Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures.

Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A(2A) and D(2) receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D(2)R-CFP or A(2A)R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D(2)R-CFP and A(2A)R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A(2A)R positive MVs were treated with the adenosine A(2A) receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A(2A)Rs were functionally competent in target cells. These findings demonstrate that A(2A) receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

Aspects on the integrative actions of the brain from neural networks to "brain-body medicine".

"Integration" is a key term in describing how nervous system can perform high level functions. A first condition to have "integration" is obviously the presence of efficient "communication processes" among the parts that have to be combined into the harmonious whole. In this respect, two types of communication processes, called wiring transmission (WT) and volume transmission (VT), respectively, were found to play a major role in the nervous system, allowing the exchange of signals not only between neurons, but rather among all cell types present in the central nervous system (CNS). A second fundamental aspect of a communication process is obviously the recognition/decoding process at target level. As far as this point is concerned, increasing evidence emphasizes the importance of supramolecular complexes of receptors (the so called receptor mosaics) generated by direct receptor-receptor interactions. Their assemblage would allow a first integration of the incoming information already at the plasma membrane level. Recently, evidence of two new subtypes of WT and VT has been obtained, namely the tunnelling nanotubes mediated WT and the microvesicle (in particular exosomes) mediated VT allowing the horizontal transfer of bioactive molecules, including receptors, RNAs and micro-RNAs. The physiological and pathological implications of these types of communication have opened up a new field that is largely still unexplored. In fact, likely unsuspected integrative actions of the nervous system could occur. In this context, a holistic approach to the brain-body complex as an indissoluble system has been proposed. Thus, the hypothesis has been introduced on the existence of a brain-body integrative structure formed by the "area postrema/nucleus tractus solitarius" (AP/NTS) and the "anteroventral third ventricle region/basal hypothalamus with the median eminence" (AV3V-BH). These highly interconnected regions operate as specialized interfaces between the brain and the body integrating brain-borne and body-borne neural and humoral signals.

C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction.

Micro-vesicles can be released by different cell types and operate as 'safe containers' mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway. In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases.

3D reconstruction of the crural and thoracolumbar fasciae.

PURPOSE: To create computerized three-dimensional models of the crural fascia and of the superficial layer of the thoracolumbar fascia. METHODS: Serial sections of these two fasciae, stained with Azan-Mallory, van Gieson and anti-S100 antibody stains, were recorded. The resulting images were merged (Image Zone 5.0 software) and aligned (MatLab Image Processing Toolkit). Color thresholding was applied to identify the structures of interest. 3D models were obtained with Tcl/Tk scripts and Paraview 3.2.1 software. From these models, the morphometric features of these fasciae were evaluated with ImageJ. RESULTS: In the crural fascia, collagen fibers represent less than 20% of the total volume, arranged in three distinct sub-layers (mean thickness, 115 µm), separated by a layer of loose connective tissue (mean thickness, 43 µm). Inside a single sub-layer, all the fibers are parallel, whereas the angle between the fibers of adjacent layers is about 78°. Elastic fibers are less than 1%. Nervous fibers are mostly concentrated in the middle layer. The superficial layer of the thoracolumbar fascia is also formed of three thinner sub-layers, but only the superficial one is similar to the crural fascia sub-layers, the intermediate one is similar to a flat tendon, and the deep one is formed of loose connective tissue. Only the superficial sub-layer has rich innervation and a few elastic fibers. DISCUSSION: Computerized three-dimensional models provide a detailed representation of the fascial structure, for better understanding of the interactions among the different components. This is a fundamental step in understanding the mechanical behavior of the fasciae and their role in pathology.

Perfluoroalkyl substances and bone health in young men: a pilot study.

