Biomedical subjects

A Benedetti

Publications and source records attributed to A Benedetti.

At least 55 records · Page 3Linked to original sources

PubMed1999

Use of biochemical indices in the mediterranean environment: comparison among soils under different forest vegetation.

In the present study, soil biomass activity, organic carbon storage, and turnover times were compared in adjacent mediterranean biotopes with different forest vegetation, to analyze the effects of litter diversity and soil management protocols on microbial decomposition rates. Samples of forest soil from four vegetation types were collected at depths of 0-20 and 20-40 cm in the 'Tenuta Presidenziale di Castelporziano' Reserve on the Tyrrhenian coast, near Rome (Italy). The samples were incubated under standard laboratory conditions (-33 kPa water tension, and 30 degrees C), in order to compare the microbial activity independently of temperature and humidity. The CO2-C accumulation curves over a 28-d incubation period showed substantially different kinetics between the samples; in particular, soils with above-ground diversity were characterised by high mineralization activity when compared with those sampled under monospecific vegetation. For all the sites, statistically significant linear correlation was observed between nitrogen concentration and potentially mineralizable carbon (r = 0.97), and microbial biomass carbon (Cmic) to total organic carbon (Corg) ratio and the microbial metabolic quotient q(CO2) (r = -0.96). The q(CO2), indicator of the stability of ecosystems, was enhanced by plant diversity, while the Cmic:Corg ratio was reduced.

Biomass↗

PubMed1999

The Na+/H+ exchanger modulates the fibrogenic effect of oxidative stress in rat hepatic stellate cells.

BACKGROUND/AIMS: Oxidative stress is associated with liver fibrosis in vivo and with hepatic stellate cell (HSC) activation in vitro, but the intracellular mechanisms mediating these effects are mostly unknown. The Na+/H+ exchanger plays a key role in regulating the cell cycle, and is involved in HSC proliferation. Its role in different HSC features, such as collagen accumulation, is still unknown. We thus evaluated if the Na+/H+ exchanger modulates the fibrogenic effect of oxidative stress in rat HSC. METHODS: HSC were incubated with 0.1 mM ferric nitrilotriacetate complex (FeNTA). Intracellular hydroperoxides and malonildialdehyde (MDA) levels in the culture media were measured by the dichlorofluorescein and TBARS method, respectively. Intracellular pH and Na+/H+ exchanger activity were measured using the fluorescent dye BCECF. Cell proliferation was measured by immunohistochemistry for bromodeoxyuridine incorporation. Collagen type I accumulation in the culture media was measured by ELISA. RESULTS: HSC incubation with FeNTA resulted in a significant production of intracellular hydroperoxides and MDA, associated with increased Na+/H+ exchange activity and baseline intracellular pH (pHi). Exposure of HSC to FeNTA significantly enhanced the number of proliferating HSC and collagen type I levels in the culture medium. All these effects were reversed by the antioxidant resveratrol and by the Na+/H+ exchanger inhibitor amiloride. CONCLUSIONS: This study indicates that the Na+/H+ exchanger might represent a common mediator of the different effects induced by oxidative stress on HSC. The reduction in cell proliferation and collagen synthesis induced by amiloride could represent a new therapeutic challenge in liver fibrosis.

Animals↗

PubMed1999

Effect of pharmacological modulation of liver P-glycoproteins on cyclosporin A biliary excretion and cholestasis: a study in isolated perfused rat liver.

