Biomedical subjects

M Giorgi

Publications and source records attributed to M Giorgi.

At least 55 records · Page 3Linked to original sources

PubMed1997

Subtype 2c of hepatitis C virus is highly prevalent in Italy and is heterogeneous in the NS5A region.

Hepatitis C virus (HCV) isolates, obtained from 50 Italian patients with community-acquired infection, that had previously been classified as subtype 2a or 2b by current rapid genotyping methods were further characterized by partial sequence analysis. All the isolates were reclassified: 45 within subtype 2c and the other 5 as subtype 1b, 3a, or 4d. Thus, subtype 2c is much more prevalent than previously recognized, with about 30% of all HCV strains detected in Italy being subtype 2c. In contrast, isolates of subtypes 2a and 2b appear to be infrequent, if not absent. Further studies showed that subtype 2c isolates are heterogeneous in the NS5A region, in that they may or may not contain a 57-nucleotide (nt) segment spanning from nt 7533 to nt 7589 of the viral genome. Partial nucleotide sequencing of the NS5B region of four 2c subtypes excluded the possibility that the isolates possessing or not possessing the 57-nt segment in the NS5A region may have resulted from recombination phenomena.

Adult↗

PubMed1996

A functional role for some Fugu introns larger than the typical short ones: the example of the gene coding for ribosomal protein S7 and snoRNA U17.

The compact genome of Fugu rubripes, with its very small introns, appears to be particularly suitable to study intron-encoded functions. We have analyzed the Fugu gene for ribosomal protein S7 (formerly S8, see Note), whose Xenopus homolog contains in its introns the coding sequences for the small nucleolar RNA U17. Except for intron length, the organization of the Fugu S7 gene is very similar to that of the Xenopus counterpart. The total length of the Fugu S7 gene is 3930 bp, compared with 12691 bp for Xenopus. This length difference is uniquely due to smaller introns. Although short, the six introns are longer than the approximately 100 bp size of most Fugu introns, as they host U17 RNA coding sequences. While four of the six U17 sequences are 'canonical', the remaining two represent diverged U17 pseudocopies. In fact, microinjection in Xenopus oocytes of in vitro synthesized Fugu transcripts containing the 'canonical' U17f sequence results in efficient production of mature U17 RNA, while injection of a transcript containing the U17 psi b sequence does not.

Animals↗

PubMed1995

Unique features in the mitochondrial D-loop region of the European seabass Dicentrarchus labrax.

We have cloned and sequenced the displacement-loop (D-loop) region of the mitochondrial DNA (mtDNA) from the European seabass Dicentrarchus labrax (Dl). This sequencing revealed the presence of four tandemly repeated elements (R1, R2, R3 and R4); the individual variation in mtDNA total length is entirely accounted for by their variable number. The individuals examined also possessed an imperfect copy of one of the tandem repeats (psi R2). At least one termination-associated sequence (TAS) is present in each of the repeats and in two copies 5' upstream from the tandem array as well. The alignment of the Dl D-loop region with D-loop sequences from four other Teleosts and one Chondrosteus showed the Dl sequence to be larger than that of other fish. The extraordinary length of the Dl D-loop sequence is also due to the 5' and 3' regions that are flanking the tandem array, the largest ones to date analyzed in fish. In this study, we also report the unique organization and localization of putative TAS and conserved-sequence block (CSB) elements, and the presence of a conserved 218-bp sequence in the Dl D-loop region.

Animals↗

PubMed1994

Differential decrease of copper content and of copper binding to superoxide dismutase in liver, heart and brain of copper-deficient rats.

Dietary copper-deficiency in rats produced a organ-specific decrease of copper content. This was paralleled by a decrease of the activity of the copper-enzyme superoxide dismutase. In liver such a decrease is partially due to the existence of an apo-form of superoxide dismutase, which can be reactivated by addition of exogenous copper to tissue extracts. These results demonstrate in vivo that superoxide dismutase is post-translationally modulated by copper in higher vertebrates as previously found for yeast and mammalian cells in culture.

Animals↗

PubMed1994

Competitive polymerase chain reaction for quantitating feline immunodeficiency virus load in infected cat tissues.

