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L Monaco

Publications and source records attributed to L Monaco.

At least 55 records · Page 3Linked to original sources
PubMed1993

Molecular characterization of a conserved, guanine nucleotide-dependent ADP-ribosylation factor in Drosophila melanogaster.

ADP-Ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin-catalyzed ADP-ribosylation in vitro. Because the functional role(s) of ARF in mammalian systems is (are) elusive, we looked for ARF in Drosophila melanogaster, and report the partial purification and molecular cloning of an ARF from Drosophila. We cloned the Drosophila ARF 1 gene without library screening by a combination of 5 polymerase chain reactions (PCRs), yielding a 546-base open reading frame encoding 182 amino acids, which are > 93% identical to those of mammalian class I ARFs. This ARF gene maps to 79F3-6 in the proximal region of the left arm of Drosophila chromosome 3. The Drosophila ARF1 gene structure, including placement of introns, is highly conserved relative to mammalian class 1 ARF genes. A single ARF mRNA species of 1.8 kb was abundant in all Drosophila body segments. Recombinant Drosophila ARF 1 synthesized in Escherichia coli had biochemical and immunochemical activities similar to those of mammalian ARF. The similarities of sequence and biochemical properties between Drosophila and mammalian ARFs contrast with their differences from Drosophila arl (ARF-like protein), which does not stimulate cholera toxin-catalyzed ADP-ribosylation, and is only approximately 52-56% identical in amino acid sequence to mammalian ARFs.

ADP-Ribosylation Factor 1↗
PubMed1993

Pro-opiomelanocortin gene is expressed in post-implantation mouse embryos and enhances growth potential of myogenic cells.

We previously showed that the adrenocorticotropin hormone (ACTH) is a mitogen for myoblasts and is present in post-implantation mammalian embryos (Cossu et al. [1989] Dev. Biol. 131:331-336; De Angelis et al. [1992] Dev. Biol. 151:446-458). In this paper, we investigated the expression of the corresponding gene, pro-opiomelanocortin (POMC), by in situ hybridization and polymerase chain reaction. In situ analysis revealed low level expression in the basal layer of 10.5 d.p.c. neural tube and in several discrete areas around the dorsal aorta. By more sensitive Reverse-transcription polymerase chain reaction (RT-PCR) analysis, expression was detected also in developing limb buds and in cultured myogenic cells, but not in fibroblasts. To investigate the possible role of POMC gene expression in myogenesis, we induced its over-expression in proliferating myoblasts. Upon sub-optimal growth conditions, over-expressing cells were found to give rise to clones larger than control cells. The differentiation potential of POMC over-expressing myogenic cells was unchanged.

Animals↗
PubMed1992

Molecular and immunological characterization of ADP-ribosylarginine hydrolases.

Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively. Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis. Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent. A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels. Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains. A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes. Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase. A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity. A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA. The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.

Amino Acid Sequence↗
PubMed1991

Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.

Animals↗
PubMed1990

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin.

ADP-ribosylation factors (ARFs) are approximately 20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known ARFs. Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone psi ARF 4, a putative ARF pseudogene, from a human genomic library in lambda phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A)+ RNA the cDNA corresponding to the expressed homolog of psi ARF 4, referred to as human ARF 4. It appears that psi ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.

ADP-Ribosylation Factors↗
PubMed1989

Signal transduction in the Sertoli cell: serum modulation of the response to FSH.

Immature Sertoli cells of the testicular seminiferous tubule maintain the expression of their differentiated phenotype when cultured in unsupplemented medium. In preliminary experiments we observed that foetal bovine serum (FBS) stimulates polyphosphoinositides (PI) hydrolysis in Sertoli cells. We then evaluated the effect of serum on the function of the immature Sertoli cell in culture, in terms of cAMP and estrogen production. Treatment of Sertoli cells for 30 min with 1-10% FBS had no effect on basal cAMP accumulation but abolished the response to FSH. The serum concentration producing half-maximal inhibition of the FSH-dependent cAMP accumulation was 0.5-1%. Comparison of the FSH-dose-response in the absence or presence of serum showed a decreased maximal response when serum was present. Sertoli cells exposed to serum were also less responsive to the beta-adrenergic agonist isoproterenol, to cholera toxin, and to forskolin. The serum inhibition was rapidly reversed upon removal of serum or incubating the cells with the phosphodiesterase inhibitor MIX (methyl-isobutyl-xanthine). Similarly to what observed with cAMP, serum affected androgen aromatization stimulated by FSH, isoproterenol, cholera toxin, forskolin and dibutyryl cAMP. These data indicate that factors present in serum can act as modulators of the Sertoli cell function in vitro by rapidly and reversibly inhibiting the cAMP and steroidogenic response of the Sertoli cell to FSH.

