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L Montanaro

Publications and source records attributed to L Montanaro.

At least 73 records · Page 4Linked to original sources
PubMed1998

Primary endocervical extraosseous Ewing's sarcoma/PNET.

A 36-year-old woman presented with intermenstrual spotting and was found to have a cystic mass involving the uterine cervix on a pelvic ultrasound examination. A necrotic and hemorrhagic tumor was excised by hysterectomy and processed for light and electron microscopic investigation and molecular analysis. Microscopic examination revealed a small round cell tumor that immunohistochemical studies (including staining for the highly restricted surface antigen p30/32MIC2) and ultrastructural studies indicated was an extraosseous Ewing's sarcoma (EES)/primitive neuroectodermal tumor (PNET). This diagnosis was established by detection of EWS/ERG fusion transcript through reverse transcription polymerase chain reaction (RT-PCR) with nested primers. Full body computed tomography failed to detect any extrauterine tumor, and the patient is clinically free of disease 18 months after hysterectomy. This case represents the first report of a primary EES/PNET arising in the uterine cervix.

Adult↗
PubMed1998

Bone-resorbing cytokines in serum of patients with aseptic loosening of hip prostheses.

Our aim was to determine if the serum levels of bone-resorbing cytokines (IL-1beta, TNF-alpha, IL-6, GM-CSF) are altered in patients with aseptic loosening of a total hip prosthesis, and if such levels are influenced by the type of implant. We determined cytokine levels in sera from 35 patients before revision for failed total hip arthroplasty and compared them with those in 25 healthy donors. We also assessed the soluble receptor of interleukin-2 (sIL-2r) in serum as an indication of a specific immune reaction against the implant. Our findings showed that the sIL-2r and TNF-alpha serum level did not change. The IL-6 level was not significantly altered, but was higher in patients with TiAIV prostheses than in those with a CrCoMo implant and in patients with cemented prostheses. The IL-1beta level was found to be higher in those with a TiAIV cemented prosthesis than in the control group (p=0.0001) and other groups of patients (p=0.003 v uncemented TiAIV, p=0.01 v cemented CrCoMo, p=0.001 v uncemented CrCoMo). The GM-CSF level significantly increased in patients compared with healthy subjects (p=0.008), and it was higher in those with cemented than with uncemented implants (p=0.01). Only patients with cementless CrCoMo prostheses had levels of GM-CSF similar to those of the control group. The highest GM-CSF concentrations were observed in patients treated with non-steroidal anti-inflammatory drugs (NSAIDs) in the last months before revision (p=0.04). In addition, when massive osteolysis was observed, the level of GM-CSF tended to decrease to that of the control group.

Acetabulum↗
PubMed1998

Nucleolar function and size in cancer cells.

We have have studied the relationship between nucleolar function and size and cell doubling time in cancer cells. Seven human cancer cell lines characterized by different proliferation rates were used. Nucleolar functional activity was evaluated by measuring RNA polymerase I activity and expression of RNA polymerase I upstream binding factor (UBF), DNA topoisomerase I, and fibrillarin, three proteins involved in synthesis and processing of rRNA. Transcriptional activity of RNA polymerase I was strictly related to cell doubling time (r = -0.97; P < 0.001). The quantitative distribution of UBF, DNA topoisomerase I, and fibrillarin was evaluated on Western blots using specific monoclonal antibodies by densitometric analysis of autoradiographic signals. It was found to be directly related to RNA polymerase I transcriptional activity (r = 0.89, P = 0.008 for UBF; r = 0.95, P = 0.001 for DNA topoisomerase I; and r = 0.91, P = 0.004 for fibrillarin) and inversely related to cell doubling time (r = -0.87, P = 0.011 for UBF; r = -0.97, P < 0.001 for DNA topoisomerase I; and r = -0.91, P = 0.005 for fibrillarin). The nucleolar areas were measured by automated image analysis on toluidine blue-stained cells. The values of the stained nucleolar structures per cell were directly related to RNA polymerase I transcriptional activity (r = 0.94, P = 0.001) and inversely related to cell doubling time (r = -0.98, P < 0.001). The same area values of the nucleolar structures stained by toluidine blue were also closely related to the amount of UBF (r = 0.92, P = 0.003), DNA topoisomerase I (r = 0.98, P < 0.001), and fibrillarin (r = 0.95, P = 0.001), and to the in situ quantitative distribution of AgNOR proteins (r = 0.98, P < 0.001). Our results demonstrated that in cancer cells rRNA transcriptional activity and nucleolar size are inversely related to cell doubling time. Quantitative distribution of nucleolar structures within the cell represents a cytohistological parameter of the rapidity of cell proliferation.

