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Biomedical subjects

G Fassina

Publications and source records attributed to G Fassina.

160 records · Page 9Linked to original sources
PubMedDate not supplied

Protein A mimetic peptide ligand for affinity purification of antibodies.

A peptide mimicking protein A for its ability to recognize the Fc immunoglobulin portion has been identified through screening of a synthetic multimeric peptide library. Screening of the multimeric library, composed of randomized synthetic tripeptide tetramers, has been carried out using a very simple assay, measuring the library ability to interfere with the interaction between protein A and biotinylated immunoglobulins, monitored on solid phase using an enzyme-linked immunosorbent assay format. The tetrameric tripeptide identified after three screening cycles was produced in larger amounts and then immobilized in high yield on preactivated solid support for the preparation of affinity columns, which proved useful for a very convenient one-step purification of antibodies directly from crude sera. Antibody purity after affinity purification was close to 95 per cent, as determined by densitometric scanning of sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels of purified fractions, and up to 2 mg of antibody could be purified from 1 ml of peptide-derivatized affinity support. The ligand was stable to treatment with a vast array of sanitation agents, such as ethanol and 0.1 M sodium hydroxide, and to repeated use, thus making the ligand applicability extremely attractive for the purification of monoclonal antibodies for therapeutic use. Column binding selectivity was similar to that of protein A-affinity columns, since immunoglobulin G from several sources (rabbit, goat, sheep, mouse) was conveniently purified, with no detection of leaked ligand fragments in the purified preparations.

Animals↗
PubMedDate not supplied

Atrial bioenergetic variations in moderate hypoxia: danger or protective defense?

The effects of hypoxia on contractile tension and on tissue adenylate pool content, nicotinamide adenine nucleotide, NAD, nicotinamide adenine dinucleotide phosphate, NADP, and creatine phosphate, CrP, were investigated in isolated, spontaneously beating, guinea pig atria. When two different degrees of hypoxia were induced by lowering oxygen tension from 95% O2 (control) to 40% (moderate hypoxia) and 20% (severe hypoxia) for 30 min, contractile tension slowly decreased to 60% and 40% of control, respectively. In 40% O2 hypoxic atria, ATP was not significantly decreased, AMP slightly increased, TAN (total adenylate nucleotides) and adenylate energy charge [(ATP + 0.5 ADP)/(ATP + ADP + AMP)] did not change and creatine phosphate was decreased down to 53%. Hypoxic atria in 20% O2 showed a significant decrease of 26% in ATP, while ADP and AMP increased four and seven times, respectively. The adenylate energy-charge value was reduced from 0.93 to 0.70. Creatine phosphate decreased to below the analytical detection limit. Moderate hypoxia (40% O2), which induced a significant decrease of contractile tension but only minor changes of energetic tissue metabolism, was further investigated 2, 5, and 10 min after low oxygen tension was applied. Two stages of variations were evident during 30 min of experimental hypoxia. Within the first 10 min, concomitantly with atrial tension decrease, ATP, NAD, NADP, ATP/AMP, ATP/ADP, and TAN decreased, CrP began to decrease, inosine and xanthine showed no significant change. During the following 20 min of hypoxia, all parameters returned to the control levels with the exception of creatine phosphate. Adenylate energy charge did not change. The electrophysiological analysis of atrial cells did not show any major change in action potential configuration and resting potential, during 40% O2 hypoxia. The differences at metabolic level between moderate and more severe hypoxia suggest that the energetic state may be extremely unbalanced, in atrial tissue, as long as hypoxia is aggravated. Moreover, the time-course study, during 30 min of 40% O2, suggests that the early decrease of contractile tension does not depend on lowered energy availability, instead it might be, at least in part, a preventive measure to maintain energy balance in myocardial tissue to counteract hypoxic damage and, in this mechanism of defense, creatine phosphate shuttle seems to play a relevant role.

Action Potentials↗
PubMedDate not supplied

Molecular diagnostics using analytical immuno high performance liquid affinity chromatography.

