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S Gaetani

Publications and source records attributed to S Gaetani.

At least 19 recordsLinked to original sources
PubMed2005

Copper regulated synthesis, secretion and degradation of ceruloplasmin in a mouse immortalized hepatocytic cell line.

Ceruloplasmin, a blue copper oxidase circulating in serum of all vertebrates, is a glycoprotein synthesized mainly in hepatocytes and secreted into plasma with six tightly bound atoms of copper per molecule. Many aspects of the mechanisms by which synthesis and secretion of this protein are regulated by copper are still not known. In HepG2 hepatocarcinoma cells this fine regulation is not maintained; we have then utilized Met-murine-hepatocytes (MMH), isolated from the liver of transgenic mice expressing a truncated form of c-Met (hepatocyte growth factor receptor), that are immortalized but not transformed. Copper deficiency was induced by treatment of cells with bathocuproine disulphonate. Experiments of metabolic labeling with 35S-methionine-cysteine and of Western blotting followed by immunostaining, demonstrated that maturation and secretion of ceruloplasmin but not its synthesis are affected by copper availability. In this paper we have shown that in copper deficiency ceruloplasmin accumulates in a pre-Golgi compartment, in which the protein is still in a Endo H sensitive form, and where presumably copper binding to the apo-protein takes place. Moreover, we found that treatment of copper-deficient cells with the proteasomal inhibitor lactacycstin leads to immature ceruloplasmin accumulation in the cell. We have optimized conditions to induce in vitro copper deficiency and found that MMH-D3 cells represent a suitable model to study in detail the molecular mechanism of copper-regulated ceruloplasmin synthesis, secretion and degradation.

Animals↗
PubMed2005

Regulation of food intake by oleoylethanolamide.

Oleoylethanolamide (OEA), the naturally occurring amide of ethanolamine and oleic acid, is an endogenous lipid that modulates feeding, body weight and lipid metabolism by binding with high affinity to the ligand-activated transcription factor, peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In the present article, we describe the biochemical pathways responsible for the initiation and termination of OEA signaling, and outline the pharmacological properties of this compound in relation to its ability to activate PPAR-alpha. Finally, we discuss the possible role of OEA as a peripheral satiety hormone.

Adipose Tissue↗
PubMed2003

Vitamin A transport: in vitro models for the study of RBP secretion.

Retinol-binding protein (RBP) is the specific plasma carrier of retinol, encharged of the vitamin transport from the liver to target cells. Ligand binding influences the RBP affinity for transthyretin (TTR), a homotetrameric protein involved in the RBP/TTR circulating complex, and the secretion rate of RBP. In fact, in vitamin A deficiency, the RBP release from the hepatocytes dramatically decreases and the protein accumulates in the cells, until retinol is available again. The mechanism is still not clear and new cellular models are needed to understand in detail how the soluble RBP can be retained inside the cell. In fish, a vitamin A transport system similar to that of higher vertebrates is emerging, although with significant differences.

Animals↗
PubMed2002

Isolation, expression and characterization of carp retinol-binding protein.

Vitamin A alcohol and its precursors carotenoids are introduced in the organism with the diet, transported to the liver and from there as retinol to target tissues by a specific carrier, the retinol-binding protein (RBP). RBP, isolated and characterized in many vertebrates, shows very high homology among the species investigated; however, very little is known in fish. In the present work RBP cDNA isolated from a carp liver library was transcribed and translated in vitro and the corresponding protein characterized. Carp RBP amino acid sequence and tertiary structure are highly conserved, but the protein shows two peculiar and unique characteristics: the signal sequence is not processed by the ER signal peptidase and two N-glycosylations are present at the N-terminus portion of the protein. It was also demonstrated that RBP glycosylation is not a feature common to all teleosts. Transfection experiments show that the green fluorescent protein (GFP) can be directed into the secretory pathway by the carp RBP N-terminal region, both in fish and in mammal cells, demonstrating that the sequence, although not processed, is recognized as a secretory signal in different species. Results obtained from different investigators indicated that in fish plasma RBP circulates without interacting with transthyretin (TTR) or other proteins, suggesting that the complex with TTR, whose postulated function is to hamper easy kidney filtration of circulating RBP, has evolved later in the evolutionary scale. This hypothesis is reinforced by the finding that carp RBP, as well as trout and other lower vertebrates in which circulating complex has never been demonstrated, lacks a short C-terminal sequence that seems to be involved in RBP-TTR interaction. In carp, carbohydrates could be involved in the control of protein filtration through the kidney glomeruli. Moreover, experiments of carp RBP expression in Cos-1 cells and in the yeast Saccharomyces cerevisiae show that glycosylation is necessary for protein secretion; in particular, additional in vitro experiments have shown it is involved in protein translocation through ER membranes.