Animals↗
PubMed1989

Regulation of c-fos messenger ribonucleic acid by fibroblast growth factor in cultured Sertoli cells.

The data reported here demonstrate that basic FGF and Sertoli-cell-derived FGF have rapid stimulatory effects on c-fos mRNA levels. Basic FGF appears to utilize a signal transduction pathway that is distinct from that used by FSH and serum but similar in its potency and transiency. This is consistent with other reports in the literature on FGFs mechanism of action. For example, a monoclonal antibody to phosphatidyl 4,5-biphosphate will block the effects of PDGF on thymidine incorporation into NIH 3T3 cells but has no effect on basic FGF. Our data support the emerging possibility of a novel pathway mediating FGF actions. A similar precedent has been established for serotonin-induced DNA synthesis in smooth muscle cells. The rat Sertoli cell, with both FSH and FGF rapidly inducing c-fos, is an excellent model system for addressing the mechanism of action of basic FGF, the specificity of the c-fos response and the role of FGF in the reproductive system. Stimulation of c-fos mRNA by basic FGF in the cultured Sertoli cell presents questions regarding the role of FGF and c-fos in the male reproductive system. Basic FGF has been shown to stimulate cell division and plasminogen activator activity in cultured immature porcine Sertoli cells. Plasminogen activator may play a critical role in the tissue remodeling required for spermiogenesis. Interestingly, fos has been shown to be expressed preferentially in pachytene spermatocytes in the mouse. These observations taken together with the finding that basic FGF mRNA levels in Xenopus oocytes are markedly elevated, suggests that basic FGF may be important for Sertoli cell function, spermatogonial cell proliferation and subsequent meiotic and sperm maturational events.

Animals↗
PubMed1989

A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells.

We have shown that the cultured Sertoli cell from the immature rat contains a fibroblast growth factor (FGF)-like factor. It behaves as a cationic peptide, is a potent competence factor for BALB/c3T3 mouse embryo fibroblasts, and displays a high affinity for heparin. Both bovine basic FGF and Sertoli cell FGF-like factor rapidly increase c-fos mRNA in cultured Sertoli cells. FSH, serum, and phorbol esters individually stimulate c-fos in cultured Sertoli cells whereas platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-I have little affect. However, unlike FSH, basic FGF does not stimulate an increase in cAMP and unlike either serum or phorbol esters, basic FGF does not stimulate phosphoinositol turnover or intracellular calcium changes. When Sertoli cell protein kinase C activity is suppressed by preexposure to phorbol ester, basic FGF continues to be a potent stimulator of c-fos, indicating that the calcium/phospholipid pathway is not involved in FGF induction. Basic FGF and FSH also increase jun-B mRNA levels in cultured Sertoli cells. In response to FGF, jun-B is more transiently increased than c-fos. In contrast, in response to FSH, jun-B persists longer than c-fos. These results indicate that cultured Sertoli cells contain a FGF-like factor that increases c-fos mRNA via a mechanism not involving cAMP and the calcium/phospholipid pathways. The different responsiveness of c-fos and jun-B to FSH and basic FGF may explain differences in the ultimate actions of these two ligands.

Animals↗
PubMed1988

Effects of calcitonin gene-related peptide on synaptic acetylcholine receptor-channels in rat muscle fibres.

Cultured myotubes and freshly dissociated muscle fibres from adult rats were exposed to calcitonin gene-related peptide (CGRP) and studied by patch-clamp recording during the peptide-induced maximal accumulation of cellular cyclic AMP (cAMP). Acetylcholine receptor- (AChR-) channel properties in myotubes were not modified by the presence of CGRP (10(-7) M). The peptide, applied to the non-patched membrane, significantly increased the variance of the AChR-channel amplitude distribution at the synaptic region of muscle fibres, and three classes of AChR-channels were resolved immediately after peptide application. AChR-channels at extrasynaptic regions of fibres from denervated muscles were unaffected by CGRP. It is suggested that CGRP may regulate the synaptic AChR-channel conductance through second messenger systems.