Analytical immuno high performance liquid affinity chromatography (analytical immuno HPLAC) was evaluated as a molecular diagnostic tool. Antibodies raised in rabbits against bovine neurophysin II were immobilized through Protein A crosslinking onto coated silica. Interaction of immobilized antibody with mobile antigen was characterized by zonal and frontal elutions of 14C-labeled bovine neurophysin II under isocratic, nondenaturing conditions. The chromatographic behavior shows that analytical immuno HPLAC with immobilized antibodies can be used to detect the number and functional nature of matrix-interacting antigens in mixtures, thus providing a quantitative chromatographic technology for "antigen mapping."

Animals↗
PubMedDate not supplied

Pharmacological effects and binding studies of new methylxanthine thioderivatives.

The effects of two methylxanthine derivatives, 6-thiocaffeine (TC) and 6-thiotheophylline (TT), were investigated in different in vitro and in vivo conditions. On guinea-pig isolated trachea, both TC and TT showed a relaxant effect (EC50 50 microM and 60 microM, respectively), more potent than theophylline (300 microM). In guinea-pig isolated atria TC (30-50 microM) was able to antagonize R-PIA (a stable agonist on adenosine receptors) negative effect in not a clearly competitive way. Higher concentration (100 microM) began to reverse that inhibitory effect. In vitro Ki of TC and TT for A1 and A2 adenosine receptors was intermediate in comparison to caffeine and theophylline. On the contrary, the two thioderivatives showed a higher affinity for [3H]-nitrendipine binding sites, in comparison to the original methylxanthines. All these data suggest a complex mechanism of action, probably at the level of adenosine extracellular receptors and L-type Ca2+ channels. In vivo experiments in mice provided evidence for a lack of CNS stimulant effects, but a loss of motor coordination was observed. Both thioderivatives showed a reduced acute toxicity. These data can be useful for the development of drugs for the therapy of asthma with reduced side effects.

Animals↗
PubMedDate not supplied

Effects of esculetin (6,7-dihydroxycoumarin) on guinea-pig tracheal chains in vitro.

Esculetin (6,7-dihydroxycoumarin) was tested in isolated guinea-pig tracheal chains from normal and ovalbumin-sensitized animals. The drug showed a concentration-dependent relaxant effect, with an EC50 of 60 microM. Pretreatment with esculetin (30-60 microM) was not able to reduce the contractile response induced by either carbachol (0.1 microM), histamine (10 microM), BaCl2 (10 mM) or K+ (80 mM). Esculetin increased the contraction evoked by PGF2 alpha and by histamine, but reduced A23187-induced contraction. Mepacrine (10 microM), a phospholipase A2 inhibitor, reduced the relaxant effect of the lower esculetin concentrations (3-60 microM). Indomethacin (0.1 microM) increased the relaxation induced by esculetin (3-10 microM). In tracheal chains from ovalbumin-sensitized guinea-pigs, esculetin (60 microM) significantly reduced the duration of the recovery phase after antigen challenge contraction. In conclusion, in our in vitro conditions, this natural coumarin derivative seems to act by interfering with the arachidonic acid cascade.

Animals↗
PubMedDate not supplied

Quantitative predictivity of the transformation in vitro assay compared with the Ames test.

For 59 chemical compounds, we have found homogeneous data on transformation in vitro, mutagenicity in the Ames test, and carcinogenicity. We have compared the potency in inducing transformation in vitro in hamster fibroblast cells with the carcinogenic potency and found a modest correlation coefficient between the two parameters (r = 0.37). For these same 59 compounds it was also possible to compare mutagenic potency in the Ames test with carcinogenic potency. The correlation level was very similar (r = 0.34). The predictivity of transformation in vitro increased significantly when only compounds for which some kind of dose-response relationship was available were utilized (r = 0.65). This result stresses the importance of the quantitative aspect of the response in predictivity studies. The present study is compared with previous studies on the quantitative predictivity of different short-term tests. Our work is not definitive, but gives an idea of the possible type of approach to the problem of comparing quantitative predictivities.

Animals↗
PubMedDate not supplied

Improvement of liver cancer detection with simultaneous assessment of circulating levels of free alpha-fetoprotein (AFP) and AFP-IgM complexes.

We assessed the presence of alpha-fetoprotein (AFP) complexed with IgM (AFP-IgM IC) in serum of patients affected by hepatocellular carcinoma (HCC), cirrhosis and chronic hepatitis as well as in healthy subjects by means of a dedicated ELISA assay. The amount of AFP-IgM IC was expressed in arbitrary units (AU) on a reference standard curve. Free AFP (FAFP) levels were determined in parallel in each sample by means of an automated immunoassay system. The mean serum concentration of AFP-IgM IC was significantly higher in HCC patients (mean +/- SD: 1378.3 +/- 2935.7 AU/mL) than in cirrhotic patients (129.8 +/- 261.4 AU/mL) and in patients with chronic hepatitis (80.9 +/- 168.9 AU/mL) (p < 0.01). HCC patients had FAFP values above the 20 ng/mL cutoff in 44% of cases (22/50) and AFP-IgM IC values above the 120 AU/mL cutoff in 60% of cases (30/50). The occurrence of the free and IgM-complexed form of circulating AFP did not overlap, and 82% of patients (41/50) were positive for at least one marker. The results indicate that AFP-IgM IC is a complementary serological marker to FAFP and that the combination of these biomarkers may be useful in the diagnosis of liver cancer.

Adult↗
PubMedDate not supplied

Detection of circulating CEA-IgM complexes in early stage colorectal cancer.

We have recently shown that alpha fetoprotein (AFP) and squamous cell carcinoma antigen (SCCA), biomarkers associated with hepatocellular carcinoma, may be detected in patient sera as circulating immune complexes with IgM, and that assessment of serum levels of AFP-IgM and SCCA-IgM may be used for the detection of liver cancer. In this study we measured the levels of carcinoembryonic antigen (CEA) as free form (FCEA) and complexed to IgMs (CEA-IgM) in sera of patients affected by colorectal carcinoma (CRC) at different stages as well as in healthy subjects. FCEA levels were above the 5 ng/mL cutoff in 43% of CRC patients (31/72) and CEA-IgM levels were above the 200 AU/mL cutoff in 38% of CRC patients (27/72). Serum levels of CEA-IgM immune complexes (IC) and FCEA did not overlap and 64% of patients (46/72) were positive for at least one marker without compromising the detection specificity (94%). Early detection of CRC was significantly improved by CEA-IgM IC assay. CRC patients at an early stage (stage 1) had elevated CEA-IgM levels in 29% of cases (7/24), while FCEA levels were elevated in only 8% of cases (2/24). These results indicate that CEA-IgM is a complementary serological marker to FCEA which is much more sensitive for early stage CRC, and that the combination of these biomarkers may be useful in the early detection of colorectal cancer.

Adult↗
PubMedDate not supplied

The role of the thiol N-acetylcysteine in the prevention of tumor invasion and angiogenesis.

We have extensively studied the effects of N-acetylcysteine (NAC), a cytoprotective drug that can prevent in vivo carcinogenesis. Here we review our findings NAC completely inhibits gelatinolytic activity of metalloproteases and chemotactic and invasive activities of tumor cells. In addition, NAC reduces the number of lung metastases when malignant murine melanoma cells are injected into nude mice. NAC treatment decreases the weight of primary tumors and produces a dose-related increase in tumor latency. Moreover, oral administration of NAC reduces the formation of spontaneous metastases. In experimental metastasis assays, we have found a synergistic reduction in the number of lung metastases after treatment with doxorubicin (DOX) and NAC in nude mice. In tumorigenicity and spontaneous metastasis assays, the combined administration of DOX and oral NAC again has shown synergistic effects on the frequency and weight of primary tumors and local recurrences and completely prevented the formation of lung metastases. The addition of NAC to endothelial cells strongly reduces their invasive activity in response to angiogenic stimuli. NAC inhibited the degradation and release of radiolabeled type IV collagen by activated endothelial cells, indicating that NAC blocks gelatinase activity. Oral administration of NAC reduces the angiogenic response induced by KS tumor cell products, confirming the ability of NAC to inhibit the invasive activity of endothelial cells in vivo and thereby blocking angiogenesis.