In different cell types P-glycoproteins (P-gp) are involved in the transport of cyclosporin A (CyA). The aim of this study was to evaluate the effect of the pharmacological modulation of the hepatic P-gp on biliary secretion of CyA and on cholestasis induced by acute administration of CyA in the isolated perfused rat liver (IPRL). Verapamil was used as a P-gp specific inhibitor and acetylaminofluorene (AAF) as a P-gp inducer. CyA biliary excretion was determined by administering in the IPRL a tracer dose of [3H]CyA with or without verapamil or AAF. The effect on bile flow was evaluated by administering increasing doses of CyA (2.8, 8, and 20 mg/kg body wt) in the IPRL. Morphological evidence of damage was evaluated by optical and electron microscopy in the liver as well as in primary culture of rat hepatocytes exposed to CyA +/- verapamil. Verapamil significantly inhibited the biliary excretion of a tracer dose of [3H]CyA (0.15+/-0.04 vs 0.33+/-0.07%; P < 0.05). In contrast, pretreatment with AAF significantly increased the biliary excretion of [3H]CyA, (0.61+/-0.10 vs 0.33+/-0.07%; P < 0.05). CyA induced a dose-dependent inhibition of bile flow with a maximal effect at 20 mg/kg CyA (-49.3+/-4.5% decrease of basal bile flow). CyA cholestasis was significantly worsened by the P-gp inhibitor, verapamil (-75.5+/-7.5%; P < 0.05), but it was unaffected by induction of P-gp via AAF pretreatment (-44.9+/-1.7%). During CyA cholestasis, the cumulative biliary excretion of [3H]CyA was lower than in the absence of cholestasis (0.22+/-0.05 vs 0.33+/-0.07%; P < 0.05), was inhibited by verapamil (0.08+/-0.01%; P < 0.05), but was unaffected by AAF (0.23+/-0.05%). No morphological evidence of damage was observed in the liver, and no evidence of cytoskeleton derangement was seen in primary cultures of rat hepatocytes exposed to CyA +/- verapamil. We demonstrated that pharmacological modulation of P-gp may influence the biliary excretion of CyA. The acute cholestatic effect of CyA is worsened by P-gp inhibitors, while it is unaffected by P-gp inducers. This indicates CyA should not be given with other P-gp substrates or inhibitors.

2-Acetylaminofluorene↗

PubMed1999

Hepatic stellate cell activation and liver fibrosis are associated with necroinflammatory injury and Th1-like response in chronic hepatitis C.

BACKGROUND/AIMS: The involvement of a direct viral cytopathic effect or an immune-mediated mechanism in the progression of hepatic damage in chronic hepatitis C is controversial. The type of immune response is itself a matter of controversy, and histological data are lacking. The aim of this study was to identify the factors associated with the progression of liver injury in 30 HCV/RNA-positive untreated patients with chronic hepatitis. METHODS: Necroinflammatory and architectural damage were evaluated using Ishak's score. Activated hepatic stellate cells (HSC) were visualized by immunohistochemistry for alpha-smooth muscle actin (alphaSMA) and quantitated by morphometry. Plasma HCV/RNA was evaluated using a competitive RT-PCR method. To study the type of immune response involved in the progression of liver injury, interferon gamma (IFNgamma)-positive cells (as expression of a Th1-like response) were evaluated by immunohistochemistry and quantitated by morphometry. RESULTS: HSC were mostly detected close to areas of lobular necroinflammation or lining fibrotic septa. The alphaSMA- and Sirius Red-positive parenchyma correlated significantly with necroinflammatory and architectural scores. IFNgamma-positive cells were detected in periportal areas associated with the inflammatory infiltrates and significantly correlated with architectural damage. No relationship was found between the histological features of liver injury and viral load. CONCLUSIONS: HSC activation and progression of liver injury are unrelated to viral load but associated with a Th1-like response, a plausible target for the treatment of chronic hepatitis C.

Actins↗

PubMed1999

The significance of apoptosis in the liver.

Apoptosis is a form of cell death which occurs in normal as well as in pathological tissues. We provide a description of the morphological changes during apoptosis and an overview of the role of apoptosis dysregulation in the pathogenesis of non-neoplastic liver diseases.

Apoptosis↗

PubMed1999

Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver.

