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E Monti

Publications and source records attributed to E Monti.

At least 37 records · Page 2Linked to original sources
PubMed1995

Purification of LamB proteins using continuous elution electrophoresis: a comparison with immunoaffinity chromatography.

LamB is a membrane protein that allows the exposition of a foreign peptide on the surface of a recombinant E. coli cells. An immunopurified hybrid LamB protein has been used to elicit high-titre antibodies to a foreign epitope. Looking for a simpler purification procedure we have compared the traditional approach, which includes affinity chromatography, to continuous elution electrophoresis, in the purification of two different hybrid LamB proteins as foreign epitopes. The results obtained showed that both methods yielded the same purification, although the electrophoretic procedure had a higher yield. Continuous-elution electrophoresis could be a useful tool for the purification of membrane proteins.

Antibodies, Monoclonal↗
PubMed1994

Cytosolic sialidase from pig brain: a 'protein complex' containing catalytic and protective units.

Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three bands using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, M(r), 180 kDa) was resolved into a catalytic unit (M(r) 30 kDa), active but very liable upon storage at 4 degrees C and freezing and thawing, and two protective units (66 kDa and 42 kDa), inactive, but capable to stabilize the catalytic unit. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a as substrate, the catalytic unit showed a Michaelis-Menten kinetics, and the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten kinetics was exhibited with MU-NeuAc in both cases. The apparent Vmax and Km values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2 x 10(-5) and 1.6 x 10(-5) M, respectively. The model we propose for cytosolic sialidase complex is one of each protective units and 2-3 catalytic units. The sialidase complex and protective units did not display any beta-D-galactosidase, beta-D-N- acetylglucosaminidase, alpha-L-fucosidase, alpha-D-glucosidase and carboxypeptidase activities.

Animals↗
PubMed1994

Delayed doxorubicin cardiomyopathy in the rat: possible role of reduced food intake.

The development of delayed doxorubicin cardiomyopathy in the rat is accompanied by profound anorexia, dramatically reducing the caloric intake. To assess the contribution of a restriction in food to the alterations in cardiac function, animals treated with doxorubicin were compared with a group of pair-fed control animals and with a second group of controls with unrestricted access to food. Prolongation of the Q alpha T interval of the electrocardiogram developed in rats treated with doxorubicin, but not in pair-fed controls. Myofibrillar ATPase activity and the contractile strength of isolated papillary muscles were depressed in rats treated with doxorubicin, but not in pair-fed rats. The reduction in ventricular weight was proportional to the reduction in the body weight in pair-fed rats, whereas a higher ratio of ventricular to body weight was observed in rats treated with doxorubicin. These results indicate that the alterations in cardiac function observed in delayed doxorubicin cardiomyopathy are not due to a reduction in the intake of food.

Adenosine Triphosphatases↗
PubMed1993

Synergistic antiproliferative effects of interleukin-1 alpha and doxorubicin against the human ovarian carcinoma cell line (NIH:OVCAR-3).

Interleukin-1 alpha (IL-1 alpha) exerts antiproliferative effects on a human ovarian carcinoma cell line, NIH:OVCAR-3, which is resistant to clinically relevant concentrations of doxorubicin (DOX) and other chemotherapeutic agents. This action of IL-1 alpha depends on the presence of type I (80 kDa) receptors, although no quantitative relationship has been established between receptor occupancy and inhibition of cell growth. When NIH:OVCAR-3 cells were exposed to IL-1 alpha and DOX in combination, a mutual potentiation of the antiproliferative effects of the two agents was observed. This synergistic effect was not due to IL-1 receptor expression up-regulation by DOX, and receptor-dependent internalization of the cytokine was also unaffected. The involvement of IL-1 receptors is supported by the observation that synergism between the two agents was diminished (but not abolished) in the presence of a specific IL-1 receptor antagonist at concentrations blocking more than 75% of IL-1 alpha binding. DOX was found to significantly increase IL-1 alpha accumulation by NIH:OVCAR-3 cells after long-term (48 hr) exposure to the cytokine at 37 degrees, which might be due to increased nonspecific fluid phase uptake or to interference with cytokine degradation and/or release processes. The potent synergy of IL-1 alpha and DOX against ovarian carcinoma cells in vitro suggests that this drug combination may be effective against this disease in the clinic.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

The hydrophilic spin trap, 5,5-dimethyl-1-pyrroline-1-oxide, does not protect the rat heart from reperfusion injury.