Animals↗
PubMed1988

Adenosine inhibition of the hormonal response in the Sertoli cell is reversed by pertussis toxin.

The rat Sertoli cell in culture expresses A1 inhibitory adenosine receptors. In this study, we have used pertussis toxin as a tool to characterize the mechanism of action of adenosine on these cells. Cells were preincubated for 18-24 h with pertussis toxin, and the responses to FSH and to the adenosine analog phenylisopropyladenosine (PIA) were measured by assaying cAMP accumulation. The effect of toxin on adenosine receptors was also evaluated by measuring binding of the adenosine agonist cyclohexyladenosine (CHA). The total number of specific CHA-binding sites was reduced 60-70% in membranes prepared from cells cultured for 24 h in the presence of pertussis toxin; the binding sites remaining after treatment displayed no apparent change in affinity for [3H]CHA. The effect of guanine nucleotides on CHA binding was also reduced after toxin pretreatment, but not abolished. PIA inhibited FSH-stimulated cAMP accumulation by 70-80%. Maximal inhibition was observed at a concentration of 10 nM PIA, and the ED50 of the dose-response curve was 1 nM. Pretreatment of the Sertoli cell with pertussis toxin completely blocked the PIA inhibition. The pertussis toxin effect was time and dose dependent. Reversal of the inhibition was observed after 6 h of treatment with a maximal dose of toxin (100 ng/ml). The dose of toxin producing a half-maximal effect was 10-30 ng/ml. In addition to this blockade of purine nucleotide inhibitory effects, exposure of the Sertoli cell to pertussis toxin concentrations ranging from 1-400 ng/ml consistently led to a potentiation of the FSH response measured as cAMP accumulation. In cell-free preparations (crude particulate fraction of the Sertoli cells, or sucrose gradient-purified plasma membranes), pertussis toxin catalyzed the incorporation of [32P]ADP ribose into a polypeptide with a molecular mass of 40-41 K. This peptide had electrophoretic mobility similar to that of a partially purified guanine nucleotide-binding protein (Gi). These data indicate that adenosine A1 inhibitory receptors are coupled to an inhibitory component (Gi) of adenylate cyclase. In the Sertoli cell, inhibitory and stimulatory signals interact in a bimodal regulation of adenylate cyclase and intracellular cAMP.

Adenosine↗
PubMed1988

Follicle-stimulating hormone modulation of phosphoinositide turnover in the immature rat Sertoli cell in culture.

The possibility that FSH modulates the Ca++-, phospholipid-dependent pathway was studied in cultured rat Sertoli cells. Cells were metabolically labeled with [3H]myoinositol, and accumulation of [3H]inositol phosphates [( 3H]inositol mono-, di-, and trisphosphates) was measured by extraction and fractionation on ion exchange chromatography. Fetal bovine serum increased Sertoli cell phosphoinositide (PI) turnover in a time and concentration-dependent manner (ED50 = 2-3% vol/vol). FSH by itself had no significant effect on the accumulation of inositol phosphates. On the other hand, FSH inhibited, in a concentration-dependent manner, the serum-stimulated accumulation of inositol phosphates by more than 50%. HPLC ion exchange chromatography of the inositol phosphates showed that all isomers present in serum-stimulated extracts were reduced to the same extent after treatment with FSH. The treatment of the cells with either forskolin, the beta-adrenergic agonist isoproterenol, or (Bu)2cAMP also inhibited the PI turnover stimulated by fetal bovine serum. FSH inhibition was not detected in mature Sertoli cells, in which FSH no longer stimulates cAMP accumulation. These results demonstrate that FSH, via formation of cAMP, exerts inhibitory effects on the PI turnover of the Sertoli cell. Thus, while stimulating the cAMP-dependent pathway, at the same time FSH inhibits the Ca++-, phospholipid-dependent pathway.