To quantitate FIV provirus copy numbers present in tissue of infected cats, we have applied a competitive polymerase chain reaction (cPCR) recently described for HIV. The method consists in coamplifying a fixed amount of the DNA to be examined with graded copy numbers of a DNA competitor incorporating a short deletion and bearing the same primer recognition sequences. These conditions ensure almost identical thermodynamic and amplification efficiency for both template species but permit a prompt recognition of the two amplification products by gel electrophoresis. Since the amounts of the two amplicons are dependent on relative initial template concentrations, the number of FIV genomes in the sample can be calculated by densitometric analysis of the electrophoretic bands. After validation, the method has been applied to study the provirus loads in the tissues of cats infected with the Pisa-M2 isolate of FIV.

Animals↗

PubMed1994

Cloning and characterization of the European seabass, Dicentrarchus labrax, mitochondrial genome.

Mitochondrial DNA (mtDNA) from the European seabass, Dicentrarchus labrax, has been cloned and characterized. Its gene organization was deduced by a comparison of the sequenced termini of different subclones obtained from European seabass mtDNA to the completely-sequenced mtDNAs from carp and freshwater loach. The difference in genome size between the European seabass mtDNA (approximately 18 kb) and most of the other characterized fish mtDNAs (approximately 16.5 kb) is accounted for by the displacement-loop (D-loop). Comparisons have been performed between the derived amino-acid sequences of three sequenced genes, cytochrome c oxidase subunit 2 (COII), NADH dehydrogenase subunit 4L (ND4L) and ATP synthase subunit 8 (ATPase8), from D. labrax, and their counterparts in other fishes and Xenopus laevis.

Amino Acid Sequence↗

PubMed1994

Activities of 3':5' cyclic nucleotide phosphodiesterases in the superior cervical ganglion of rat: characterization, compartmentalization and observations in young and old animals.

We investigated the presence and features of "low Km" 3'-5' cyclic nucleotide phosphodiesterase activity in the homogenates and extracts of rat superior cervical ganglion. The DEAE chromatographic elution profile of a Triton X-100 extract showed two peaks of cAMP phosphodiesterase activity eluted at 280 and 600 mM sodium acetate and two peaks of cGMP phosphodiesterase activity eluted at 300 and at 500 mM sodium acetate. The activity was poorly stimulated by calcium-calmodulin and neither stimulated or inhibited by cGMP. Both cGMP PDE peaks were inhibited by zaprinast, with IC50's of 1.4 microM and 0.28 microM: their Km values were 4.4 and 3.8 microM, respectively. These features, together with cGMP binding activity, indicate that both enzymes belong to the phosphodiesterase V family. The Km values of the first and second cAMP phosphodiesterase peaks were 1.7 and 3.8 microM. Although both peaks displayed a cAMP specific hydrolysis, only the second peak was inhibited by RO 20-1724, with an IC50 of 8 microM. Preganglionic denervation indicated that the bulk of phosphodiesterase activity is localized in ganglion cells. In order to investigate possible effects of aging on the ganglionic function, phosphodiesterase activity was assayed in the ganglia of young (3 months) and old (25 months) male Fisher rats. The chromatographic profiles and kinetic features revealed no significant differences between young and old rats.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1994

Effects of Di-(2-ethylhexyl)phthalate on peroxisomes of liver, kidney and brain of lactating rats and their pups.

Di-(2-ethylhexyl)phthalate administered to adult lactating rats, from delivery to weaning, induces modifications of the peroxisomal enzymatic pattern in the liver, kidney and brain of both dams and pups. These modifications are age- and organ-dependent. Biochemical analysis shows that: 1) catalase specific activity is two-fold increased in the liver of both adult and newborn animals, in the kidney of newborns and in the brain of adults. 2)D-amino acid oxidase doubles in all newborn organs and in adult brain; it increases, although to a lesser extent, also in adult kidney, while it is half-reduced in adult liver. 3) Dihydroxyacetone phosphate acyl transferase only doubles in newborn liver, remaining fairly unchanged in all the other tested tissues. 4) Palmitoyl-CoA oxidase is greatly induced in the liver of both dams and litters, doubled in the kidneys and slightly increased or not at all in the brain of pups and mothers, respectively. The effect of the drug on enzyme activities is reversible, with different time courses depending on the considered enzyme and organ. Western blottings confirm the biochemical data. Electron microscopy shows proliferated peroxisomes in the liver and kidney of treated animals but not in the brain, where high catalase-like immunoreactivity is observed in the cytosol of neurons. Taken together, our data demonstrate that the response of peroxisomal enzymes to DEHP treatment is age- as well as tissue-dependent and specific for each enzyme studied.