The role and site of free radical generation in myocardial ischemia/reperfusion injury were investigated using the hydrophilic spin trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). DMPO (40 mM) proved ineffective in preserving the contractile performance and energy metabolism of Langendorff-perfused rat hearts following ischemia and reperfusion. This result, which is in contrast with the cardioprotection observed with hydrophobic spin traps, suggests that free radicals are generated intracellularly under these conditions.

Animals↗
PubMed1993

A monoclonal antibody to the NH2-terminal segment of human IFN-gamma selectively interferes with the antiproliferative activity of the lymphokine.

To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.

Amino Acid Sequence↗
PubMed1993

Synergistic interactions of etoposide and interleukin-1 alpha are not due to DNA damage in human melanoma cells.

Several possible mechanisms of the synergistic interactions of IL-1 alpha and VP-16 against A375-C6 human melanoma cells were investigated. Studies indicate that IL-1 alpha did not increase topoisomerase II-dependent VP-16-mediated DNA damage, nor did IL-1 alpha inhibit the repair of VP-16-induced DNA damage in these cells. Furthermore, IL-1 alpha by itself or in combination with VP-16 did not cause significant fragmentation of cellular DNA into oligomers, indicating programmed cell death (apoptosis) was not involved in the mechanism of synergy. In contrast, an IL-1-specific receptor antagonist significantly decreased IL-1 alpha toxicity toward the melanoma cells and nearly eliminated the synergistic interactions of IL-1 alpha with VP-16. These results strongly indicate that synergism of IL-1 alpha with VP-16 was dependent upon an IL-1-receptor-mediated processes. DNA-strand breakage was unlikely to be a primary intracellular target for IL-1 alpha cytotoxicity and synergism with VP-16.

Cytotoxins↗
PubMed1993

Purification of interleukin-2 antibodies from healthy individuals.

By covalent binding of recombinant interleukin-2 (rIL-2) to Sepharose, it was possible to immunopurify specific human anti-IL-2 antibodies from a pool of immunoglobulins obtained from healthy subjects. Since low quantities of the ligand released by the matrix could interfere with the evaluation of the biological activity of anti-IL-2 antibodies, the antibody preparation was subjected to pepsin digestion which is known to destroy the IL-2 molecule. Purified human anti-IL-2 antibodies were found to be mostly IgG1 and able to neutralize IL-2 induced peripheral blood lymphocytes (PBL) proliferation in vitro. The availability of purified anti-IL-2 antibodies, obtained from healthy individuals, able to modulate IL-2 activity, could be important in several therapeutic approaches.

Blotting, Western↗
PubMed1993

High-titre antibodies to a foreign epitope elicited by affinity-purified hybrid LamB proteins.

A monoclonal antibody to the LamB protein, named LBS-1, was developed and characterized. It was then covalently bound to Sepharose and used to purify hybrid LamB proteins from Escherichia coli crude extracts. A peptide of the interferon-gamma (IFN-gamma) NH2-terminal region, inserted within the LamB protein, was used as a model to assess immune response in mice injected with sonicated E. coli extract or with affinity-purified hybrid LamB protein. None of the mice immunized with the whole bacterial extract produced antibodies to IFN-gamma. On the other hand, all the mice immunized with the purified protein developed high-titre anti-IFN-gamma antibodies. These results might be due to the presence of bacterial components capable of masking the LamB protein to the immune system. The use of affinity-purified LamB proteins may constitute in some instances a more effective way of generating an immune response against foreign epitopes as opposed to whole bacterial antigens.

Amino Acid Sequence↗
PubMed1993

The detection and biological activity of human antibodies to IL-2 in normal donors.

Antibodies to interleukin-2 (IL-2) have been reported in the sera of patients under therapies involving this cytokine and in the sera of patients infected with human immunodeficiency virus (HIV). Our study proves that auto-antibodies to IL-2 are present also, at a lower titre, in healthy individuals. These antibodies were affinity purified and studied for their capability to interfere with the in vitro biological activity of IL-2. Data obtained show that human anti-IL-2 antibodies can interfere with lymphocyte proliferation both in the lymphokine activated killer (LAK) cell assay and in the mixed lymphocyte reaction (MLR). However, the kinetics of inhibition by anti-IL-2 antibodies differs from LAK cell assay to MLR as the former are always inhibited in a time-independent manner, and the latter only by adding antibodies at the outset of culture. The neutralizing activity observed in vitro suggests that such antibodies play a part in the elaborate cytokine network by which the immune system regulates the amplitude and duration of its response.

Autoantibodies↗
PubMed1992

Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts.