PURPOSE: Perfluoroalkyl substances (PFAS) are a class of endocrine-disrupting chemicals. Toxicological studies indicate that PFAS accumulate in bone tissue and could cause alterations in bone metabolism. The primary objective of this study was to examine the association between PFAS exposure and bone status in a cohort of young men resident in a well-defined area with high PFAS environmental pollution. METHODS: Bone status was assessed in 117 subjects aged 18-21 by quantitative ultrasound (QUS) at the heel. Subjects underwent an accurate medical visit. Socio-demographic characteristics, lifestyle, and medical histories were collected. We also verified the interaction between PFAS and hydroxyapatite by computational modelling. The organic anion-transporting peptide (OATP), the putative transporter of PFAS, was evaluated by qPCR in bone biopsies from femoral heads discarded during arthroplasty in three male subjects. RESULTS: Exposed subjects showed significantly lower stiffness index, which resulted in lower t-score and higher prevalence of subjects at medium-high risk of fracture (23.6%) compared with controls (9.7%). Data from computational modelling suggested that PFOA exhibits a high affinity for hydroxyapatite, since the estimated change in free energy is in the order of that exhibited by bisphosphonates. Finally, we observed consistent expression of OATP1A2 gene in primary human osteoblasts. CONCLUSIONS: This is the first study reporting increased osteoporosis risk in young men exposed to PFAS and provide preliminary information on molecular mechanisms that could explain this observation, in agreement with previous studies on animal models and humans. However, these results must be interpreted with caution given the cross-sectional study design and the small number of cases.

Perfluorooctanoic acid alters progesterone activity in human endometrial cells and induces reproductive alterations in young women.

Female fecundity is finely regulated by hormonal signaling, representing a potential target for endocrine-disrupting chemicals. Among the chemicals of most concern are the perfluoroalkyl substances (PFAS), widely used in consumer goods, that are associated with adverse effects on reproductive health. In this context, the endometrium clearly represents an important fertility determining factor. The aim of this study was to investigate PFAS interference on hormonal endometrial regulation. This study was performed within a screening protocol to evaluate reproductive health in high schools. We studied a cohort of 146 exposed females aged 18-21 from the Veneto region in Italy, one of the four areas worldwide heavily polluted with PFAS, and 1080 non-exposed controls. In experiments on Ishikawa cells included UV-Vis spectroscopy, microarray analysis and qPCR. We report a significant dysregulation of the genetic cascade leading to embryo implantation and endometrial receptivity. The most differentially-expressed genes upon PFOA coincubation were ITGB8, KLF5, WNT11, SULT1E1, ALPPL2 and G0S2 (all p < 0.01). By qPCR, we confirmed an antagonistic effect of PFOA on all these genes, which was reversed at higher progesterone levels. Molecular interference of PFOA on progesterone was confirmed by an increase in the intensity of absorption spectra at 250 nm in a dose-dependent manner, but not in the presence of ß-estradiol. Age at menarche (+164 days, p = 0.006) and the frequency of girls with irregular periods (29.5% vs 21.5%, p = 0.022) were significantly higher in the exposed group. Our results are indicative of endocrine-disrupting activity of PFAS on progesterone-mediated endometrial function.

Implications of the 'Energide' concept for communication and information handling in the central nervous system.

Recently a revision of the cell theory has been proposed, which has several implications both for physiology and pathology. This revision is founded on adapting the old Julius von Sach's proposal (1892) of the Energide as the fundamental universal unit of eukaryotic life. This view maintains that, in most instances, the living unit is the symbiotic assemblage of the cell periphery complex organized around the plasma membrane, some peripheral semi-autonomous cytosol organelles (as mitochondria and plastids, which may be or not be present), and of the Energide (formed by the nucleus, microtubules, and other satellite structures). A fundamental aspect is the proposal that the Energide plays a pivotal and organizing role of the entire symbiotic assemblage (see Appendix 1). The present paper discusses how the Energide paradigm implies a revision of the concept of the internal milieu. As a matter of fact, the Energide interacts with the cytoplasm that, in turn, interacts with the interstitial fluid, and hence with the medium that has been, classically, known as the internal milieu. Some implications of this aspect have been also presented with the help of a computational model in a mathematical Appendix 2 to the paper. Finally, relevances of the Energide concept for the information handling in the central nervous system are discussed especially in relation to the inter-Energide exchange of information.

Common key-signals in learning and neurodegeneration: focus on excito-amino acids, beta-amyloid peptides and alpha-synuclein.

In this paper a hypothesis that some special signals ("key-signals" excito-amino acids, beta-amyloid peptides and alpha-synuclein) are not only involved in information handling by the neuronal circuits, but also trigger out substantial structural and/or functional changes in the Central Nervous System (CNS) is introduced. This forces the neuronal circuits to move from one stable state towards a new state, but in doing so these signals became potentially dangerous. Several mechanisms are put in action to protect neurons and glial cells from these potentially harmful signals. However, in agreement with the Red Queen Theory of Ageing (Agnati et al. in Acta Physiol Scand 145:301-309, 1992), it is proposed that during ageing these neuroprotective processes become less effective while, in the meantime, a shortage of brain plasticity occurs together with an increased need of plasticity for repairing the wear and tear of the CNS. The paper presents findings supporting the concept that such key-signals in instances such as ageing may favour neurodegenerative processes in an attempt of maximizing neuronal plasticity.