Bile duct damage and/or loss is limited to a range of duct sizes in cholangiopathies. We tested the hypothesis that CCl4 damages only large ducts. CCl4 or mineral oil was given to bile duct-ligated (BDL) rats, and 1, 2, and 7 days later small and large cholangiocytes were purified and evaluated for apoptosis, proliferation, and secretion. In situ, we measured apoptosis by morphometric and TUNEL analysis and the number of small and large ducts by morphometry. Two days after CCl4 administration, we found an increased number of small ducts and reduced number of large ducts. In vitro apoptosis was observed only in large cholangiocytes, and this was accompanied by loss of proliferation and secretion in large cholangiocytes and loss of choleretic effect of secretin. Small cholangiocytes de novo express the secretin receptor gene and secretin-induced cAMP response. Consistent with damage of large ducts, we detected cytochrome P-4502E1 (which CCl4 converts to its radicals) only in large cholangiocytes. CCl4 induces selective apoptosis of large ducts associated with loss of large cholangiocyte proliferation and secretion.

Animals↗

PubMed1998

Heterogeneity of glucose transport in rat liver microsomal vesicles.

Glucose transport across the membrane of rat liver microsomal vesicles was studied by a rapid filtration method in three different experimental systems: (i) inward transport in the presence of extravesicular glucose, (ii) efflux from passively preloaded vesicles, and (iii) efflux of glucose generated intravesicularly by glucose-6-phosphatase upon addition of glucose 6-phosphate were investigated. The apparent intravesicular glucose space estimated with the rapid filtration method was lower than the total microsomal glucose accessible space both the in the steady-state phase of uptake and at the starting point of efflux: 0.5 versus 2.3 microl/mg protein. The initial rate of influx/efflux was dependent on the extravesicular/intravesicular glucose concentration and was much lower than the rate of influx estimated previously by the light-scattering technique. Both influx and efflux could be inhibited by N-ethylmaleimide and possibly became saturable at high (>100 mM) glucose concentration. Known inhibitors of GLUT transporters (genistein, cytochalasin B, phloretin, and hexoses) did not affect glucose influx. The time course of glucose efflux from vesicles preincubated in the presence of glucose 6-phosphate was similar to that from glucose-loaded vesicles. These data together with that obtained previously (by a light-scattering technique; Marcolongo, P., Fulceri, R., Giunti, R., Burchell, A., and Benedetti, A. (1996) Biochem. Biophys. Res. Commun. 219, 916-922) indicate that microsomal vesicles are heterogeneous regarding their glucose-transporting properties and that glucose transport is bidirectional and its feature meets the requirements of a facilitative transport.

Animals↗

PubMed1998

Structure and mutation analysis of the glycogen storage disease type 1b gene.

Glycogen storage disease (GSD) 1b is the deficiency of endoplasmic reticulum glucose-6-phosphate (G6P) transport. We here report the structure of the gene encoding a protein likely to be responsible for G6P transport, and its mapping to human chromosome 11q23.3. The gene is composed of nine exons spanning a genomic region of approximately 4 kb. Primers based on the genomic sequence were used in single strand conformation polymorphism (SSCP) analysis and mutations were found in six out of seven GSD 1b patients analysed.

Antiporters↗

PubMed1998

On the mechanism of action of econazole, the capacitative calcium inflow blocker.

The ability of bovine serum albumin to reverse the inhibitory action of econazole and the unsaturated fatty acid oleate on store-dependent Ca2+ inflow was examined in Ehrlich ascites tumour cells. We report that inhibition of Ca2+ inflow by both compounds is reversed immediately upon addition of bovine serum albumin. It is concluded that the inhibitory action of econazole resembles that of unsaturated fatty acids. The mechanism appears to be one pertaining to nonspecific events at the plasma membrane, possibly involving alterations in plasma membrane fluidity/structure.

Animals↗

PubMed1998

Gulonolactone oxidase activity-dependent intravesicular glutathione oxidation in rat liver microsomes.

The orientation of gulonolactone oxidase activity was investigated in rat liver microsomes. Ascorbate formation upon gulonolactone addition resulted in higher intravesicular than extravesicular ascorbate concentrations in native microsomal vesicles. The intraluminal ascorbate accumulation could be prevented or the accumulated ascorbate could be released by permeabilising the vesicles with the pore-forming alamethicin. The formation of the other product of the enzyme, hydrogen peroxide caused the preferential oxidation of intraluminal glutathione in glutathione-loaded microsomes. In conclusion, these results suggest that the orientation of the active site of gulonolactone oxidase is intraluminal and/or the enzyme releases its products towards the lumen of the endoplasmic reticulum.