Acyltransferases↗

PubMed1994

A preparative method for isolation of peroxisomes from rat kidney.

A new method for the isolation of peroxisomes from rat kidney cortex is described. The L fraction obtained according to Wattiaux-De Coninck et al. (1965) was layered on a discontinuous Nycodenz gradient (density = 1.15-1.21 g/ml) and then centrifuged in a fixed angle rotor for 45 min. at 136,000 g. On the basis of the morphological and biochemical analysis, the fraction recovered at the bottom of the tube was composed mainly by peroxisomes enriched in fatty acyl beta-oxidation system, whereas lower enrichment was found for other peroxisomal marker enzymes. Negligible contamination by mitochondria (marker enzyme cytochrome oxidase), lysosomes (marker enzyme acid phosphatase) and microsomes (marker enzyme NADPH cytochrome c reductase) was found.

Animals↗

PubMed1993

Characterization of 3':5' cyclic nucleotide phosphodiesterase activities of mouse neuroblastoma N18TG2 cells.

Characterization of 'low Km' 3':5' cyclic nucleotide phosphodiesterase activities (PDE) expressed in mouse N18TG2 neuroblastoma cells is reported. At least 3 peaks of activity were isolated by DEAE chromatography, none of which was calcium-calmodulin stimulated and cGMP stimulated or inhibited. A first peak elutes at 200 mM sodium acetate; it specifically hydrolyzes cGMP with a Km of 4.7 microM and shows sensitivity to zaprinast [M&B 22948] (1.8 microM). A second peak eluting at 410 mM sodium acetate hydrolyzes both cyclic nucleotides. A third peak, specific for cAMP hydrolysis, elutes at 580 mM sodium acetate, has a Km of 3.2 microM and is sensitive to RO 20 1724 (7.6 microM) and rolipram (2 microM). Hydrodynamic analysis showed for the first peak a Stokes radius of 5.3 nm with a sedimentation coefficient of 8.1 S, a frictional ratio (f/fo) of 1.41 and a native molecular mass of 182 kDa. The same analysis for peak 3 showed a Stokes radius of 4.1 nm with a sedimentation coefficient of 3.2 S, a frictional ratio of 1.63 and a native molecular mass of 56 kDa. The biochemical features reported for the enzyme eluting in the first peak, and its cGMP-binding activity stimulated by inhibitors of phosphodiesterase activity, demonstrate that it belongs to the PDE V subfamily; on the other hand the cAMP specific enzyme eluting in the third peak can be assigned to the 'RO 20 1724 inhibited' form. The significance of these findings is discussed in relation to the functional characteristics of the N18TG2 cell line.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1993

Purification of peroxisomal fraction from rat brain.

A purification procedure to obtain peroxisomes (microperoxisomes) from the brain of suckling rats is reported. A P2 fraction, (crude light mitochondria) frozen and thawed seven times, was subfractionated yielding a P4 fraction, 4-fold enriched in catalase activity with respect to the cytoplasmic extract S1. The P4 fraction was used for further purification of peroxisomes by isopicnic centrifugation on Nycodenz gradient (1.10-1.20 g/ml). When the cerebellum was not included in the starting material, the equilibrium density of peroxisomes was 1.152-1.162 g/ml. In this case the overall yield of catalase in the most enriched fraction was 7% and its relative specific activity more than 50. When the cerebellum was included in the total homogenate, the equilibrium density shifted towards higher values (1.177 g/ml) and in this case the catalase relative specific activity in the peroxisomal enriched fraction was extremely high (> 100). The biochemical results, together with the electron microscope examination of the purified fractions, demonstrate that our procedure allows the best purification of brain peroxisomes so far obtained. The different equilibrium densities of peroxisomes observed in the two sets of experiments are interpreted in terms of size heterogeneity of these organelles in different brain portions and cell types.