Amino Acid Sequence↗
PubMed2001

An anorexic lipid mediator regulated by feeding.

Oleylethanolamide (OEA) is a natural analogue of the endogenous cannabinoid anandamide. Like anandamide, OEA is produced in cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis, suggesting a function in cellular signalling. However, OEA does not activate cannabinoid receptors and its biological functions are still unknown. Here we show that, in rats, food deprivation markedly reduces OEA biosynthesis in the small intestine. Administration of OEA causes a potent and persistent decrease in food intake and gain in body mass. This anorexic effect is behaviourally selective and is associated with the discrete activation of brain regions (the paraventricular hypothalamic nucleus and the nucleus of the solitary tract) involved in the control of satiety. OEA does not affect food intake when injected into the brain ventricles, and its anorexic actions are prevented when peripheral sensory fibres are removed by treatment with capsaicin. These results indicate that OEA is a lipid mediator involved in the peripheral regulation of feeding.

Animals↗
PubMed2001

Unique biochemical nature of carp retinol-binding protein. N-linked glycosylation and uncleavable NH2-terminal signal peptide.

Retinol transport and metabolism have been well characterized in mammals; however, very little is known in fish. To study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with transthyretin in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensus N-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a nonconserved NH(2)-terminal hydrophobic domain, which functions as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP.

Amino Acid Sequence↗
PubMed2001

Neurofunctional effects of developmental alcohol exposure in alcohol-preferring and alcohol-nonpreferring rats.

The neurofunctional effects of developmental alcohol exposure (3% v/v solution from day 15 of gestation to day 7 after parturition) have been investigated in Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rat lines, selectively bred for opposite alcohol preference and consumption. Alcohol exposure significantly decreased the rate of ultrasonic emission in sP male pups; whereas, it did not affect this indicator of emotional reactivity in sNP animals. Perinatal alcohol intake did not influence either learning of an active avoidance task or hippocampal long-term potentiation in both offspring lines. Significant differences in time spent exploring novel objects were observed between control sP and sNP rats subjected to the novel exploration object test. Alcohol exposed sP rats, but not alcohol exposed sNP rats, apparently lost the capacity to discriminate between the novel and the familiar object, even though this difference is difficult to interpret because of the large differences in the respective responses to the novel objects. Neurochemical experiments have shown that basal levels of dopamine (DA) and homovanillic acid (HVA) were significantly higher in the nucleus accumbens (NAC) of sP rats with respect to sNP animals. Perinatal alcohol did not affect basal DA and HVA concentrations or amphetamine-induced DA increase and HVA decrease in the NAC of either sP or sNP offspring. These results suggest that subtle behavioral alterations induced by developmental exposure to low doses of alcohol, which do not cause malformations and/or overt neurotoxicity, may be associated with genetic factors, although not necessarily those responsible for differences in alcohol preference.

Alcohol-Induced Disorders, Nervous System↗
PubMed2000

In vivo neurochemical effects of the acetylcholinesterase inhibitor ENA713 in rat hippocampus.

Oral ENA713 (0.5, 1.5 and 4.5 mg/kg), an acetylcholinesterase inhibitor (AChEI), dose-dependently enhanced extracellular acetylcholine concentrations in the hippocampus of freely moving rats. This effect was paralleled by changes in both noradrenergic and dopaminergic transmission. In particular, ENA713 significantly decreased noradrenaline concentrations, whereas it significantly increased homovanillic acid levels, without affecting dopamine concentrations. Neither serotonin nor gamma-aminobutyric acid levels were modified by ENA713. These findings extend the neurochemical profile of ENA713 and suggest that it could be useful for the treatment of Alzheimer-type dementia which is associated with multiple neurotransmitter abnormalities in the brain.

Acetylcholine↗
PubMed1999

MMH cells: An in vitro model for the study of retinol-binding protein secretion regulated by retinol.

The untransformed stable cell line Met murine hepatocytes (MMH), generated from liver explants of transgenic mice expressing a constitutively active truncated form of the human hepatocyte growth factor receptor (cyto-Met), represents an innovative tool for in vitro studies of liver function. In the present report, we show that the MMH-D3 line isolated from the liver of a 3-day-old mouse is a useful model to investigate the regulation of the synthesis and secretion of retinol-binding protein (RBP) by retinol (vitamin A alcohol). Experiments with Northern blot hybridization, metabolic labeling of cellular proteins followed by immunoprecipitation, and Western blot analysis demonstrated that, similarly to the in vivo situation, in MMH-D3 cells the presence of retinol does not affect transcriptional and translational rate of the RBP gene but is essential for regulating the secretion rate of the protein. Unlike HepG2 human hepatocarcinoma cells used thus far in studies of retinoid metabolism, including the synthesis and secretion of RBP, vitamin A deficiency causes, in MMH-D3 cells, the inhibiton of RBP secretion and the protein accumulation in the cell, whereas retinol repletion promptly results in RBP secretion. This model will be very useful in future studies on vitamin A distribution in the organism.