Animals↗
PubMed1987

Acetylcholine receptor channel properties in rat myotubes exposed to forskolin.

Rat myotubes exposed to forskolin were studied by patch-clamp technique in cell-attached single channel recording configuration. Channel open time and opening frequency of the main class of acetylcholine receptor- (AChR-) channels (accounting for more than 90% of all unitary events) decreased in the presence of forskolin (20-100 microM). The forskolin-induced action on the AChR function fully developed with a delay of 30-60 minutes from the peak of cytosolic cyclic AMP (cAMPi) concentration. Both cAMP (1 mM), applied intracellularly for 10 min, and dibutyryl cAMP (0.5 mM), applied extracellularly for 90 min, did not accelerate the rate of desensitization of myotubes studied in whole-cell patch-clamp recording configuration. It was concluded that the action of forskolin on AChR-channel function of rat myotubes could be not associated with the cAMPi-dependent phosphorylation of AChR.

Animals↗
PubMed1987

A recombinant apoA-1--protein A hybrid reproduces the binding parameters of HDL to its receptor.

We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Inhibition by phorbol esters and other tumor promoters of the response of the Sertoli cell to FSH: evidence for dual site of action.

Several tumor promoters exert their effects by activating a Ca2+-phospholipid-dependent protein kinase (protein kinase C). To study the role of this protein kinase in the regulation of Sertoli cell function, we have evaluated the effect of phorbol esters, mezerein, and teleocidin on the response of the Sertoli cell to FSH. Cells were treated for different time intervals with the tumor promoters, and cell response was measured by stimulating the cell with FSH. 12-O-Tetradecanoylphorbol 13-acetate (TPA) had no significant effect on basal cAMP production but markedly inhibited the cAMP response to FSH. Significant inhibition of cAMP accumulation was observed after 15 min treatment with 100 nM TPA, and maximal inhibition developed within 1 h. The decrease in cAMP accumulation was dependent on the dose of phorbol ester used, with an estimated ED50 of 10-20 nM TPA. In a manner similar to TPA, mezerein and teleocidin also inhibited the cAMP response of the Sertoli cell, while the phorbol ester 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), inactive as a tumor promoter and unable to stimulate protein kinase C activity, was devoid of effect. The promoters that inhibited cAMP response also inhibited the FSH-stimulated androgen aromatization. The dose of TPA producing half-maximal inhibition of estrogen accumulation was again 10-20 nM TPA, mezerein, and teleocidin inhibited estrogen accumulation whether FSH, forskolin or cholera toxin was used to stimulate the Sertoli cell. In contrast, only FSH-dependent cAMP accumulation was inhibited by the tumor promoters, while forskolin and cholera toxin stimulations were not affected. These data suggest that tumor promoters which activate protein kinase C act at two sites of the Sertoli cell response. They alter receptor-mediated signal transduction across the membrane and affect steroidogenesis at a site distal to cAMP accumulation.

Androstenedione↗
PubMed1986

Cyclic AMP regulates the life time of acetylcholine-activated channels in cultured myotubes.

'Giga-seal' patch-clamp recording was performed in embryonic chick myotubes at day 3 to 4 of culture. Myotubes were exposed to agents that enhance the concentration of cytosolic cyclic AMP (cAMPi) and their action on acetylcholine- (ACh) activated channels was investigated. While the conductance and the closed time was unaffected by forsokolin, cholera toxin, dibutyryl cyclic AMP and 8-bromo-cyclic AMP, these agents lengthened the ACh-activated channel life time with efficacy that paralleled with their capability to increase the cAMPi.

Acetylcholine↗
PubMed1986

Expression of the human apolipoprotein AI gene fused to the E. coli gene for beta-galactosidase.

The human apoAI gene was expressed in E. coli by in-frame fusion to a modified beta-galactosidase gene present in plasmid pUR291. The fused beta-galactosidase-apoAI gene product was expressed at a high level and was recognized by an anti-human apoAI antiserum. Besides the fused protein, at least one degradation product having an Mr similar to that of beta-galactosidase was present in high amounts in bacterial extracts. These results and those of a pulse-chase experiment indicate that degradation took place only in the apoAI moiety of the chimeric protein.

Apolipoprotein A-I↗