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L Scarabelli

Publications and source records attributed to L Scarabelli.

At least 19 recordsLinked to original sources
PubMed2004

Effects of carnitines on rat sertoli cell protein metabolism.

The influence of carnitine on male reproductive function is well established. However, the molecular mechanism by which carnitines control male fertility have not yet been clearly identified. In the present study, we studied the role of carnitines at testis level by examining their effects on protein metabolism in cultured rat Sertoli cells. Our results demonstrate that L-carnitine and, in particular, L-acetylcarnitine administration affects overall protein synthesis, inducing a dose-dependent decrease in amino acid incorporation and consequently an increase of the intracellular amino-acid pool useful to speed up Sertoli cell energy metabolism. We also observed significant effect on the expression of specific proteins involved in the regulation of glucose uptake and Insulin-like Growth Factor (IGF) biological activity. Actually, an increased expression of the mRNA coding for the membrane Glucose Transporter-1 (GLUT-1) and a decreased expression of mRNA coding for the negative modulator insulin-like growth factor-binding protein-4 (IGFBP-4) were induced by in vitro treatment with L-carnitine and L- acetylcarnitine separately and in combination; in fact, the effects observed were far more pronounced following concomitant treatment with both compounds. Taken together, these data indicate that the influence of carnitines on male fertility could be partly mediated by an activity at Sertoli cell level.

Acetylcarnitine↗
PubMed2003

Prolactin effect on pre-pubertal Sertoli cell proliferation and metabolism.

Direct effects of PRL on Sertoli cell proliferation were investigated by using Sertoli cell primary cultures isolated from both prepubertal rat and porcine testes. PRL metabolic effects were analyzed in rat Sertoli cell primary cultures. Exposure to physiological doses of PRL resulted in a significant increase (+50-60%) of basal DNA synthesis, as reflected by the pattern of [3H] thymidine incorporation during culture; significant increases in lactate secretion (about 50%), androgen binding protein (ABP) production (about 30%) and basal protein synthesis (25-30%), as reflected in the augmented [14C] valine incorporation, were also evident. Taken together, our present findings, indicating significant effects of PRL on Sertoli cell proliferation and metabolism, demonstrate that Sertoli cells are a potential target for PRL action at testicular level during pre-pubertal development.

Androgen-Binding Protein↗
PubMed2001

Effect of simulated microgravity conditions on poly(ADP-ribose) polymerase activity in primary cultures of adult rat hepatocytes.

The possible involvement of poly(ADP-ribose) polymerase [PARP; E.C. 2.4.2.30] in the adaptive response to low-g conditions was studied in cultured adult rat hepatocytes exposed to simulated microgravity produced by the random positioning machine (RPM-3D-clinostat). Four different poly(ADP-ribose) polymerases (PARPs) have been identified recently. The best-studied member of this family is PARP-1, a highly conserved, multimodular 113 kDa protein. In multicellular organisms PARPs catalyze poly(ADP-ribose) synthesis from NAD+ to a number of structural and catalytic proteins. Moreover, PARP-1 can control its protein and DNA interactions by catalyzing its automodification with poly(ADP-ribose) molecules that can include up to 200 ADP-ribose residues and several branching points; by these polymers, PARP-1 may nocovalently interact with other proteins and alter their functions. PARP-1 binds to DNA and is activated by free ends interacting with several other DNA damage checkpoint proteins. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions. Poly(ADP-ribosyl)ation plays a central role in genome stability and is involved in DNA replication and repair, gene expression, cell differentiation and transformation. We have shown that a loss of PARP-1 activity is a critical event in the early molecular steps of the hepatocarcinogenesis process. Moreover, a prompt increase in this enzymatic activity is linked not only to the presence of DNA free ends but is linked also to the start of DNA synthesis. More recently, we have reported that PARP-1 is involved in hormone-mediated gene expression in vitro and in vivo during rat liver regeneration.