Acetylcysteine↗
PubMedDate not supplied

Antiapoptotic and antigenotoxic effects of N-acetylcysteine in human cells of endothelial origin.

N-Acetylcysteine (NAC) is a drug bearing multiple preventive properties that can inhibit genotoxicity and carcinogenicity. NAC also inhibits invasion and metastasis of malignant cells, as well as tumor take. We recently demonstrated the effects of NAC on Kaposi's sarcoma cells supernatant-induced invasion in vitro and angiogenesis in vivo. Many anticancer agents act through cytotoxicity of rapidly proliferating cells and several antineoplastic drugs induce apoptosis of cancer cells. Since endothelial cells are the target for the inhibition of angiogenesis, we wanted to verify that NAC, while inhibiting tumor vascularization and endothelial cell invasion would not induce endothelial cell apoptosis. We tested the ability of NAC to modulate apoptosis and cytogenetic damage in vitro and to promote differentiation on a reconstituted basement membrane (matrigel) in two endothelial cell lines (EAhy926 and HUVE). Treatment with NAC protected endothelial cells from TGF-beta-induced apoptosis and paraquat-induced cytogenetic damage. Therefore, NAC acts as an antiangiogenic agent and, at the same time, appears to prevent apoptosis and oxygen-related genotoxicity in endothelial cells.

Acetylcysteine↗
PubMedDate not supplied

Effect of dipyridamole, 5'-(N-ethyl)-carboxamidoadenosine and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine on LOVO cell growth and morphology.

The effects of stable compounds acting on adenosine receptors, 5'-(N-ethyl)-carboxamidoadenosine (NECA: A2 and A1 adenosine receptor agonist) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX: selective A1 adenosine receptor antagonist) were evaluated in vitro on doxorubicin-resistant LoVo (LoVo-R) and doxorubicin-sensitive LoVo (LoVo-S) human metastatic cell lines by using the neutral red test for cell growth. The effect of dipyridamole, an adenosine uptake inhibitor, was also evaluated. The drugs had an inhibitory effect on LoVo cell growth. The association of the drugs with doxorubicin enhanced the inhibition of cell growth, particularly for NECA and PACPX on LoVo-R cells. Morphological observation with scanning electron microscopy indicated cytotoxicity of the tested compounds, alone or in association with doxorubicin both in LoVo-R or LoVo-S cells, supporting the hypothesis of inhibitory effect on tumor cell growth.

Adenosine↗
PubMedDate not supplied

Peptide-based assay for the identification of endothelin-converting enzyme inhibitors.

A simple, solid-phase assay usable for the detection of endothelin-converting enzyme activity and inhibitors has been developed. It uses a multimeric peptide immobilized on microtiter plates that is able to specifically recognize the Big Endothelin fragment 16-32 derivatized with biotin. This fragment is cleaved between residues 21-22 by alpha-chymotrypsin with almost the same proteolysis rate as Big Endothelin, and after enzyme treatment it does not bind to the multimeric peptide adsorbed on the microtiter plates. The amount of uncleaved peptide bound to the plate is detected by subsequent treatment with streptavidin conjugated to peroxidase, followed by a chromogenic reaction. Model inhibitor profiles generated for alpha-chymotrypsin, a protease known to convert Big Endothelin in endothelin, demonstrated the utility of this assay as a rapid high-throughput aid in the study of Big Endothelin enzymatic processing and possibly in the identification of putative enzyme inhibitors.

Amino Acid Sequence↗