Alamethicin↗

PubMed1998

Histones and basic polypeptides activate Ca2+/cation influx in various cell types.

Histone H2A (1-10 microg/ml) added to Ehrlich ascite cell suspensions promoted: (i) Ca2+ influx, but no apparent intracellular Ca2+ mobilization; (ii) plasma-membrane depolarization and Na+ influx in Ca2+-free medium, which were recovered by Ca2+ readmission; (iii) influx of other cations such as Ba2+, Mn2+, choline+ and N-methyl-d-glucamine+, but not of propidium+, ethidium bromide and Trypan Blue. H2A-induced Ca2+ influx and cell depolarization were: (i) blocked by La3+ and Gd3+, but not by various inhibitors of receptor-activated Ca2+-influx pathways/channels; (ii) mimicked by various basic polypeptides, with Mr>4000; (iii) prevented or reversed by polyanions such as polyglutamate or heparin; (iv) present in other cell types, such as Jurkat, PC12 and Friend erythroleukaemia cells, but virtually absent from rat hepatocytes and thymocytes. We conclude that cationic proteins/polypeptides, by interacting in a cell-specific manner with the cell surface, can activate in those cells putative non-selective Ca2+ channels and membrane depolarization.

Animals↗

PubMed1998

Dehydroascorbate and ascorbate transport in rat liver microsomal vesicles.

Ascorbate and dehydroascorbate transport was investigated in rat liver microsomal vesicles using radiolabeled compounds and a rapid filtration method. The uptake of both compounds was time- and temperature-dependent, and saturable. Ascorbate uptake did not reach complete equilibrium, it had low affinity and high capacity. Ascorbate influx could not be inhibited by glucose, dehydroascorbate, or glucose transport inhibitors (phloretin, cytochalasin B) but it was reduced by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and by the alkylating agent N-ethylmaleimide. Ascorbate uptake could be stimulated by ferric iron and could be diminished by reducing agents (dithiothreitol, reduced glutathione). In contrast, dehydroascorbate uptake exceeded the level of passive equilibrium, it had high affinity and low capacity. Glucose cis inhibited and trans stimulated the uptake. Glucose transport inhibitors were also effective. The presence of intravesicular reducing compounds increased, while extravesicular reducing environment decreased dehydroascorbate influx. Our results suggest that dehydroascorbate transport is preferred in hepatic endoplasmic reticulum and it is mediated by a GLUT-type transporter. The intravesicular reduction of dehydroascorbate leads to the accumulation of ascorbate and contributes to the low intraluminal reduced/oxidized glutathione ratio.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

Fibrogenic effect of oxidative stress on rat hepatic stellate cells.

Oxidative stress is associated with liver fibrosis and with hepatic stellate cell (HSC) activation in vivo. However, it remains controversial whether oxidative stress contributes to HSC activation either directly or through a paracrine stimulation by damaged hepatocytes. A medium containing products released from cells undergoing oxidative stress was obtained after incubation of hepatocytes with (HCM/Fe) or without (HCM) 0.1 mmol/L ferric nitrilotriacetate complex (FeNTA). Exposure of HSC to HCM/Fe for 24 hours significantly increased the number of proliferating HSC compared with HCM and to controls at all dilutions tested. The simultaneous coincubation of HSC with HCM/Fe and desferrioxamine (50 micromol/L) did not reduce the observed increase in cell proliferation, thus excluding a role for eventually contaminating iron in HCM/Fe. HCM/Fe induced also a significant increase in collagen type I accumulation in HSC culture media. To study the cellular mechanism underlying HCM/Fe effects, we evaluated the activity of the Na+/H+ exchanger, which plays a role in regulating HSC proliferation. The incubation of HSC for 24 hours with HCM/Fe significantly increased baseline intracellular pH (pHi) and Na+/H+ exchanger activity, indicating a plausible role of this antiport in mediating cell response. In conclusion, hepatocytes undergoing oxidative stress release factors which are fibrogenic for HSC, thereby, confirming what has been only hypothesized in vivo. In addition, HSC proliferation is associated with changes in the Na+/H+ exchanger activity, thus providing a useful target for the evaluation of inhibitors of this pathway for the treatment of hepatic fibrosis.