Adaptation, Physiological↗
PubMed2000

Poly(ADP-ribose) polymerase is affected early by thyroid state during liver regeneration in rats.

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA synthesis, DNA repair, and cell replication and transformation, also plays a role in the early steps of liver regeneration induced by partial hepatectomy (PH). PARP and DNA topoisomerase I (Topo I) activities and de novo DNA synthesis were studied during liver regeneration in rats with altered thyroid state. Hepatic PARP activity, evaluated as [(32)P]NAD incorporated into isolated liver nuclei, was inhibited in hyperthyroid rats and increased in hypothyroid animals. In both euthyroid and hyperthyroid rats PARP activity was rapidly stimulated, peaking 6 h after PH. In hypothyroid animals, an early decrease in activity was found, at a minimum of 6 h after PH, followed by an early onset of DNA synthesis. An inverse relationship between PARP and Topo I activities was a shared feature among euthyroid, hypothyroid, and hyperthyroid rats. Together these data show that, in replicating hepatocytes, thyroid hormones exert a regulatory role on PARP activity, which reflects the control of a number of nuclear proteins involved in DNA metabolism.

Animals↗
PubMed1994

Hepatic poly(ADP-ribose) polymerase activity in rat is controlled by thyroid hormones.

The in vivo effect of the thyroidal state on poly(ADP-ribose) polymerase activity was investigated in eu- and hypothyroid rats after treatment with L-triiodothyronine. Untreated hypothyroids showed an increased basal rate of the enzyme. The treatment of both eu- and hypothyroid rats with L-triiodothyronine induced a prompt drop of the endogenous activity not due to a reduction of the catalytic protein. This decrease well evident 1 h after treatment was transient, returning to controls values within 8 h. In isolated liver nuclei from euthyroids the in vitro exposure to increasing L-triiodothyronine concentrations from 10(-18) to 10(-6) M resulted in a progressive inhibition of the enzyme. This loss in activity was not derived from a reduction of the total level of the catalytic protein. The pretreatment with the antagonist amiodarone suppressed the hormone effect, suggesting that nuclear receptors could mediate poly(ADP-ribose) polymerase activity.

Amiodarone↗
PubMed1993

Relationship between poly(ADP-ribose) polymerase activity and DNA damage induced by zinc dithiocarbamates in mouse and rat liver.

The genotoxic effects due to in vivo treatment with zinc dithiocarbamates were evaluated in rat and mouse liver. The two pesticides Zineb and Ziram, belonging to this chemical class, induced an increase in single-strand DNA breaks, as measured by the alkaline elution technique. The nuclear enzyme poly(ADP-ribose) polymerase (pADPRP), a chromatin-bound catalytic protein, utilizing NAD+ as a substrate, was tested by a radiometric procedure. A close relationship between the increased extent of DNA damage and the enhanced level of endogenous pADPRP activity was obtained in rat liver, whereas both parameters remained unchanged in mouse liver.

Animals↗
PubMed1992

Influence of poly(ADP ribose) polymerase depletion on promotion of liver carcinogenesis.

In previous studies we demonstrated that liver poly(ADP ribose) polymerase (pADPRP) activity was lost in animals exposed to N-2-acetylaminofluorene (2AAF) according to the Teebor and Becker experimental model (Cancer Res 31:1-3, 1971). In addition, we used the resistant hepatocyte model of Solt and Farber (Nature 263:702-703, 1976) to further investigate pADPRP activity during the multistep process of liver carcinogenesis. A marked depletion of the catalytic protein was evidenced after 2AAF exposure, confirming previous results and indicating a specific effect of 2AAF on this nuclear enzyme that controls conformational changes of chromatin and regulates several catalytic activities in the nucleus. The levels of pADPRP mRNA, measured by northern blot analysis using both experimental models, indicate that the enzyme depletion is not due to a loss of transcript. Moreover, these data indicate that pADPRP depletion, caused by 2AAF, was also maintained during liver compensatory growth, which is known to induce a rapid and marked increase in pADPRP activity and protein level. Treatment of 2AAF-exposed animals with N-acetyl-L-cysteine not only efficiently protected against DNA damage, but also prevented a rapid depletion of the catalytic protein. Interestingly, these data indicate that the marked loss of liver pADPRP occurred during the promotion step induced by 2AAF feeding and that this loss was observed using different models for experimental hepatocarcinogenesis. This phenomenon can be ascribed to a highly defective transcript that cannot be correctly translated into the specific protein or to a rapid degradation of the translated protein.

