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Biomedical subjects

V Rizzo

Publications and source records attributed to V Rizzo.

At least 73 records · Page 4Linked to original sources
PubMed1989

Correlation between growth inhibition and intranuclear doxorubicin and 4'-deoxy-4'-iododoxorubicin quantitated in living K562 cells by microspectrofluorometry.

Intranuclear drug concentration in cells treated with doxorubicin (DXR) or with 4'-deoxy-4'-iododoxorubicin (IDX) was measured by means of a quantitative microspectrofluorometric technique recently developed by us. Resolution of free and bound drug contributions in fluorescence emission spectra, as collected from a microvolume of single living cell nuclei, provided concentration data with about 10% indetermination. Uptake of DXR and IDX into the nucleus of K562 cells and DXR-resistant K562/DXR cells could then be studied with a sensitive, nondestructive technique. Growth inhibitory concentrations of K562 and K562/DXR cells, when measured with respect to drug content in the medium, differed by a factor of 25 in the case of DXR and by a factor of three in the case of IDX. By contrast, intranuclear drug concentrations measured at corresponding growth inhibitory concentrations are found to be nearly constant, i.e., independent of cellular-resistant phenotype and of anthracycline structure. This result supports an identical mechanism of action for the two drugs, most probably targeted to the nucleus, and ascribes to intracellular transport the different potency of the two drugs in the two cell lines.

Algorithms↗
PubMed1989

Kinetic studies of anthracycline-DNA interaction by fluorescence stopped flow confirm a complex association mechanism.

The kinetics of association and dissociation between calf thymus DNA and five anthracyclines, including doxorubicin, daunorubicin, and three synthetic analogues, were investigated with stopped flow using fluorescence detection. The sensitivity of this technique allowed us to work with submicromolar drug concentrations, thus excluding formation of aggregates, and with ratios of DNA base pairs to drug in the range 10-250, where site exclusion effects could be taken into account with a simple correction of DNA concentration and pseudo-first-order conditions were nearly fulfilled. In all cases, both association and dissociation reactions required a sum of three exponential terms to be fitted. However, satisfactory interpretation of reciprocal relaxation times as functions of DNA concentration was only achieved with kinetic models comprising a total of five steps. One of the extra steps was tentatively assigned to formation of a weakly bound, probably nonintercalated species. Another step was deduced from a comparison between results of association and dissociation experiments. The five steps are arranged, for convenience, in an association mechanism with two branches, though other mechanisms cannot be definitely ruled out. Correlation of cytotoxicity data with both association and dissociation rates is not found to be significant. This suggests that other factors must be involved in modulating the different biological properties of the investigated anthracyclines.

Animals↗
PubMed1989

Association of anthracyclines and synthetic hexanucleotides. Structural factors influencing sequence specificity.

The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.

Antibiotics, Antineoplastic↗
PubMed1988

Specificity in formation of triple-stranded nucleic acid helical complexes: studies with agarose-linked polyribonucleotide affinity columns.

The binding of a variety of deoxyribo and ribo homo- and copolynucleotide complementary duplexes to agarose-linked homopolynucleotide affinity columns has been studied. The results provide information concerning the specificity of recognition of complementary base pairs of nucleic acids through a mechanism that involves triple-helix formation under physiological conditions of ionic strength, pH, and temperature. The method employed made it possible, for the first time, to survey the full range of base triplets conceivable from the canonical nucleic acid bases and, in addition, hypoxanthine and thereby to differentiate between those triplets which can and cannot form. Certain previously observed features of the stereochemistry of double-helical targets for third-strand binding are confirmed, and some unrecognized features are elaborated. These include a general requirement for clusters of purine residues in one strand, protonation of third-strand C residues, the ability of natural third-strand residues to distinguish between A.T/U and G.C base pairs, and a capacity of third-strand (unnatural) I residues to recognize all base pairs within such clusters. Thus, the basis for a third-strand binding code is demonstrated.

Base Composition↗
PubMed1988

Studies of anthracycline--DNA complexes by circular dichroism.