The sulfatide fluorescent analogue N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate was administered in the form of albumin complex to normal human skin fibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.

Adolescent↗
PubMed1992

Protective activity of the spin trap tert-butyl-alpha-phenyl nitrone (PBN) in reperfused rat heart.

The aim of this work was to ascertain whether free radicals play a causal role in the injury occurring in myocardial ischemia and reperfusion. To this purpose we observed whether spin-trapping compounds protect the heart when used at a concentration capable of reacting with free radicals. The lipophilic spin trap alpha-phenyl-t-butyl nitrone (PBN) was used because it is taken up by the myocites. Isolated Langendorff rat hearts were subjected to ischemia according to two schemes: "Model A" = 30 min zero-flow ischemia followed by 30 min reperfusion; "Model B" = 60 min of low-flow ischemia (10% of the individual value; N2 saturated) followed by 30 min reperfusion. Treated groups received in addition 5.0 mM PBN which was supplied continuously. The following parameters were measured throughout the experiment: contractile performance (RPP); coronary flow (CF); CPK; phosphocreatine (PCr), ATP, inorganic phosphate (Pi), intracellular pH (pHi). The pathology obtained by "Model A" is more severe than that of Model B, and partly irreversible. During the ischemic phase in "Model A", contractility, PCr and ATP dropped to near zero; during initial reflow CPK rose about 13-fold and Pi rose 2.5-fold, while pHi decreased to 6.1. During reperfusion, a partial recovery of PCr, Pi and pHi was observed, while RPP and ATP did not increase; PBN treatment improved significantly PCr and CPK, while the other parameters were unaffected. During ischemia, "Model B" hearts showed a drop of contractility to near zero, of PCr to 35%, of ATP to 50%; CPK rose 7-fold and Pi 1.5-fold; pHi was not modified. During reperfusion, all parameters recovered in part, with exception of Pi. PBN developed a marked protective activity on all tested parameters, which gained a nearly normal value. The results of the present investigations show that the lipophilic spin trap PBN partly protects the heart from the ischemia/reperfusion injury, thus confirming that free radicals play a causal role in this pathology; the continuous loading of the tissue with the drug can be an important factor for obtaining the protective effect.

Adenosine Triphosphate↗
PubMed1992

Inhibition of the biological activity of human interferon-gamma by antipeptide antibodies.

To study the domain(s) responsible for the different biological activities of human interferon-gamma (HuIFN-gamma), rabbits were immunized with peptides corresponding to the five most hydrophilic amino acid sequences of the lymphokine. The resulting antisera were able to recognize both the immunizing peptide and the native protein. Antibodies to the carboxy-terminal region (amino acids 125-137) of HuIFN-gamma were able to interfere with the immunomodulating, antiviral, and antiproliferative activities of the lymphokine. Inhibition of the antiproliferative and antiviral activity was also observed using antibodies raised against the amino-terminal region (amino acids 4-16) and amino acids 80-95, respectively, but to a lesser extent than that observed with antibodies to the carboxyl terminus. The capability of these antibody preparations to partially interfere with only one of the lymphokine's biological activities might be explained by a mechanism of steric hindrance. The use of polyclonal antibodies allowed us to limit the presence of epitopes responsible for recombinant (r)HuIFN-gamma biological activities to the carboxy-terminal region.

Antibodies↗
PubMed1992

Subcellular distribution of two spin trapping agents in rat heart: possible explanation for their different protective effects against doxorubicin-induced cardiotoxicity.

Previous investigations, performed on isolated rat atria, showed that the lipophylic spin-trapping agent N-tert-butyl- alpha-phenylnitrone (PBN) is able to prevent the acute cardiotoxic effects produced by doxorubicin (DXR), whereas the hydrophylic compound 5,5-dimethyl-pyrroline-N-oxide (DMPO) is inactive. The present study was designed to ascertain whether differences in the pharmacological effects of the two spin traps are related to their different subcellular distribution. Langendorff rat hearts were perfused for 60 minutes with [14C]-DXR and either PBN or DMPO. The subcellular mapping of the three compounds was performed by measuring DXR by liquid scintillation counting, PBN by GC/MS, and DMPO by HPLC in the following isolated fractions: nuclei, mitochondria, sarcoplasmic reticulum, sarcolemma, cytosol. DMPO was shown to accumulate in the cytosolic compartment; both PBM and DXR are taken up by nuclei and mitochondria, while only trace amounts of DXR were detected in the sarcoplasmic reticulum. These results suggest that mitochondrial (and not sarcoplasmic) enzymes are mainly involved in DXR-induced free radical production, which is thought to cause the acute cardiotoxic effects of DXR. An involvement of DXR-induced free radical generation in the nuclear compartment seems unlikely in the short-term "in vitro" effects observed with the experimental model adopted for these studies, although it may play a role in the delayed pathology.

