Biomedical subjects

Michele Papa

Publications and source records attributed to Michele Papa.

9 recordsLinked to original sources

PubMed2006

Genetically modified pigs produced with a nonviral episomal vector.

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

Animals↗

PubMed2005

Multi-transgenic pigs expressing three fluorescent proteins produced with high efficiency by sperm mediated gene transfer.

Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins.

Animals↗

PubMed2004

Neuronal vulnerability following inhibition of mitochondrial complex II: a possible ionic mechanism for Huntington's disease.

An impaired complex II (succinate dehydrogenase, SD) striatal mitochondrial activity is one of the prominent metabolic alterations in Huntington's disease (HD), and intoxication with 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial complex II, mimics the motor abnormalities and the pathology of HD. We found that striatal spiny neurons responded to this toxin with an irreversible membrane depolarization/inward current, while cholinergic interneurons showed a hyperpolarization/outward current. Both these currents were sensitive to intracellular concentration of ATP. The 3-NP-induced depolarization was associated with an increased release of endogenous GABA, while acetylcholine levels were reduced. Moreover, 3-NP induced a higher depolarization in presymptomatic R6/2 HD transgenic mice compared to wild-type (WT) mice, showing an increased susceptibility to SD inhibition. Conversely, the hyperpolarization did not significantly differ from the one recorded in WT mice. The diverse membrane changes induced by SD inhibition may contribute to the cell-type-specific neuronal death in HD.

Acetylcholine↗

PubMed2003

Fibrillogenesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F.

The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing beta-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.

Anilino Naphthalenesulfonates↗

PubMed2003

Expression pattern of the ether-a-gogo-related (ERG) K+ channel-encoding genes ERG1, ERG2, and ERG3 in the adult rat central nervous system.

Voltage-dependent K(+) channels play a pivotal role in controlling cellular excitability within the nervous system. The aim of the present study was to investigate the expression in the adult rat brain of the three ether-a-gogo-related gene (ERG) family members ERG1, ERG2, and ERG3, encoding for K(+) channel subunits. To this aim, the distribution of ERG transcripts was studied by means of reverse-transcription polymerase chain reaction (RT-PCR) and nonradioactive in situ hybridization histochemistry (NR-ISH). Furthermore, ERG1 subunit distribution was studied by immunohistochemical analysis. RT-PCR analysis revealed ERG1, ERG2, and ERG3 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. NR-ISH experiments detected transcripts encoded by all three ERG genes in the cerebral cortex and in all CA subfields and in the granular cell layer of the dentate gyrus of the hippocampus; strong ERG1 signals were also detected in scattered large elements throughout the oriens, pyramidal, and radiatum layers, and in the hilus of the dentate gyrus. In the thalamus, positively labeled neurons were detected in the reticular nucleus with ERG1 and ERG3 and in the anterodorsal nucleus with ERG2 riboprobes. Transcripts for ERG1 and, to a lesser degree, also for ERG3, were detected in the basal ganglia and in several brainstem nuclei. All three ERG genes appeared to be expressed in cerebellar Purkinje cells. Finally, ERG1 expression was also revealed in non-neuronal elements such as ependymal and subependymal cells along the ventricular walls and hippocampal astrocytes. These results suggest that the K(+) channel isoforms of the ERG family appear to be expressed in different central nervous system regions where they might differentially control the firing of neurons engaged in several networks.

Action Potentials↗

PubMed2003

Differential expression of the Na+-Ca2+ exchanger transcripts and proteins in rat brain regions.

In the central nervous system (CNS), the Na(+)-Ca(2+) exchanger plays a fundamental role in controlling the changes in the intracellular concentrations of Na(+) and Ca(2+) ions. These cations are known to regulate neurotransmitter release, cell migration and differentiation, gene expression, and neurodegenerative processes. In the present study, nonradioactive in situ hybridization and light immunohistochemistry were carried out to map the regional and cellular distribution for both transcripts and proteins encoded by the three known Na(+)-Ca(2+) exchanger genes NCX1, NCX2, and NCX3. NCX1 transcripts were particularly expressed in layers III-V of the motor cortex, in the thalamus, in CA3 and the dentate gyrus of the hippocampus, in several hypothalamic nuclei, and in the cerebellum. NCX2 transcripts were strongly expressed in all hippocampal subregions, in the striatum, and in the paraventricular thalamic nucleus. NCX3 mRNAs were mainly detected in the hippocampus, in the thalamus, in the amygdala, and in the cerebellum. Immunohistochemical analysis revealed that NCX1 protein was mainly expressed in the supragranular layers of the cerebral cortex, in the hippocampus, in the hypothalamus, in the substantia nigra and ventral tegmental area, and in the granular layer of the cerebellum. The NCX2 protein was predominantly expressed in the hippocampus, in the striatum, in the thalamus, and in the hypothalamus. The NCX3 protein was particularly found in the CA3 subregion, and in the oriens, radiatum, and lacunoso-moleculare layers of the hippocampus, in the ventral striatum, and in the cerebellar molecular layer. Collectively, these results suggest that the different Na(+)-Ca(2+) exchanger isoforms appear to be selectively expressed in several CNS regions where they might underlie different functional roles.

Animals↗

PubMed2002

Tryptophanyl substitutions in apomyoglobin determine protein aggregation and amyloid-like fibril formation at physiological pH.

Myoglobin is an alpha-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of beta-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.

Amyloid↗

PubMed2002

Brain distribution of the Na+/Ca2+ exchanger-encoding genes NCX1, NCX2, and NCX3 and their related proteins in the central nervous system.

In the central nervous system, the Na(+)/Ca(2+) exchanger plays a fundamental role in controlling changes in the intracellular concentrations of Na(+) and Ca(2+) ions that occur in physiologic conditions such as neurotransmitter release, cell migration and differentiation, gene expression, as well as neuro-degenerative processes. Three genes, NCX1, NCX2, and NCX3, encoding for Na(+)/Ca(2+) exchanger isoforms have been cloned. In this review, by using non-radioactive in situ hybridization and light immunohistochemistry with NCX isoform-specific riboprobes and antibodies, respectively, a systematic brain mapping for both transcripts and proteins encoded by all three NCX genes is described. Intense expression of NCX transcripts and proteins was detected in the cerebral cortex, hippocampus, thalamus, metathalamus, hypothalamus, brainstem, spinal cord, and cerebellum. In these areas, NCX transcripts and proteins were often found with an overlapping distribution pattern, although specific brain areas displaying a peculiar expression of each exchanger isoform were also found. Furthermore, immunoelectron and confocal microscopy revealed the expression of the NCX1 isoform of the exchanger at both pre- and postsynaptic sites as well as in association with membranes of the endoplasmic reticulum. Collectively, these data suggest that the different isoforms of the Na(+)/Ca(2+) exchanger appear to be selectively expressed in several CNS regions where they might underlie different functional roles.

Amygdala↗

PubMedDate not supplied

Pietro Anzolino da Eboli and the thermal therapy of renal pathologies.

In the Campi Flegrei area of Campania, a region of Southern Italy, some thermal baths still exist, known for the therapeutical properties of their waters. Utilized since remote ages for the treatment of a variety of pathologies. In "De Balneis Terrae Laboris", an Italian poet of the Middle Ages and scientist, Pietro Anzolino da Eboli, reports every detail about more than thirty different thermal sites in the Campi Flegrei, including all therapeutic effects of their waters. Based on Pietro's manuscript, we report in the current paper all the sites whose waters were - among other indications - also recommended for the treatment of some urinary diseases.

Balneology↗