Animals↗

PubMed1992

Purification and characterization of a low-Km 3':5'-cyclic adenosine phosphodiesterase from post-meiotic male mouse germ cells.

We describe the purification and the study of the kinetic and hydrodynamic properties of a 'low Km' cAMP phosphodiesterase specifically expressed in haploid male germ cells of the mouse. The enzyme has been purified approx. 13,000-fold with respect to the activity in total cell homogenate. The purified enzyme hydrolyzed specifically cAMP with a Km of 3.3 microM and with a Vmax of 10.5 mumol of cAMP hydrolyzed/min per mg of protein. The hydrolytic activity was neither stimulated nor inhibited by cGMP, whereas it was inhibited by RO 20-1724 and Rolipram. The enzyme showed a Stokes radius of 3.8 nm and a sedimentation coefficient of 3.1 S, corresponding to a native molecular mass of 50 kDa and a frictional ratio of 1.53. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of sucrose gradient fractions of the purified enzyme showed a major band of 43 kDa copeaking with enzyme activity.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1992

A new continuous optical assay for maltase and sucrase.

A new method for the assay of maltase and sucrase is reported. The method makes use of mutarotase, hexokinase and glucose 6-phosphate dehydrogenase as ancillary enzymes. The reaction is linear at least up to a delta E/min of 0.13.

Cell Membrane↗

PubMed1991

Expression of the c-kit proto-oncogene in the murine male germ cells.

The proto-oncogene c-kit encodes a transmembrane protein tyrosine kinase receptor. The c-kit gene has recently been shown to be allelic with the W locus. Mutations at the white spotting locus (W) affect various aspects of hematopoiesis, melanogenesis and gametogenesis during development and in the adult animal. We have investigated the expression of the proto-oncogene c-kit in mouse testicular cell populations. The c-kit mRNA was found to be expressed at high levels in spermatogonia, and at lower levels in meiotic pachytene spermatocytes. Moreover, two novel testis-specific c-kit transcripts of 3.5 and 2.3 kb are present in postmeiotic haploid germ cells. These results suggest a role of c-kit not only during testis development in the embryo, but also throughout all stages of male germ cell development after birth.

Animals↗

PubMed1990

Fibrinogen induces endothelial cell adhesion and spreading via the release of endogenous matrix proteins and the recruitment of more than one integrin receptor.

We have previously shown that fibrinogen (fg) acts as a subendothelial matrix protein in promoting human endothelial cell (EC) adhesion and cytoskeletal organization. In this study we report that EC spreading on fg, at variance with other matrix proteins, requires endogenous matrix protein synthesis and secretion. ECs, upon seeding on fg, promptly released and organized a fibronectin (fn) matrix. Fg was more effective than vitronectin (vn) in promoting the deposition of this protein. ECs treated with monensin to block matrix protein secretion still adhered to fg but did not properly organize their cytoskeleton and adhesion structures. In contrast, monensin did not affect EC spreading either on vn or on fn. Using antibodies to the alpha and beta chains of fn (alpha 5 beta 1) and vn (alpha v beta 3) receptors, it was found that ECs adherent to fg show clustering and organization in adhesion structures of both type of receptors. A faint staining of adhesion structures with alpha 2 but not alpha 3 and alpha 6 antibodies was also observed. Antibodies either to vn or fn receptors were able to disrupt the EC monolayer and to induce EC retraction and detachment, thus indicating that both receptors are important in maintaining a sustained EC adhesion to fg. However, when ECs were treated with monensin only the vn receptor was organized in adhesion structures while the fn receptor was diffusely distributed. This suggests that clustering of the fn receptor is mediated by the release of endogenous matrix proteins induced by the exposure to fg. In conclusion, fg has a peculiar and complex type of interaction with ECs since it requires endogenous matrix protein release and the recruitment of more than one adhesive receptor. This suggests a specific way of response of ECs to each extracellular matrix component.

Actins↗

PubMed1990

HIV serology among Italian male military recruits at entrance and discharge.