Randomized placebo-controlled trial on local applications of opioids after hemorrhoidectomy.

BACKGROUND: Hemorrhoidectomy is associated with considerable postoperative pain. This study assessed whether a small dose of morphine or oxycodone administered in the embedded sponge set in the anus at the end of a hemorrhoidectomy intervention reduced postoperative pain. METHODS: The presence of opioid receptors was assessed in the anal mucosa excised from ten patients with perianal condyloma acuminata and 19 patients with symptomatic third-fourth degree hemorrhoids. A double-blind prospective randomized placebo-controlled trial was then conducted in 135 patients with hemorrhoids. Hemorrhoidectomy patients were randomized to morphine (MG), oxycodone (OG), or control (CG) groups, each patient having an absorbable sponge dressing left in the anus embedded with 1 mg of morphine, 1 mg oxycodone, or vehicle, respectively. The mean time for the first dose of analgesic drugs, the use of analgesics, and the mean time to void bladder was evaluated. RESULTS: The presence of kappa- and delta-opioid receptor immunoreactivity was detected in the anal mucosa excised from patients with perianal condyloma acuminata and hemorrhoids. Furthermore, there was a significant (P < 0.001) upregulation of kappa receptor immunoreactive-like material in hemorrhoidectomy patients. The mean time for the first analgesic administration was significantly increased (P < 0.001) in MG versus CG. A further significant increase (P < 0.001) was observed in the OG patient group. The mean time for voiding was significantly higher in CG when compared to the MG and OG patient groups. CONCLUSION: The local administration of very low doses of kappa-opioid agonist decreased hemorrhoidectomy postoperative pain through the interaction with specific opioid receptors located on anal mucosa.

Structural plasticity in G-protein coupled receptors as demonstrated by the allosteric actions of homocysteine and computer-assisted analysis of disordered domains.

Structural plasticity of G-protein coupled receptors (GPCRs) is of basic importance for their interactions with ligands, in particular with other proteins such as receptors or receptor-modifying proteins that can lead to different functions for the same GPCR. In the present paper, structural plasticity of GPCRs has been investigated discussing allosteric modulatory actions of Homocysteine (Hcy) on D2 receptors together with data obtained by computer-assisted analysis of the presence of disordered domains in GPCRs. Previous evidence for a modulatory action of Hcy on D2 receptors has been further extended by means of experiments on the effects of Hcy local intrastriatal injection on rotational behaviour. Altogether the present data allow considering under a new angle the well known proposal of A2A antagonists as new therapeutic agents in Parkinson's disease (PD). Furthermore, they point out to not only the importance of drugs capable of reducing Hcy brain levels, but also to the potential therapeutic impact of drugs capable of regionally blocking (for PD) or enhancing (for some schizophrenic syndromes) Hcy allosteric action on D2 receptors. As far as the investigations on GPCR plastic domains, extracellular, intracellular and transmembrane domains of 14 GPCRs have been considered and propensity of each of these domains for a structured or unstructured conformation has been evaluated by means of ad hoc computer programs. It has been shown that the N- and C-terminals as well as intracellular loop 3 have a high propensity towards an unstructured conformation, hence they are potentially very plastic domains, which can undergo easily to interactions with other ligands, particularly with other protein domains. This aspect is obviously of the greatest importance not only for the function of single GPCRs, but also for their interactions either with other receptors (receptor-receptor interactions) or, more generally, for formation of clusters of membrane associated proteins, hence of "protein mosaics", where the GPCRs could represent the input unit of the supra-molecular device.

From the hierarchical organization of the central nervous system to the hierarchical aspects of biocodes.

The quite recent (at least on the evolutionary time scale) emergence of nervous systems in complex organisms enabled the living beings to build a wide-ranging model of the external world in order to predict and evaluate the outcomes of their actions. Such a process likely represents a real coding activity, since, by proper handling of information, it generates a mapping between the external environment and internal cerebral activity patterns. The patterns of neural activity that correspond to the final maps, however, emerge from the holistic assembly of a multilevel functional organization. Nerve tissue components, indeed, appear organized in compartments, also called functional modules (FM), that contain system components and circuits of different miniaturizations not only arranged to work together either in parallel or in series but also nested within each other. At least three levels can be recognized in a functional module and it is possible to point out that such a hierarchical organization of the brain circuits could be mirrored by a corresponding hierarchical organization of biocodes. This feature can also suggest the hypothesis that the same logic could operate also at system level to integrate FM into functional brain areas and to associate areas to generate the final map used by humans to image the external world and to imagine untestable worlds.

Anatomic distribution of apoptosis in medulla oblongata of infants and adults.

The aim of the study was to evaluate the distribution of apoptosis in the medullary nuclei of infants and adults who died of hypoxic-ischaemic injury. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) in brainstems from 22 adults (7 subjects who died of opiate intoxication, 15 who died of other hypoxic-ischaemic injury) and 10 infants. The nuclei examined included the hypoglossal, dorsal motor nucleus of the vagus, nucleus tractus solitarii, nucleus of the spinal trigeminal tract, cuneate, vestibular and inferior olivary nuclei. A morphometric analysis with the optical disector method was performed to calculate the mean percentages (+/- standard deviation) of TUNEL-positive neuronal and glial cells for the sample populations. Opiate deaths did not have higher apoptotic indices than other adult hypoxic-ischaemic deaths. Statistically significant differences between adults and infants were found in the neuronal apoptotic indices of the cuneate (28.2 +/- 16.3% vs. 6.9 +/- 8.7%), vestibular (24.7 +/- 15.0% vs. 11.3 +/- 11.4%), nucleus tractus solitarii (11.2 +/- 11.2% vs. 2.3 +/- 2.4%), dorsal motor nucleus of the vagus (6.8 +/- 8.5% vs. 0.1 +/- 0.2%) and hypoglossal (6.6 +/- 5.7% vs. 0.1 +/- 0.2%), indicating higher resistance of the neuronal populations of these infant medullary nuclei to terminal hypoxic-ischaemic injury or post-mortem changes. Differences in neuronal apoptotic index were also statistically significant among nuclei, suggesting differential characteristics of survival. Nuclei with higher neuronal apoptotic indices were the cuneate, vestibular and nucleus of the spinal trigeminal tract, which are located in the lateral medullary tegmentum and share the same vascular supply from the posterior inferior cerebellar artery.

Intra-articular 500-730 kDa hyaluronan (Hyalgan®) therapy in the management of osteoarthritis. Can a specific therapeutic profile be defined?

OBJECTIVE: Intra-articular hyaluronic acid (HA) injections for the symptomatic relief of pain have been available for treatment since the 1980s. Practitioner experience and real-world evidence have been accumulated to suggest that HA injections are effective and well tolerated in patients. Treatment guidelines issued by different professional medical societies, however, do not point in a single direction. This appears mainly due to conflicting results of the proposed meta-analyses at least in part associated with a variability between different HA preparations on different outcome parameters, suggesting that intra-articular HA products should not be treated as a group, as there are differences between them influencing both efficacy and safety. PATIENTS AND METHODS: The present review is focused on the quite relevant amount of preclinical and clinical studies (the first studies dating back to thirty years ago) concerning a specific HA-based preparation (500-730 kDa native HA) and supporting its use as a tool for intra-articular therapy. They also include comparative studies to other HA preparations. RESULTS: The analysis of this experience allows to define a specific profile for 500-730 kDa HA as a tool for the management of osteoarthritis in terms of main mechanism of action, kinetics features and interaction with joint tissues, subpopulation of patients expected to obtain the highest benefit from the treatment, safety issues and impact on disease-cost. CONCLUSIONS: The abovementioned factors may also represent useful criteria to better characterize the specificities of each HA-based preparation and to achieve a more stratified categorization of this class of therapeutic tools.

Phytochemistry, antioxidant potential and antifungal of Byrsonima crassifolia on soil phytopathogen control.

