to transduce in vivo pancreatic islets after systemic injection required additional physical measures, such as direct pancreatic injection or hepatic vessel clamping. Because pancreatic islet uptake of the human polyomavirus family member BK virus was previously reported in hamsters after systemic administration, we hypothesized that SV40, a polyomavirus member remarkably similar to BK virus, may also infect the pancreas.

Glomerular CD34 expression in short- and long-term diabetes.

Aging and diabetes are associated with exacerbated expression of adhesion molecules. Given their importance in endothelial dysfunction and their possible involvement in the alteration of glomerular permeability occurring in diabetes, we have evaluated expression of the sialomucin-type adhesion molecule CD34 in renal glomerular cells of normal and diabetic animals at two different ages by colloidal gold immunocytochemistry and immunoblotting. CD34 labeling was mostly assigned to the plasma membranes of glomerular endothelium and mesangial processes.

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats.

Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding AMPK subunit beta(2).

Modification of kidney barrier function by the urokinase receptor.

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of alphavbeta3 integrin.

Biological role, protein expression, subcellular localization, and oxidative stress response of paraoxonase 2 in the intestine of humans and rats.

Oxidative stress is a cardinal manifestation of various intestinal disorders. However, very little knowledge is available on the intestine's inherent defense mechanisms against free radicals. This study was designed to determine the protein expression, subcellular localization and oxidative stress response of paraoxonase 2 (PON2), a member of a powerful antioxidant family in human and rat intestine.

Transcytosis of gastric leptin through the rat duodenal mucosa.

Leptin is secreted into the gastric juice by epithelial Chief cells and reaches the duodenum in a biologically intact active form. We assessed the possibility that this gastric leptin crosses the intestinal mucosa by transcytosis through enterocytes to reach blood circulation. Endogenous gastric leptin secretion was triggered by cholinergic stimulation.

Localization of P-glycoprotein at the nuclear envelope of rat brain cells.

P-glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines.

Localization and role of NPC1L1 in cholesterol absorption in human intestine.

Recent studies have documented the presence of Niemann-Pick C1-Like 1 (NPC1L1) in the small intestine and its capacity to transport cholesterol in mice and rats. The current investigation was undertaken to explore the localization and function of NPC1L1 in human enterocytes. Cell fractionation experiments revealed an NPC1L1 association with apical membrane of the enterocyte in human jejunum.

In situ localization of P-glycoprotein (ABCB1) in human and rat brain.

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial.