Biomedical subjects

M Mazzotti

Publications and source records attributed to M Mazzotti.

At least 19 recordsLinked to original sources

PubMed2001

Supercritical fluid simulated moving bed chromatography II. Langmuir isotherm.

The simulated moving bed (SMB) technology offers the possibility of scaling up single column, batch chromatographic separations to continuous operation. This has proved particularly effective for the separation of enantiomers, and has been applied in the liquid and gas phase, as well as using a supercritical fluid as eluent (SF-SMB). In the last case, the main performance improvements are due to the possibility of tuning the elution strength of the mobile phase, by changing the pressure in the four sections of the SMB unit. Thus a SF-SMB can be operated in two modes, i.e. the isocratic and the pressure gradient mode. In this research, design criteria for these two operating modes have been developed, which can be applied to systems described by linear as well as nonlinear Langmuir adsorption isotherms. The role and effect of an appropriate modifier in increasing solubility and reducing retention times have been investigated. The results reported allow to identify the operating conditions, including the pressure levels and the modifier concentration, which lead to optimal separation performance in terms of productivity and desorbent requirement.

Adsorption↗

PubMed2000

On-line monitoring of enantiomer concentration in chiral simulated moving bed chromatography.

Simulated moving bed chromatography is a key technology for the pilot and production scale separation of enantiomers of chiral chemical species. Product quality control is probably the most important issue in this kind of separation at both scales, and for this it is clear that on-line monitoring of absolute enantiomer concentrations plays a major role. In this work, an on-line system consisting of a UV detector and a polarimeter in series is used to monitor the composition of the extract and raffinate streams of a laboratory SMB unit. The model system adopted is the separation of the enantiomers of the Tröger's base on microcrystalline cellulose triacetate (CTA) using ethanol as mobile phase. The technique is effective and accurate, thus providing promising perspectives for SMB process control and dynamic optimization.

Chromatography, Liquid↗

PubMed2000

Design and optimisation of a simulated moving bed unit: role of deviations from equilibrium theory.

The design of a simulated moving bed involves thermodynamic, kinetic and hydrodynamic aspects and requires the optimisation of several variables: plant design variables, such as the column length and diameter, and operating variables, among them four independent flow-rates, the feed concentration and the switch time. In this work we develop an algorithm to design both the unit and its operating conditions, with an overall view on equilibrium properties, efficiency and hydrodynamics, using a simple equilibrium stage model. In this way we determine the parameters leading to the highest possible productivity for a given separation, only requiring the knowledge of the equilibrium isotherms, the Van Deemter equation and a correlation for pressure drop. The algorithm has been used to investigate the effect on the separation performance of some parameters, such as particle size and required product purity, which are not considered by equilibrium theory. The results have been compared with the predictions of equilibrium theory and the observed deviations have been put in evidence and discussed.

Algorithms↗

PubMed2000

Simulated moving-bed chromatography and its application to chirotechnology.

The increased awareness of the differences in biological activity of the two enantiomers of a chiral drug has raised the demand for enantiomerically pure products, particularly in the pharmaceutical industry. Simulated moving-bed chromatography can be used for the separation of the two enantiomers of a chiral molecule, which is feasible at all production scales, from laboratory to pilot to production plant. The use of non-enantioselective synthesis of racemic mixtures and simulated moving-bed enantiomer separation might make the development process of a new chiral drug substantially shorter and cheaper.

Chromatography↗

PubMed1999

Enantiomer separation of alpha-ionone using gas chromatography with cyclodextrin derivatives as chiral stationary phases.

The gas chromatographic enantiomer separation of alpha-ionone was studied with three different chiral stationary phases using as chiral selectors: (1) heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin, dissolved in polysiloxane PS-086, (2) octakis(2,6-di-O-pentyl-3-O-trifluoroacetyl)-gamma-cyclodextrin and (3) octakis(2,6-di-O-pentyl-3-O-butanoyl)-gamma-cyclodextrin, both dissolved in polysiloxane SE-54. The influence of the concentration of the chiral selector in the polysiloxane, coated on Chromosorb P AW-DMCS 80-100 mesh, is described and discussed, as well as the effect of Chromosorb loading. The feasibility of the preparative gas chromatographic separation of the enantiomers of alpha-ionone is considered; in order to provide a term of comparison, the estimated performances are compared with those achieved in the separation of the enantiomers of the inhalation anaesthetic enflurane.

Anesthetics, Inhalation↗

PubMed1998

Simulated moving bed chromatographic resolution of a chiral antitussive.

The behavior of a laboratory simulated moving bed (SMB) unit for continuous chromatographic separation of enantiomers has been considered. This was applied to the resolution of a chiral antitussive agent, guaifenesin, on Chiralcel OD, during an experimental campaign involving nineteen runs. The application of recently developed criteria for the design and optimization of SMB units allows us to understand and rationalize the experimental results, as well as to indicate how to optimize the separation performances. A three-step procedure to determine the adsorption isotherms needed to apply these criteria is proposed; it is reliable and may be applied also where pure components are not available.

Adsorption↗

PubMed1996

Antisense epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma cells.

Human rhabdomyosarcoma cells express membrane epidermal growth factor receptor (ECF-R), which could confer responsiveness to EGF and transforming growth factor-alpha (TGF-alpha) of autocrine or paracrine origin. To study the role played by this growth factor circuit in the proliferation and differentiation of myogenic neoplastic cells, human rhabdomyosarcoma EGF-R-expressing cells (RD/18 clone) have been transfected with a plasmid containing a fragment of the EGF-R cDNA in the antisense orientation. In vitro growth and differentiative ability were studied on six antisense-transfected clones (AS) in comparison to parental RD/18 cells and to cells transfected with the plasmid containing only the neomycin resistance gene (NEO). A reduced EGF-R membrane expression was found in AS clones by decreased immunofluorescence with an anti-EGF-R monoclonal antibody. All AS transfectants had a greatly impaired proliferative ability, even when cultured in fetal bovine serum-containing medium. Proliferation of AS clones was completely blocked in medium supplemented with 2% horse serum. The differentiation ability of AS clones was heterogeneous, ranging from clones with a percentage of myosin-positive cells higher than controls to clones with a negligible myosin expression. Therefore, the growth impairment determined by the loop interruption is not sufficient to switch on the differentiation program. The role played by EGF-R in the proliferation of human rhabdomyosarcoma cells suggests that this receptor could constitute a target for a therapeutic approach.

Animals↗

PubMed1996

Epidermal growth factor receptor interaction with clathrin adaptors is mediated by the Tyr974-containing internalization motif.

The carboxyl-terminal regulatory domain of the epidermal growth factor (EGF) receptor is essential for its endocytosis and interaction with the clathrin-associated protein complex AP-2. To identify AP-2 binding motif in the receptor, several single and multiple-point mutations within the region between residues 966 and 977 of the human EGF receptor were made, and the mutant receptors were expressed in NIH3T3 cells. Mutation of tyrosine 974 alone or together with surrounding residues and the deletion of residues 973-975 essentially eliminated AP-2 co-immunoprecipitation with the EGF receptor. Furthermore, a synthetic peptide corresponding to receptor residues 964-978 blocked AP-2 association with the wild-type EGF receptor. These data suggest that AP-2 has only one high-affinity binding site in the EGF receptor composed of Tyr974-containing motif. Receptor mutants that did not bind AP-2 displayed a lower rate of internalization, down-regulation, and turnover compared to wild-type receptors when expressed at high levels. However, similar receptor mutants expressed at low levels were internalized and down-regulated as efficiently as wild-type receptors. Internalization of the mutant receptors lacking the high-affinity binding site for AP-2 was inhibited by K+-depletion of the cells, indicating that their endocytosis required intact coated pits. We suggest that whereas one mechanism of EGF receptor recruitment into coated pits involves high-affinity binding of AP-2 to Tyr974-containing motif, another pathway may be mediated by weak receptor/AP-2 interactions or by proteins other than AP-2.

3T3 Cells↗

PubMed1995

SHC and GRB-2 are constitutively by an epidermal growth factor receptor with a point mutation in the transmembrane domain.

A single point mutation, Glu627--> Val, equivalent to the activating mutation in the Neu oncogene, was inserted in the transmembrane domain of the human epidermal growth factor (EGF) receptor. Unlike the wild type, Glu627-EGF receptor, transfected in NIH3T3 cells, gave rise to focal transformation and growth in agar even in the absence EGF. Constitutive activity of mutant EGF receptor amounted to 20% of that of wild type receptor stimulated by EGF. In addition, the mutant receptor was more sensitive to EGF, reaching maximum transforming activity at 5 ng/ml EGF. NIH3T3 cells expressing Glu627-EGF receptor showed a transformed phenotype and were not arrested in G0 upon serum deprivation. The mutant receptor was constitutively autophosphorylated, and several other cellular proteins were phosphorylated on tyrosine in absence of the ligand. Among these, the SHC adaptor protein was phosphorylated in absence of EGF, the other adaptor, GRB-2 was constitutively associated with the Glu627-EGF receptor in vivo and in vitro, and mitogen-activated protein kinase was constitutively phosphorylated. In contrast, other EGF receptor substrates, like phospholipase C gamma, were not phosphorylated in absence of EGF. The mutant receptor showed a higher sensitivity to cleavage by calpain both in absence and presence of EGF, appeared as a 170- and 150-kDa doublet in cell extracts, and a specific calpain inhibitor blocked the appearance of the 150-kDa form. Since the calpain cleavage site is located in the receptor cytoplasmic tail, this finding suggests that the Glu627 mutation induces a slightly different conformation in the EGF receptor intracellular domain. In conclusion, our data show that a point mutation in the EGF receptor transmembrane domain was able to constitutively activate the receptor and to induce transformation via constitutive activation of the Ras pathway.

3T3 Cells↗

PubMed1993

27-Hydroxycholesterol modulation of low density lipoprotein metabolism in cultured human hepatic and extrahepatic cells.

27-Hydroxycholesterol**, 25-hydroxycholesterol and cholesterol suppressed LDL uptake and degradation in human extrahepatic and hepatic cell lines in a concentration-dependent manner. Cholesterol was the least potent, and the inhibitory effect of oxysterols was more pronounced in skin fibroblasts and in endothelial cell line EAhy 926 than in hepatoma HepG2 cells. Shorter incubations were required for oxysterols to achieve 50% inhibition of LDL uptake and degradation in all cultured cells. The inhibition of LDL catabolism in extrahepatic cells by 27-hydroxycholesterol occurred at concentrations close to those observed in human plasma (0.2-0.6 microM). The results support a possible role of 27-hydroxycholesterol, a physiological oxysterol, in the regulation of cellular cholesterol homeostasis in non-hepatic tissues.

Cells, Cultured↗

PubMed1993

Relationship between mevalonate pathway and arterial myocyte proliferation: in vitro studies with inhibitors of HMG-CoA reductase.

The role of mevalonate and its products (isoprenoids) in the control of cellular proliferation was examined by investigating the effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (vastatins) on growth and on cholesterol biosynthesis of cultured arterial myocytes (SMC). Simvastatin (S) and fluvastatin (F), but not pravastatin (P), decreased the rate of growth of rat vascular SMC. The inhibition, evaluated as cell number, was dose-dependent with IC50 values of 2.8 and 2.2 microM for S and F, respectively; P (1-500 microM) was inactive. The inhibition of cell growth induced by 3.5 microM S (70% decrease) was prevented completely by the addition of 100 microM mevalonate, partially (70-85%) by the addition of 10 microM geraniol, 10 microM farnesol and 5 microM geranylgeraniol, but not by the addition of squalene, confirming the specific role of isoprenoid metabolites in regulating cell proliferation. All the tested vastatins inhibited the incorporation of [14C]acetate into cholesterol but P had 800 times lower potency than S and F. Similar results were obtained in SMC from human femoral artery. At least 80% inhibition of cholesterol synthesis was necessary to induce a decrease in SMC proliferation. To further investigate the relationship between cholesterol synthesis and cell growth, two enantiomers of F were investigated. The enantiomer more active on HMG-CoA reductase was 70- and 1.6-fold more potent on arterial myocyte proliferation than its antipode and the racemic mixture, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyclic Monoterpenes↗

PubMed1992

Characterization of a family with moderate hypercholesterolemia and binding defective low density lipoprotein.

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder presenting with hypercholesterolemia and abnormal low density lipoprotein (LDL) that binds poorly to LDL receptors. This disease appears to be caused by a mutation in the apo B gene. In the present study thirteen members of a family with moderate hypercholesterolemia (250-350 mg/dl) were investigated. Biochemical studies on cultured skin fibroblasts ruled out classical familial hypercholesterolemia (receptor deficiency). LDL from nine affected members displayed, in an "in vitro" cell binding assay, a reduced affinity (2.5 fold) for the receptor, and had normal electrophoretic mobility, size and chemical composition. Lp(a) levels in family members were comparable to those present in normolipidemics and lower than those observed in primary hypercholesterolemia. The disorder is transmitted over three generations as an autosomal codominant trait and all the affected members are heterozygotes and hypercholesterolemic.

Adolescent↗

PubMed1992

Ability of the LDL receptor from several animal species to recognize the human apo B binding domain: studies with LDL from familial defective apo B-100.

To verify whether the LDL receptors from different animal species recognize the binding domain of human apo B-100 we studied the interaction of LDL from control and familial binding defective apo B-100 (FDB) with cultured cells. Human, monkey, bovine, guinea pig and rabbit LDL receptors distinguish between normal and binding defective LDL with a displacement ratio (defective/normal) of 3.3, 2.6, 3.4, 3.1 and 2.0, respectively. Guinea pig and rabbit receptors, however, showed affinities 2-3-fold lower than the human receptor. Hamster, rat and mouse cells failed to differentiate between normal and FDB LDL with a ratio of 1.2, 0.8, and 1.4; the apparent affinities were 4-8 times lower than that of the human receptor. The data from the latter species suggest that the LDL receptor recognizes an area of human apo B different from the human receptor binding domain. The ability of antibody Mb47 to inhibit the binding of human LDL to human, rabbit and guinea pig but not to mouse cells further stresses this concept. Moreover, in 17 alpha-ethinyl estradiol-treated rats the rate of disappearance from plasma of FDB and control 125I-labelled LDL was identical, thus confirming the in vitro observations. These data suggest that the binding domain of the LDL receptor is functionally conserved in man, monkey, cow, rabbit and guinea pig, but is quite distinct in rat, mouse and hamster.

Animals↗

PubMed1991