Animals↗

PubMed1998

Delineating a putative phobic-anxious temperament in 126 panic-agoraphobic patients: toward a rapprochement of European and US views.

BACKGROUND: The current US official position, since DSM-III, is that panic attacks represent the hallmark of panic disorder and play a major role in the development of the agoraphobic syndrome. The more favoured view in the European tradition is that neurotic personality and/or prodromal features such as mild depression and excessive worries precede the illness. METHOD: We studied 126 consecutive cases of panic disorder with or without agoraphobia by DSM-III-R criteria, evaluated by relevant structured and semi-structured interviews. RESULTS: We provide evidence that characterological and prodromal antecedents represent a putative phobic-anxious temperamental substrate occurring in at least 30% of our sample. This temperament consists of three or more of the following traits: (1) increased sympathetic activity with repeated sporadic and isolated autonomic manifestations; (2) marked fear of illness; (3) hypersensitivity to separation; (4) difficulty to leave familiar surroundings; (5) marked need for reassurance; (6) oversensitivity to drugs and substances. Our data further suggest that these attributes are of familial origin, as a result of which the illness tends to declare itself earlier. LIMITATION: The present investigation is largely correlational without a prospective component; however, the key validating familial data were obtained blindly. CONCLUSION: Our data support a pathogenetic model whereby genetic diathesis unfolds from subclinical to clinical manifestations along temperamental, panic, phobic and avoidant patterns. We submit that the delineation of the phobic-anxious temperament will be useful in more completely charting the life course of the panic-agoraphobic spectrum; avoidant and dependent (Axis II) patterns appear more distal in the pathogenetic chain and, in many cases, can be conceptualized to be epiphenomenal to the disease process.

Adolescent↗

PubMed1998

Liver microsomal transport of glucose-6-phosphate, glucose, and phosphate in type 1 glycogen storage disease.

The transport of glucose-6-phosphate (G6P), glucose, and orthophosphate into liver microsomes, isolated from six patients with various subtypes of type 1 glycogen storage disease (GSD), was measured using a light-scattering method. We found that G6P, glucose, and phosphate could all cross the microsomal membrane, in four cases of type 1a GSD. In contrast, liver microsomal transport of G6P and phosphate was deficient in the GSD 1b and 1c patients, respectively. These results support the involvement of multiple proteins (and genes) in GSD type 1. The results obtained with the light-scattering method are in accordance with conventional kinetic analysis of the microsomal glucose-6-phosphatase system. Therefore, this technique could be used to directly diagnose type 1b and 1c GSD.

Adult↗

PubMed1998

Differential expression of cyclin-dependent kinase 6 in cortical thymocytes and T-cell lymphoblastic lymphoma/leukemia.