Blotting, Western↗
PubMed1998

A polymerase chain reaction (PCR) method for the identification of collagen adhesin gene (CNA) in Staphylococcus-induced prosthesis infections.

Staphylococci are well-recognized pathogens of foreign body-associated infections. The pathogenesis of such infections involves an initial step of contact between the colonizing bacterium and the biomaterial, with subsequent colony formation. Several studies have been devoted to identify adhesion mechanisms for these bacteria. Slime in particular has been extensively investigated. Recently, considerable attention has been given to the host protein receptors that have been shown in in vitro assays to serve as substrates for bacterial adherence. To determine the importance of the collagen adhesin as virulence factor in Staphylococcus-induced prosthesis infection, a simple and reliable method using a polymerase chain reaction (PCR) was devised to identify collagen adhesin gene (cna). By using lysates of ten strains from orthopedic prostheses (5 Staphylococcus aureus and 5 Staphylococcus epidermidis) and two 20-oligonucleotides as primers, a 192-bp region of the cna gene was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were in accordance with those obtained by the in vitro phenotypic characterization of binding ability to collagen of Staphylococcus strains.

Adhesins, Bacterial↗
PubMed1997

N-myc amplification and cell proliferation rate in human neuroblastoma.

In neuroblastoma, N-myc amplification has been found to be strikingly associated with rapid tumour progression and poor prognosis. Recent studies have demonstrated that cell proliferative activity also significantly predicts the clinical outcome in patients with neuroblastoma. In order to define the correlation between N-myc amplification and cell proliferation rate, in the present investigation the two parameters were first assessed in 48 neuroblastoma tumours. N-myc amplification was evaluated in frozen specimens by Southern-blot analysis using the NB 19-21 probe and it was detected in nine patients. Cell proliferative activity was determined by measuring the AgNOR protein area in histological sections selectively stained by silver. The mean AgNOR protein area value of neuroblastomas with N-myc amplification (3.63 +/- 1.62 microns2) was not significantly different from that of neuroblastomas without N-myc amplification (2.46 +/- 1.57 microns2; P = 0.30). On the other hand, both N-myc amplification and AgNOR protein expression were found to be significantly related to the clinical outcome of the disease (P < 0.001 and P = 0.0143, respectively; median follow-up time = 47 months; range 18-106 months). In a second set of experiments, the relationship between N-myc amplification and cell proliferation rate was assessed in seven established human neuroblastoma cell lines. N-myc amplification was found to be completely independent of the population doubling time (DT), which, on the contrary, was strictly related to the quantitative expression of AgNOR protein (r = -0.947; P < 0.001). Altogether, the present results indicate that N-myc amplification and cell proliferation rate are not interrelated in neuroblastoma, each representing an independent biological parameter of cancer cells associated with the clinical behaviour of the disease.

Antigens, Nuclear↗
PubMed1997

The RNA-N-glycosidase activity of Shiga-like toxin I: kinetic parameters of the native and activated toxin.

Shiga toxin and Shiga-like toxins are ribosome-inactivating proteins with RNA-N-glycosidase activity which remove a specific adenine from 28S RNA. The toxins are composed of an A subunit non-covalently associated to a multimer of receptor-binding B subunits. Near the COOH-terminus of the A subunit, a disulfide-bonded loop contains two trypsin-sensitive arginine residues. Proteolytic nicking at these sites, followed by reduction, removes from the A subunit the C-terminal end together with the associated B subunits. The requirement of such cleavage for biological activity of Shiga toxin and Shiga-like toxins has been recently questioned. The present paper reports the kinetic constants of the adenine release from highly purified Artemia salina ribosomes catalysed by Shiga-like toxin I and by its A subunit before and after treatment with trypsin, urea and dithiothreitol or urea and dithiothreitol alone. All reactions had approximately the same Km (1 microM). The Kcat was 0.6 min-1 for the untreated holotoxin and 6 min-1 for the isolated A subunit, respectively. The trypsin treatment increased 1000-fold the Kcat of the holotoxin (770 min-1) and 100-fold the Kcat of the A subunit (640 min-1). The same Kcat (693 min -1) was also observed when the A subunit was treated only with urea and dithiothreitol. Thus the full activity of Shiga-like toxin I required not only removal of the B subunits but also activation of the A subunit itself. Such activation could be largely induced in vitro by drastic loosening of the molecule induced by urea and dithiothreitol, but in vivo would probably require a proteolytic cleavage of the toxin. Inactivation of ribosomes by Shiga-like toxin I did not require sensitization of ribosomes by ATP and macromolecular cofactors present in postribosomal supernatants.