Animals↗
PubMed1998

In vitro and in vivo studies on transthyretin oligomerization.

The oligomerization of transthyretin has been studied in vitro and in vivo. The results showed that wild-type transthyretin synthesized in vitro in the absence of microsomes did not form dimers and tetramers, but in the presence of microsomes the mature transthyretin which had been translocated into the microsomal lumen formed dimers and a small amount of tetramers which could be detected only by using a cross-linking reagent. Efficiency of tetramer formation depends upon the source of microsomes; in fact the amount of tetramer formed in liver microsomes was much higher than that in pancreas microsomes. Transthyretin synthesized in HepG2 cells appeared after SDS-PAGE analysis in mostly tetrameric form, while that synthesized in transfected COS-1 cells appeared mainly as dimers. Brefeldin A treatment and pulse-chase experiments in HepG2 cells showed that transthyretin tetramer was formed in the endoplasmic reticulum. These results strongly indicate that transthyretin tetramer is formed most efficiently in the endoplasmic reticulum lumen of hepatocytes. Transthyretin without the signal peptide [S(-)] and transthyretin with a mutation that prevents processing of the signal peptide Msc failed to form dimers and tetramers in vitro. In the transfected COS-1 cells, however, S(-) transthyretin did form dimers while Msc transthyretin failed to oligomerize. These results show that the cleavage of the signal peptide and some cellular factors are required for transthyretin oligomerization.

Animals↗
PubMed1998

Effects of retinoids on gene expression in different epithelial models in vivo and in vitro.

We have previously reported that the induction of Vitamin A deficiency results in a threefold decrease in the hepatic expression of cellular retinol binding protein I (CRBP I) mRNA in vivo and that the treatment of intestinal cell lines in vitro with retinoids leads to the induction of CRBP I transcription. In the present paper we extend the analysis to retinoid-dependent gene expression in the testicular epithelium in vivo and in the intestinal cell line FRIC B. In rat testis excess Vitamin A results in the reduced production of mature spermatozoa and in the premature release of immature germ cells in the lumen, while Vitamin A deficiency leads to almost complete degeneration of the germinal epithelium. We show reduced level of expression of CRBP I mRNA in vitamin A deficient testis. Retinoid treatment of cultured intestinal cells, which induces a reorganization of the actin cytoskeleton, has no effect on the expression of the differentiation induced gene Dri 42. The results show that even though unable to trigger by themselves the differentiation process, retinoids exert a direct effect on the expression of specific genes.

Animals↗
PubMed1996

Retinol binding protein and transthyretin are secreted as a complex formed in the endoplasmic reticulum in HepG2 human hepatocarcinoma cells.

Retinol binding protein (RBP), the retinol-specific carrier, circulates in blood as a 1:1 complex with the homotetrameric protein transthyretin (TTR). Both RBP and TTR are synthesized and secreted by the hepatocyte. In this work we have demonstrated, using HepG2 cells as a model system, that the association between the two proteins occurs inside the cell before secretion. The intracellular complex was detected only when metabolically labeled cells were lysed under mild detergent conditions (1.5% octylglucoside), followed by immunoprecipitation and SDS-PAGE. Alternatively, the immunoprecipitates from unlabeled cells lysed with the same buffer were analyzed by Western blotting. This finding was confirmed using the cross-linking agent dithiobis(succinimidyl) propionate before cell lysis. Moreover, we found that in cells treated with brefeldin A to block the exit of proteins from the endoplasmic reticulum (ER), the complex was present in the microsomal fraction. Thus, we can conclude that the RBP-TTR complex is formed inside the cell, more precisely within the ER. As RBP and TTR both lack an ER retention signal, we considered the possible involvement of chaperones in RBP and TTR retention in the ER and in complex formation. We found that calnexin, an ER integral membrane protein which functions as a chaperone, coprecipitates with RBP and TTR when cell lysis and immunoprecipitation are performed under mild conditions (1% Triton X-100). This result strongly suggests that calnexin may be involved in RBP and TTR retention in the ER, in TTR tetramer assembly, and possibly in complex formation.

Brefeldin A↗
PubMed1994

Subtractive hybridization cloning of novel genes differentially expressed during intestinal development.