Cyclin-dependent kinase-6 (CDK6) is the earliest inducible member of the CDK family in human T lymphocytes, involved in growth factor stimulation and cell cycle progression. CDK6 is one of the targets of p16 and p15, CDK inhibitors encoded by MTS1 and MTS2, two tumor suppressor genes that are frequently deleted in T-cell leukemia. In this study we have investigated CDK6 expression in normal and neoplastic lymphoid tissues using immunohistochemistry and flow cytometry. In normal (six samples) and hyperplastic (four samples) thymuses, strong CDK6 expression was observed in a discrete proportion of cortical thymocytes (10 to 15%), mainly located in the peripheral (subcapsular) zone of the cortex. All tested cases of T-cell lymphoblastic lymphoma/leukemia (T-LBL/ALL) showed strong CDK6 expression in the majority (up to 100%) of neoplastic lymphoid cells. Western blot analysis confirmed the expected CDK6 protein size (40 kd). According to Southern blot analysis, CDK6 overexpression in neoplastic T lymphoblasts was not due to gene amplification. In all other lymphomas investigated (28 peripheral T-cell non-Hodgkin's lympohomas (T-NHLs), 7 CD30+ anaplastic NHLs, 22 high-grade B-NHLs, 15 low-grade B-NHLs, 25 B-cell precursor ALLs), CDK6 was not expressed or expressed at low levels, with the only exception of three nasal angiocentric T-NHLs, all exhibiting CDK6 immunoreactivity comparable to that observed in T-LBL/ALL. These data provide evidence that CDK6 is abnormally expressed in T-LBL/ALL and may be involved in the pathogenesis of this malignancy. In addition, the quantitative difference of CDK6 expression between neoplastic and non-neoplastic cortical thymocytes can be potentially useful in the differential diagnosis of thymic neoplasms on histological and cytological specimens.

Blotting, Southern↗

PubMed1998

Differential expression of p57kip2, a maternally imprinted cdk inhibitor, in normal human placenta and gestational trophoblastic disease.

Evidence has recently been provided to support a role for genomic imprinting in the regulation of embryonic implantation and development and placental growth, as well as in the pathogenesis of proliferative trophoblastic diseases. The cyclin-dependent kinase inhibitor p57KIP2 has recently been recognized as a maternally imprinted gene. We investigated p57KIP2 expression in first-trimester normal placentas from interrupted pregnancy, spontaneous abortions, and different types of proliferative trophoblastic diseases using single- and double-marker immunohistochemical techniques. In normal placenta, nuclear p57KIP2 expression was observed at high frequency (up to 100%) in extravillous trophoblast, cytotrophoblast, and implantation-site interstitial trophoblast, but was absent in syncytiotrophoblast. p57KIP2 was also expressed in the stromal cells of maternal decidua, which was one of the few adult tissues retaining p57KIP2 expression (most other adult tissues investigated were negative). p57KIP2 expression was either absent or low in all cases of diploid/tetraploid complete moles (20 cases) and in three cases of gestational choriocarcinoma. On the other hand, all spontaneous abortions (12 cases) and triploid partial moles (19 cases) showed p57KIP2 levels comparable to those observed in normal placenta. These findings are in line with the hypothesis that deregulation of genomic imprinting, particularly the loss of cell-cycle inhibitors such as p57KIP2, is involved in the abnormal development of androgenetic trophoblastic proliferations. In addition, this simple immunohistochemical analysis could provide a useful diagnostic marker in difficult cases.

Choriocarcinoma↗

PubMed1998

Observer agreement in endoscopic assessment of ulcerative colitis.

BACKGROUND: Colonoscopy is the investigation of choice to evaluate ulcerative colitis, but the reliability of the assessment of endoscopic signs is not clear. AIMS: The aim of this study was to evaluate interobserver agreement for the identification of endoscopic lesions typical of ulcerative colitis, and the influence of training. MATERIAL AND METHODS: Four experienced observers and 11 endoscopists under training assessed 49 still images selected from endoscopic video recordings. RESULTS: The agreement rate between experienced observers was excellent or good (k > 0.39) for recognition of 10 out of 14 signs or patterns (loss of vascular pattern, erythema, oedema, granular mucosa, blood, pseudopolyp, erosion, ulcer, normal pattern, severe activity), and was poor for pus, stricture, mild activity, moderate activity. The rates between endoscopists under training were excellent or good for 6 items (loss of vascular pattern, erythema, oedema, pseudopolyp, normal pattern, severe activity). CONCLUSIONS: Trained observers can reproducibly record most endoscopic signs of ulcerative colitis. A reliable overall scoring of severity should be based on a simple three-grading scale, i.e. normal pattern, moderate activity, severe activity. Acceptable agreement rates can be obtained by endoscopists under training on some well-defined endoscopic appearances.

Colitis, Ulcerative↗