To evaluate the usefulness of anti-HIV mass screenings, we examined 662 new military recruits and 1353 soldiers being discharged. None of the former and only one of the latter (0.07%) resulted seropositive on repeated ELISA and Western-blot. For comparison we also report the results of routine anti-HIV antibody testing in our diagnostic laboratory: the highest proportion of seropositive subjects was found among symptomatic patients (79%), followed by haemophiliac patients (31%), drug addicts (24%), sexual partners of seropositive subjects (21%), prisoners (5%) and homosexual men (5%). Health care personnel and prison guards were all negative. These data confirm that in Italy HIV infection is still relatively confined to the classic risk groups. While generalized screening during military service seems to be excessive, periodic sample surveys could be very useful to follow the evolution of HIV epidemiology.

Blotting, Western↗

PubMed1990

Stress at work and mental health status among female hospital workers.

Relations between working conditions and mental health status of female hospital workers were studied in a sample of 1505 women: 43% were nurses, 32% auxiliaries, and 7% ancillary staff; 13% were other qualified health care staff, mainly head nurses; 5% had occupations other than direct health care; 63% worked on the morning, 20% on the afternoon, and 17% on the night shift. Data were collected at the annual routine medical visit by the occupational health practitioner, using self administered questionnaires and clinical assessments. Five health indicators were considered: a high score to the general health questionnaire (GHQ); fatigue; sleep impairment; use of antidepressants, sleeping pills, or sedatives; and diagnosis of psychiatric morbidity at clinical assessment. Four indices of stress at work were defined: job stress, mental load, insufficiency in internal training and discussion, and strain caused by schedule. The analysis was conducted by multiple logistic regression, controlling for type of occupation, shift, number of years of work in hospital, daily travel time to work, age, marital status, number of children, and wish to move house. Sleep impairment was mostly linked to shift and strain due to schedule. For all other indicators of mental health impairment and especially high GHQ scores, the adjusted odds ratios increased significantly with the levels of job stress, mental load, and strain due to schedule. This evidence of association between work involving an excessive cumulation of stress factors and mental wellbeing should be considered in interventions aimed at improving the working conditions of hospital workers.

Adult↗

PubMed1990

Endothelial cell motility, integrin receptor clustering, and microfilament organization are inhibited by agents that increase intracellular cAMP.

Adhesion of human umbilical vein endothelial cells (ECs) to various extracellular matrix proteins is mostly mediated by receptors of the integrin family. The interaction of ECs with extracellular matrix proteins is accompanied by cell spreading, cytoskeletal organization, and clustering of the specific integrin receptors in complex supramolecular structures known as adhesion plaques or focal contacts. Little is known on the functional role of focal contacts in EC adhesion and motility and on the possibility to modulate their organization. In this article we report that an increase in intracellular cAMP levels severely impaired focal contact formation. This process did not affect cell attachment, but increased cell adhesion and strongly inhibited cell motility. ECs were treated with the cAMP-increasing agents forskolin and 2-chloro-adenosine or with the cAMP analogue 8-bromo-cAMP. When treated cells were seeded on purified vitronectin, fibrinogen, or fibronectin little modification in the number of attached cell was observed. In contrast ECs showed impaired organization of microfilaments and poorly developed clusters of beta 3- and beta 1-integrin receptors. On a vitronectin substrate, vinculin followed the distribution of beta 3-receptors. It was typically enriched at the focal contacts in control cells but was fragmented in small dots at the cell periphery in treated cells, as were bundles of actin stress fibers. Similarly, when forskolin was added to ECs spread on vitronectin or on fibrinogen, there was a progressive but reversible disruption of actin microfilaments and diffusion of beta 3 receptors. This was accompanied by a tighter adhesion of the cells to substrata. Migration of ECs in response to different matrix proteins was severely inhibited by cAMP-increasing agents. These data indicate that EC adhesion can occur very efficiently in the absence of fully developed beta 3- or beta 1-integrin receptor-containing focal contacts but suggest that the capacity to normally assemble focal contacts and cytoskeletal proteins is required for full cell spreading and migration.

2-Chloroadenosine↗