Intestinal genes whose expression is regulated during development and differentiation were identified and cloned from a rat villi cDNA library using a subtracted cDNA probe. The isolated clones are transcribed in the fully differentiated intestinal epithelium 21 days after birth and absent or poorly expressed in the fetal gut at 15 days of gestation. Two of the DRI (differentially-expressed in rat intestine) genes are novel, while the others encode the microvillar protein ezrin and intracellular carrier proteins for retinol and fatty acids. Expression of the newly isolated DRI27 and DRI42 clones parallels epithelial differentiation during development and it is more pronounced in the distal portions of the small intestine. In situ hybridization experiments indicate that the DRI mRNAs are expressed in the differentiated cell types of the gut epithelium. Moreover, the expression of DRI27 and DRI42 is strongly related to the stage of epithelial differentiation during gut development. This relationship holds true also for the expression of DRI42 in other tissues. These clones will be a valuable tool to identify regulatory sequences and factors responsible for confining gene expression to the differentiated epithelial cell types in mammalian small intestine.

Animals↗
PubMed1994

First-trimester human trophoblast is class II major histocompatibility complex mRNA+/antigen.

Lack of expression of the polymorphic class I and class II MHC antigens in the cytotrophoblast is one of the major factors determining the privileged immunologic status of the placenta. In this report, we show that first-trimester human placental cytotrophoblast cells display moderate to strong expression of class II MHC (HLA-DR alpha and -DR beta) and Ii chain transcripts, apparently in absence of detectable class II antigens and Ii chain. In addition, DR alpha, DR beta, and Ii mRNAs, but not antigens, are consistently upregulated by IFN-gamma. Constitutive expression and upregulation of mRNAs are detectable in trophoblast cells kept in short term as well as prolonged (2-3 weeks) culture. These results are reminiscent of an analogous mRNA+/antigen- dissociation occurring, in the case of class I MHC gene products, in a subpopulation of first-trimester cytotrophoblast cells. Thus, analogous mechanisms prevent the expression of potentially hazardous class I and II allodeterminants at early stages of semiallogeneic pregnancy.

Blotting, Northern↗
PubMed1993

Metallothionein gene is expressed in developing rat intestine and is induced by zinc but not by corticosteroids.

The expression of metallothionein (MT) mRNA during perinatal development of rat intestine and its induction by zinc and corticosteroids were studied. Pregnant rats from d 17 to 22 of gestation and rats at 2, 4, 13 and 21 d of postnatal life were injected with saline solution (control) or with zinc (10 mg/kg body wt) or corticosteroids (1 mg/kg body wt). After 6 h, tissues were removed for analysis. Northern hybridization of polyA + RNA to 32P-MT-cDNA revealed that MT was expressed already at d 17 of fetal life, increased afterwards (reaching the maximal expression around birth) and decreased soon after until weaning. Metallothionein mRNA was markedly induced by zinc at d 18 of fetal life to a level that remained constant throughout postnatal life. Corticosteroids were ineffective in inducing MT gene expression during prenatal and postnatal development. In 21-d-old adrenalectomized rats the level of MT mRNA was similar to that of control rats of the same age and was not changed by hormone treatment. The results indicate that MT gene expression can be induced by zinc during fetal life and that its expression without exogenous inducers cannot be ascribed to circulating corticosteroids.

Adrenal Cortex Hormones↗
PubMed1991

Vitamin A intake and in vivo expression of the genes involved in retinol transport.

Two different metabolic alterations in vitamin A status are known to cause changes in the amount of circulating retinol-binding protein (RBP) and cellular retinol-binding protein (CRBP) in experimental animals; namely vitamin A deficiency, characterized by depleted retinol-liver stores and hypervitaminosis A, characterized by hepatic accumulation of retinyl esters. We have induced vitamin A deficiency and hypervitaminosis A in two groups of rats with the aim of determining whether the expression of the genes coding for these two proteins might be directly regulated by retinol. Using human RBP and CRBP cDNAs as probes, we measured the rate of transcription of the two genes in liver nuclei from control and treated rats by run-on transcription assays, and the steady-state level of the mRNAs by Northern blot analysis of total liver RNA. The distribution profile of RBP and CRBP mRNAs on fractionated liver polysomes was also examined. We have found a threefold decrease in the hepatic level of CRBP mRNA in vitamin-A-deficient animals, while the RBP mRNA is not affected by this nutritional deprivation. The decreases does not correspond to a lower transcription rate of the gene and therefore it is likely to result from lower stability of the CRBP mRNA. In hypervitaminosis A, we do not observe any differences in both the steady-state level of the mRNAs and in the rate of transcription of the two genes. The results are discussed in terms of retinol-dependent stabilization of the mRNA coding for CRBP.

Animals↗