2-Acetylaminofluorene↗
PubMed1992

Effect of N-2-acetylaminofluorene on poly(ADP-ribose) polymerase activity and DNA synthesis in cultured rat hepatocytes exposed to epidermal growth factor.

The nuclear enzyme poly(ADP-ribose) polymerase is involved in basic cellular processes such as DNA replication and repair, cell differentiation and transformation, gene expression. We have studied the effect of 2AAF, a genotoxic aromatic amine, on pADPRP activity during DNA synthesis stimulated by EGF, using the cultured rat hepatocytes model. DNA synthesis was measured as [3H]thymidine incorporated/microgram DNA while pADPRP activity was expressed in pmol[32P]NAD incorporated/min/microgram DNA. Our results show that 2AAF treatment of EGF-stimulated rat hepatocytes induces a full block of DNA replication which is preceded and accompanied by a net inhibition of endogenous and total pADPRP activity, respectively. A block in pADPRP activity in normal hepatocytes, exposed to 2AAF in vitro or in vivo, could play a key role in cell transformation. Our data add further information on the possible involvement of this nuclear catalytic activity during DNA replication.

2-Acetylaminofluorene↗
PubMed1991

Relationship between DNA topoisomerase I activity and DNA synthesis in cultured hepatocytes: effects of orotic acid.

Previous studies have demonstrated that DNA topoisomerase I activity can be closely related to DNA replication and active transcription in different experimental models. This relationship was further investigated by studying the time course of DNA topoisomerase I activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. DNA topoisomerase I activity was assayed quantifying ATP-independent relaxation of a negatively supercoiled plasmid, substrate for the enzyme. Incubation of cellular extracts was carried out at 30 degrees C for 15'. Products were electrophoresed and analyzed by densitometry. DNA synthesis was measured as [3H]thymidine incorporation in the hepatocytes, after a 2 hrs pulse labelling. An increase in DNA topoisomerase I activity was observed early after epidermal growth factor addition, before the onset of DNA synthesis. Concomitant administration of orotic acid, which has recently shown mitoinhibitory effect, abolished the EGF-induced activity as well as DNA synthesis. Taken together these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by DNA topoisomerase I with a precise time schedule. Orotic acid administration abolishes this response and causes an overall loss in DNA topoisomerase I activity.

Animals↗
PubMed1990

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration.

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.

Animals↗
PubMed1990

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes.

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.

Animals↗
PubMed1990

Differential assay and biological significance of poly(ADP-ribose) polymerase activity in isolated liver nuclei.

Poly(ADP-ribosyl)ation of nuclear proteins is catalyzed by poly(ADP-ribose) polymerase. This enzyme is involved in the regulation of basic cellular functions of DNA metabolism. DNA breaks induced by DNA-damaging agents trigger the activation of poly(ADP-ribose) polymerase increasing its endogenous level. This increase modifies the pattern of poly(ADP-ribosyl)ated chromatin proteins. In this paper we describe a procedure for the isolation of intact nuclei from rat liver to be used for the endogenous activity assay. Artifactual activation of the enzyme was avoided since a very low level of DNA-strand breaks occurs during the isolation of nuclei. We present a series of experiments which prove the ability of this procedure to detect increases in endogenous liver activity without modification of the total level. The application of this technique can be useful for a better understanding of the role of early changes in poly(ADP-ribose) polymerase level in physiological conditions and during exposure to DNA-damaging agents.