The use of chemical defensives to control fungal diseases has by consequence to impact negatively over the environment and human health, this way, the use of plant extracts with antifungal properties along with proper cultural management makes viable an alternative plant production control, specially for familiar and organic cultures. The objective of this study was to perform phytochemical and antioxidant analysis of Byrsonima crassifolia (canjiqueira) barks and evaluate its antifungal potential over Fusarium solani and Sclerotinia sclerotiorum mycelial growth. The ethanol extract from plants collected in Pantanal, Mato Grosso do Sul, Brazil was submitted to phytochemical prospection, total phenol and flavonoids quantification and antioxidant activiy determination (DPPH). To evaluate antifungal activity concentrations of 800, 1200, 1600, 2000 and 2400 µg 100 mL-1 of ethanol extract were used. Which concentration was separately incorporated in agar (PDA) and shed in Petri dishes, followed by the fungi mycelial disc where the colonies diameter was measured daily. Negatives control with agar without extract and agar with an ethanol solution were used. The B. crassifolia ethanol extract presented inhibitory activity over the fungi studied where concentrations of 800 and 1600 µg 100 mL-1, inhibited 38% of the mycelial growth of F. solani; to S. sclerotiorum the best concentration was 2400 µg 100 mL1, reducing 37.5%. The antifungal bark extract potential of this specie is attributed to phenolic compounds and to triterpenes derivatives.

Role of cooperativity in protein folding and protein mosaic assemblage relevance for protein conformational diseases.

Biological systems are organized in intricate and highly structured networks with hierarchies and multiple scales. Cells can be considered as "meso-scale level" systems placed between the "macro-scale level" (systems of cellular networks) and the "micro-scale level" (systems of molecular networks). In fact, cells represent complex biochemical machineries made by networks of molecules connected by biochemical reactions. Thus, the brain should be studied as a system of "networks of networks". Recently, the existence of a Global Molecular Network (GMN) enmeshing the entire CNS was proposed. This proposal is based on the evidence that the extra-cellular matrix is a dynamic molecular structure capable of storing and releasing signals and of interacting with receptors and proteins on the cell membranes. Proteins have a special role in molecular networks since they can be assembled into high-order molecular complexes, which have been defined as Protein Mosaics (PM). Protein monomers in a PM (the "tesserae" of the mosaic) can interact via classical and non-classical cooperativity behaviour involving allosteric interactions. In the present paper, new features of allostery and cooperativity for protein folding, assemblage and topological features of PM will be discussed. Against this background, alterations in PM via allosteric modulations and non-classical cooperativity mechanisms may lead to protein aggregates like beta amyloid fibrils. Such aggregates cause pathological changes in the GMN structure and function leading to neurodegenerative diseases such as Alzheimer's disease. Thus, a novel view of the so called Protein Conformational Diseases (PCD) is proposed.

Fine ultrastructure of chromaffin granules in rat adrenal medulla indicative of a vesicle-mediated secretory process.

Observation by transmission electron microscopy, coupled with morphometric analysis and estimation procedure, revealed unique ultrastructural features in 25.94% of noradrenaline (NA)-containing granules and 16.85% of adrenaline (A)-containing granules in the rat adrenal medulla. These consisted of evaginations of the granule limiting membrane to form budding structures having different morphology and extension. In 14.8% of NA granules and 12.0% of A granules, outpouches were relatively short, looked like small blebs emerging from the granule surface and generally contained electron-dense material. A proportion of 11.2% of NA granules and 4.9% of A granules revealed the most striking ultrastructural features. These secretory organelles presented thin, elongated, tail-like or stem-like appendages, which were variably filled by chromaffin substance and terminated with spherical expansions of different electron density. A cohort of vesicles of variable size (30-150 nm in diameter) and content was found either close to them or in the intergranular cytosol. Examination of adrenal medullary cells fixed by zinc iodide-osmium tetroxide (ZIO) revealed fine electron dense precipitates in chromaffin granules, budding structures as well as cytoplasmic vesicles. These data indicate that a common constituent is revealed by the ZIO histochemical reaction in chromaffin cells. As catecholic compounds are the main tissue targets of ZIO complexes, catecholamines are good candidates to be responsible for the observed ZIO reactivity. This study adds further to the hypothesis that release of secretory material from chromaffin granules may be accomplished by a vesiclular transport mechanism typical of piecemeal degranulation.

Ganglioside treatment and improved axonal regeneration capacity in experimental diabetic neuropathy.