Animals↗
PubMed1992

Synergistic antiproliferative effects of the combination of interleukin-1 alpha and doxorubicin against human melanoma cells.

We have investigated the antiproliferative effects of recombinant human interleukin-1 alpha (IL-1) combined with the cytotoxic antitumor drug doxorubicin against A375 human melanoma IL-1-sensitive (C6) and IL-1-resistant (C5) clonal cell lines. Growth inhibition was assessed by the MTT assay, and C5 cells were 10-fold less sensitive to IL-1 than the C6 cells, but both cell lines were equally sensitive to doxorubicin. Synergistic antitumor activity between the two agents was evaluated by median effects/combination index analysis, and IL-1 and doxorubicin were strongly synergistic over a broad range of drug concentrations. The strongest synergism occurred when C6 cells were exposed to IL-1 prior to doxorubicin, and when C5 cells were pretreated with doxorubicin for 6 hr prior to IL-1 additions. An examination of various ratios of the two agents revealed a maximum 20-fold potentiation of doxorubicin, and a 30-fold potentiation of IL-1 median dose values in the combination compared to the median dose values obtained with doxorubicin or IL-1 alone. Doxorubicin treatment enhanced the binding and internalization of [125I]IL-1 after 24 and 48 hr at 37 degrees C, but IL-1 binding to cells incubated on ice was increased only marginally by doxorubicin pretreatment. Treatment of C6 cells with IL-1 for 24 hr did not alter the cellular accumulation of doxorubicin. A recombinant protein IL-1 receptor antagonist that binds to both the 80 kDa type I and the 65 kDa type II IL-1 receptors, blocked the cytostatic effects of IL-1 and abrogated the synergism with doxorubicin. In cells synchronized following release from aphidicolin block, doxorubicin caused a G2 + M accumulation, IL-1 alone had no effect, and the combination of both agents resulted in a G2 + M block similar in magnitude to that caused by doxorubicin alone. These results provide preclinical evidence that doxorubicin combined with IL-1 may be beneficial in the clinical treatment of malignant melanoma and possibly other types of solid tumors.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1992

A novel fluorescent pH indicator for the acidic range.

Reaction of a commercial preparation of lissamine-rhodamine B sulfonyl chloride with primary amines gives rise to two sulfonamide derivatives, of which one behaves as a pH indicator with maximal fluorescence emission in the acidic range and a pKa of about 4.6. The structure of this derivative and the mechanism of fluorescence dependence on pH, elucidated by means of NMR, is presented.

Acids↗
PubMed1991

Prevention of doxorubicin-induced cardiomyopathy by reduced glutathione.

The aim of the present investigation was to evaluate the potential cardioprotective effect of reduced glutathione (GSH) against the delayed cardiomyopathy induced by doxorubicin (DXR) in a well-documented rat model. DXR was administered i.v. at a weekly dose of 3 mg/kg for a total of 4 doses; 250 or 500 mg/kg of GSH was given i.v. 10 min before and 2 h after each DXR injection, resulting in a total weekly dose of 500 or 1000 mg/kg, respectively. The development of cardiotoxicity was monitored in vivo by means of electrocardiography (QaT duration), and was evaluated by measuring the contractile performance of isolated atria and by light and electron microscopy of left ventricular samples excised 5 weeks after the last DXR administration. DXR was found to impair body weight gain and to produce an irreversible and time-dependent prolongation of QaT, a decrease in myocardial contractility of isolated atria and typical morphologic alterations, including myocyte vacuolization and myofibrillar loss. Pretreatment with GSH at a dose of 500 mg/kg x 2, but not at 250 mg/kg x 2, partially prevented the impairment of body weight gain, QaT prolongation in ECG and the decrease in myocardial contractility of isolated atria induced by DXR. Alterations of the morphologic pattern were also significantly reduced in animals receiving the higher dose of GSH. Determinations of the cardiac non-protein sulfhydryl group content showed that GSH, at doses higher than or equal to 500 mg/kg, significantly increased this parameter, irrespective of the presence of DXR. In conclusion, the present data indirectly support the hypothesis that oxidative damage is involved in DXR cardiotoxicity and indicate that maintenance of the reduced thiol pool could be an important issue in myocardial protection.

Animals↗