Animals↗
PubMed1996

Qualitative and quantitative analysis of AgNOR proteins in chemically induced rat liver carcinogenesis.

A qualitative and quantitative analysis of silver-stained nuclear organizer regions (AgNOR) proteins was performed during hepatocarcinogenesis induced in rats initiated by diethylnitrosamine (DENA) using the resistant-hepatocyte model. Nuclear proteins from control hepatocytes, hyperplastic nodules, and hepatocellular carcinomas (HCC) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were transferred to nitrocellulose membranes and specifically silver-stained for AgNOR proteins. No difference was observed in the distribution pattern of the silver-stained bands among control, hyperplastic, or cancer cells. The same was true if human cirrhosis and HCC were compared. The evaluation of individual AgNOR protein amounts by computerized densitometric analysis showed that 1) the integrated optical density value of the total AgNOR proteins was greatest in cancer cells, lesser in hyperplastic hepatocytes, and lowest in control hepatocytes, and 2) the amount of the two major silver-stained proteins, nucleolin (105 kd) and protein B23 (39 kd), was always a constant percentage of total AgNOR proteins. An experiment using bromodeoxyuridine incorporation showed that, during hepatocarcinogenesis, AgNOR protein quantity progressively increased and was significantly related to the increased hepatocyte labeling index. These results show that AgNOR protein distribution changes during hepatocarcinogenesis are caused neither by the synthesis of new AgNOR proteins nor by an unbalanced synthesis of individual AgNOR proteins, but to an increased synthesis of nucleolin and protein B23, which is associated with a progressive increased hepatocyte proliferation rate.

Animals↗
PubMed1996

tRNA(Trp) as cofactor of gelonin, a ribosome-inactivating protein with RNA-N-glycosidase activity. Features required for the cofactor activity.

Purified beef and rabbit liver tRNA(Trp), but not yeast tRNA(Trp), increase the in vitro inactivation of eukaryotic ribosomes by gelonin, a ribosome-inactivating protein with RNA-N-glycosidase activity on 28S rRNA. Aminoacylation and stepwise trimming of the 3'-end of bovine tRNA(Trp) affect the cofactor activity, the most active species being that shortened by the last two nucleotides. ATP only moderately increases the activity of purified mammalian tRNA(Trp) and in this increase the cognate aminoacyl-tRNA synthetase apparently has no role. Mobility shift experiments indicate that bovine tRNA(Trp) binds both to ribosomes and to gelonin and favours the dissociation of gelonin from ribosomes.

Animals↗
PubMed1995

Differential up-regulation by tRNAs of ribosome-inactivating proteins.

Some plant ribosome-inactivating proteins (RIPs) with RNA-N-glycosidase activity on 28S RNA require, for the inactivation of ribosomes, the presence of macromolecular cofactors present in post-ribosomal supernatants. In the case of gelonin one of the cofactors is tRNATrp lacking one or two nucleotides at the 3'-CCA end [Brigotti, M., Carnicelli, D., Alvergna, P., Pallanca, A., Lorenzetti, R., Denaro, M., Sperti, S. and Montanaro, L. (1995) Biochem. J. 310, 249-253]. In the present study it is shown that tRNAs are involved in the up-regulation of all the cofactor-requiring RIPs up to now identified (agrostin, barley RIP, PAP and tritin, besides gelonin). Polyacrylamide gel electrophoresis shows that tRNA fractions with different mobilities stimulate different RIPs. With the identification of agrostin, the cofactor-requiring RIPs (italics) add to five out of a total of thirteen investigated: barley RIP, bryodin-R, gelonin, lychnin, momordin, momorcochin-S, PAP, saporin-6, tritin [Carnicelli, D., Brigotti, M., Montanaro, L. and Sperti, S. (1992) Biochem. Biophys. Res. Commun. 182, 579-582], agrostin, luffin, trichokirin and trichosanthin (present study).

Animals↗
PubMed1995

3'-immature tRNA(Trp) is required for ribosome inactivation by gelonin,a plant RNA N-glycosidase.

Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3'-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.

Animals↗
PubMed1995

Proportionally constant quantitative transmission of nucleolin and protein B23 in cycling cancer cells.

Objective-To investigate whether and to what extent the two major AgNOR proteins, nucleolin and protein B23, are maintained after one cell division in proliferating cells.Design-Using three asynchronously growing human cancer cell lines, TG, SJNKP, and CHP 212 cells, nucleolin and protein B23 were first identified on SDS-polyacrylamide separated nucleolar proteins, transferred to nitrocellulose and silver stained for AgNOR proteins. Measurement of doubling time indicated a period very close to 24h for each of the cell lines. To quantify the percentage of nucleolin and protein B23 maintained in daughter cells after duplication, cells were labelled with [(35)S]-methionine and a 24h cold chase performed. Nucleolin and protein B23 labelling was evaluated by densitometric analysis on nitrocellulose autoradiograms.Results-The radioactivity relative to nucleolin and protein B23 bands maintained in the daughter cells was a constant fraction of that present before cell duplication. In the three cell lines the percentage of residual radioactivity measured in the nucleolin bands was 42.2, 40.6, and 41.2 and in protein B23 bands 48.0, 46.2, and 44.1.Conclusions-After one cell division the nucleolin and protein B23 quantity present in cells may be highly variable, depending on the amount of the two proteins present in the mother cell. This is important in relation to the correct utilisation of AgNOR protein quantity as an index for evaluating cell kinetics.

Journal Article↗
PubMed1994

RNA present in post-ribosomal supernatants makes ribosomes susceptible to inactivation by gelonin and alpha-sarcin.

The remarkable resistance of isolated ribosomes to gelonin is overcome by cofactors present in post-ribosomal supernatants. In rat liver post-ribosomal supernatant RNA is the cofactor responsible of the sensitization of ribosomes. Isolated RNA, which consists mostly of deacylated tRNA, accounts for less than 10 per cent of the activity of the original supernatant. The activity of the supernatant is completely destroyed by micrococcal nuclease and RNAase A and also by proteinase K, suggesting that some protein enhances the effect of RNA. RNA has a role also in the sensitization of ribosomes to alpha-sarcin, an RNAase which inactivates ribosomes by hydrolyzing a single phosphodiester bond in the same region of 28S rRNA which is the target of the N-glycosidase activity of gelonin.

Animals↗
PubMed1993

Partial purification of two proteins which sensitize ribosomes to gelonin: sensitization is not linked to phosphorylation of ribosomal proteins.

Inactivation of ribosomes by gelonin, from Gelonium multiflorum, requires ATP and extraribosomal protein(s) present in the rabbit reticulocyte lysate [SPERTI, S. et al. (1991) Biochem. J. 277, 281-284]. On the anion exchanger Mono Q the activity responsible for the sensitization of ribosomes to gelonin resolves in two peaks which both display a kinase activity on ribosomal proteins. However, staurosporin, an inhibitor of several protein kinases, strongly inhibits phosphorylation of ribosomal proteins without affecting the gelonin-promoting activity of Mono Q peaks. All the evidence collected contradicts a direct link between sensitization to gelonin and phosphorylation of ribosomes.

Animals↗
PubMed1993

Oligonucleotides complementary to the alpha-sarcin domain of 28S rRNA inhibit cell-free protein synthesis.

Oligodeoxynucleotides complementary to the alpha-sarcin domain of rat 28S rRNA inhibit cell-free protein synthesis. The poly(U) translation system containing Artemia salina ribosomes was more sensitive to inhibition than the system containing rat liver ribosomes. The 21-mer, which was the most effective of the 7 oligonucleotides tested, hybridized with naked 28S rRNA. Hybridization with whole ribosomes, assayed by S1 nuclease protection, occurred only at high ionic strength or with ribosomes actively engaged in protein synthesis.

Animals↗
PubMed1993

Inactivation of ribosomes by an inhibitor of protein synthesis from Salmonella enteritidis.

Sonic extracts of Salmonella enteritidis contain a factor which inhibits protein synthesis in cell-free systems by irreversibly inactivating ribosomes. The extent of the inactivation of ribosomes depends on the system used to assay protein synthesis, natural mRNA translation being more strongly inhibited than poly(U) translation. The inhibitory power of the Salmonella factor is destroyed by trypsin and by 5% mercaptoethanol. Placental RNase inhibitor is unable to protect ribosomes from inactivation.

Animals↗