The efficacy of gangliosides in enhancing axonal regeneration and maturation in the early stages of diabetic neuropathy was assessed by quantitative analysis of immunostained serial sections of the sciatic nerve. Sprague-Dawley rats were made diabetic with a single injection of alloxan (100 mg/kg). One week later they were injected daily intraperitoneally with either a highly purified ganglioside mixture (10 mg/kg) or sterile saline for 4 wk. At the end of the treatment, sciatic nerves were crushed and allowed to regenerate for 1 wk without ganglioside treatment. The animals were then killed, and the nerves were frozen and processed for immunohistochemistry and electron microscopy. The number of regrowing axons was counted with a computerized image-analysis system on cross sections taken at predefined distances along the regenerating stump and stained with monoclonal antibody iC8 specific for the 145,000-Mr subunit of the neurofilaments. In untreated diabetic animals the number of axons able to regenerate and sustain elongation for greater than or equal to 13 mm from the crush point was reduced by 40% with respect to control rats. Ganglioside treatment was effective in compensating almost completely for this dramatic reduction. Electron microscopy confirmed that the immunofluorescence counts corresponded to regenerating axons containing neurofilaments. These results suggest that gangliosides are able to compensate for the derangements of axonal transport of cytoskeletal proteins reported in experimental diabetic neuropathy.

Ca2+ channels and intracellular Ca2+ stores in neuronal and neuroendocrine cells.

Changes in [Ca2+]i are essential in modulating a variety of cellular functions. In no other cell type does the regulation of [Ca2+]i reach the level of sophistication observed in cells of neuronal origin. Because of its physicochemical characteristics, the fluorescent Ca2+ indicator Fura-2 has become extremely popular among neuroscientists. The use of this probe, however, has generated a number of problems, in particular, extracytosolic trapping and leakage from intact cells. In the first part of this contribution we briefly discuss the practical application of Fura-2 to the study of [Ca2+]i in primary cultures of neurons and astrocytes. In the second part, we review some recent data (mainly from our laboratories) obtained in neurons and neuroendocrine cells, concerning the regulation of different types of Ca2+ channels and the role and mechanism of intracellular Ca2+ mobilization. The experimental evidence supporting the existence of a previously unrecognised organelle, the calciosome, that we hypothesize represents the functional equivalent in non-muscle cells of sarcoplasmic reticulum, will also briefly be discussed.

Colocalization of low- and high-affinity NGF receptors on PC12 cells, C6 glioma cells and dorsal root ganglion neurons.

The biological responsiveness of neural cells to nerve growth factor (NGF) appears to require expression and ligand binding to both the low-affinity NGF receptor (LNGFR) and the proto-oncogene product trk, the latter being a receptor tyrosine kinase. Immunolocalization of the LNGFR and the high-affinity component of the NGF receptor, trk (HNGFR) was studied by electron microscopic morphometric analysis on cultured PC12 pheochromocytoma cells, C6 glioma cells and neonatal rat dorsal root ganglia neurons using a double immunogold labeling technique. Two receptor-specific antibodies, anti-LNGFR monoclonal antibody 192-IgG and a polyclonal antibody against the 14 carboxy-terminal amino acids of the Trk protein, were utilized in conjunction with immunoglobulin conjugated to colloidal gold particles of different sizes. All cells treated with NGF (50 ng/ml) displayed significant colocalization of LNGFR/HNGFR-like immunoreactivity. Gold particles associated with LNGFR (LNGFR-like immunoreactivity) were frequently seen near 2 or 3 (or more) particles delineating the HNGFR on all cell surfaces. Positive Trk-like immunoreactivity (HNGFR) thus seems to localize in close proximity to LNGFRs in at least these cell types.

Dendritic spine loss in hippocampus of aged rats. Effect of brain phosphatidylserine administration.

Dendritic spine density of pyramidal cells in region CA1 of the hippocampus has been evaluated in young (3 months), old (27 months) and old phosphatidylserine (BC-PS)-treated rats. BC-PS (50 mg/kg, suspended in tap water) was administered daily, starting at the age of 3 months until 27 months. Spine density was analyzed on Golgi-stained pyramidal neurons by a computerized analysis system. In 27-month-old rats, spine density showed with respect to 3-month-old animals, a significant decrease in both basal and apical dendrites (p less than 0.01; one-way ANOVA), with a mean loss of 12.11% in the basal dendrites and of 10.64% in the apical ones. In 27-month-old rats treated with BC-PS, values of spine density were not statistically different when compared to those of 3-month-old animals. The mechanisms underlying the beneficial effect of BC-PS treatment on neuronal connectivity might be explained on the basis of its pharmacological actions on neuronal membranes [9], neurotransmission [43] and/or interaction with NGF [7].