Animals↗
PubMed1988

Depletion of adenosine diphosphate-ribosyl transferase activity in rat liver during exposure to N-2-acetylaminofluorene: effect of thiols.

The exposure of rats to a feeding regimen containing N-2-acetylaminofluorene (2AAF) causes an accumulation of lesions on liver DNA and a progressive impairment in DNA repair capacity. We used the in vivo experimental model of Teebor and Becker (Cancer Res., 31:1-3, 1971) with the carcinogen given to rats during four consecutive cycles, each one composed of 3 weeks of treatment and 1 week of recovery. The extent of DNA damage and repair was determined during each cycle by the alkaline elution technique. The results obtained showed that the number of alkalilabile sites in DNA is significantly enhanced after the first cycle and remains increased during following cycles. Since ADP-ribosyl transferase (ADPRT) is known to play a central role in the response to DNA damage, we investigated the effect of 2AAF on this enzyme during the carcinogenic process. The activity and the structure of ADPRT were analyzed using the activity gel and Western blot techniques. The catalytic band with a molecular weight of 116,000, clearly evident in liver extracts of control rats, was no longer detectable after one cycle of exposure to 2AAF returning progressively to an almost normal level within the last two cycles. When the aminothiol N-acetyl-L-cysteine (NAC) was added to the 2AAF diet, the extent of DNA damage was drastically reduced, and DNA repair activity preserved for a longer period. In addition, the loss of ADPRT was not observed after the first cycle, but delayed to the end of the second, indicating that NAC exerts a protective effect on DNA and on ADPRT. Such effect was not evident when NAC was substituted by glutathione. The analysis of liver extracts on Western blot showed that the ADPRT immunoreactive band was almost undetectable after the first cycle suggesting that the loss in enzyme activity could be due to a block in de novo synthesis of the enzyme and not to an inhibition of its activity.

2-Acetylaminofluorene↗
PubMed1986

Selective adsorption of serum proteins by chrysotile and crocidolite.

Two distinct and contemporaneous adsorption mechanisms occur when serum proteins are incubated with chrysotile and crocidolite. The first one appears to be reversible and nonspecific, independent of the characteristics of the protein and the fiber surface. The second process seems to depend on fiber type and to involve only some protein species that are strongly concentrated on the fiber surface. This latter adsorption mechanism appears to be electrostatic in nature.

Adsorption↗
PubMed1986

Effect of glutathione and N-acetylcysteine on hepatocellular modifications induced by 2-acetylaminofluorene.

The exposure of rats to a dietary regimen containing 2-acetylaminofluorene induces a sequence of hepatocellular alterations leading to the development of preneoplastic nodules. Groups of 2-acetylaminofluorene-treated rats were given glutathione or N-acetylcysteine to evaluate the effects of these different thiols on the sequence of events that originate transformed cells. It is well known that intracellular thiols protect biological macromolecules from scavenging free radicals and electrophilic compounds produced by the metabolism of chemical agents. Male Wistar rats were maintained on a feeding regimen containing 0.05% 2-acetylaminofluorene. The diet of 2 groups of 2-acetylaminofluorene-treated animals was supplemented with either 0.1% glutathione or N-acetylcysteine. The effects in the liver of the exogenously supplied thiols during 2-acetylaminofluorene treatment were assessed evaluating DNA damage, glutathione levels, activity of marker enzymes glucose-6-phosphatase, gamma-glutamyltranspeptidase, and glutathione-S-transferase, survival rates, and development of salivary gland tumors. Our results demonstrate that the mortality due to 2-acetylaminofluorene exposure was reduced or completely abolished by thiols and that the development of salivary gland tumors was inhibited. Exogenously supplied thiols significantly reduced DNA damage as assessed by alkaline elution. At the doses employed, glutathione and N-acetylcysteine induce early stimulation of glutathione-S-transferase, had little effect on the loss of glucose-6-phosphatase activity and scanty influence on the net increase in gamma-glutamyltranspeptidase activity.

2-Acetylaminofluorene↗