A series of doxorubicin and daunorubicin analogues have been investigated in aqueous solution and as DNA-bound forms by means of circular dichroism (c.d.) spectroscopy. The structural variants comprise modifications on the amino sugar, on the aliphatic ring and the side chain of the aglycone moiety, and of the substitution pattern of the anthraquinone chromophore. Results with compounds having conformational constraints interfering with optimal fitting to DNA indicate that stereochemistry and conformation of the aliphatic ring predominantly affect c.d. spectra of anthracyclines in DNA-bound as well as in free form. Conformational correspondence with the known structure of the daunorubicin-oligonucleotide complex is inferred from the spectra of derivatives with modifications at position 6 or 11 in the anthraquinone chromophore. On the other hand, a different binding geometry is postulated for compounds either lacking the 4-methoxy group of daunorubicin (idarubicin and derivatives) or having a phenolic function in its place (carminomycin and derivatives). A possible relation with cytotoxic activity is discussed at a speculative level.

Antibiotics, Antineoplastic↗
PubMed1987

Alamethicin incorporation in lipid bilayers: a thermodynamic study.

Interaction of the peptide antibiotic alamethicin with phospholipid vesicles has been monitored by changes in its circular dichroic and fluorescent properties. The data are consistent with an incorporation of the peptide in the lipid bilayer. Aggregation of alamethicin in the membrane phase is evident from a characteristic concentration dependence of the incorporation, reflecting the existence of a critical concentration. The data can be fully understood in terms of a theoretical approach that includes aggregation and thermodynamic nonideality. Thermodynamic parameters of the peptide-lipid interaction have been evaluated under a variety of conditions of temperature, ionic strength, and lipid type (saturated and unsaturated fatty acid chains). From the results obtained in this study, one can extrapolate to the incorporation behavior of alamethicin at low concentrations, as they are typical for measurements of conductance across planar lipid films. This leads to a simple explanation of the voltage-gating mechanism of alamethicin in a straightforward way.

Alamethicin↗
PubMed1987

Incorporation kinetics in a membrane, studied with the pore-forming peptide alamethicin.

The reaction of fluorescence-labeled alamethicin with unilamellar phospholipid vesicles (DOPC and DMPC) has been investigated in a stopped-flow apparatus. Clearly single exponential time functions have been observed at temperatures above the phase transition of the bilayer. This can be interpreted in terms of an essentially one-step incorporation process. The pseudo first-order forward rate is found to be quite fast, falling in a range somewhat below the diffusion controlled upper bound. The data are quantitatively very well described on the basis of a simple mechanism. This comprises diffusion of peptide into the bilayer accompanied by a more or less slower change of the secondary structure. Aggregation of the incorporated molecules at higher concentrations is indicated to be comparatively rapid.

Alamethicin↗
PubMed1986

Thermodynamic analysis of incorporation and aggregation in a membrane: application to the pore-forming peptide alamethicin.

Interaction of the pore-forming antibiotic alamethicin with small unilamellar vesicles of dioleoylphosphatidylcholine has been studied by means of circular dichroism. The data strongly suggest that alamethicin does not bind to the surface of the vesicles but incorporates into the lipid phase to a fairly large extent. Furthermore, aggregation of the peptide in the membrane is apparent from the existence of a 'critical concentration'. Quantitative evaluation and interpretation of the data rest on a quite generally applicable thermodynamic analysis. The underlying phenomenon is treated in terms of a partition equilibrium between the aqueous and lipid media. In the bilayer phase non-ideal interactions (described by appropriate activity coefficients) as well as aggregate formation are considered. Using this approach the relevant parameters of the alamethicin-lipid system have been determined (yielding, in particular, a partition coefficient of 1.3 X 10(3) for the monomeric peptide and a critical aqueous concentration of 2.5 microM). Finally, the possible relevance of these results for the voltage-dependent gating of alamethicin is briefly pointed out.

Alamethicin↗
PubMed1986

Effects of L-5HTP with and without carbidopa on plasma beta-endorphin and pain perception: possible implications in migraine prophylaxis.

L-Tryptophan (L-TP) has been used in migraine and other pain conditions. The mechanism underlying the analgesic effect is still partly undefined. In this study the effects of subchronic administration of L-5-hydroxytryptophan (L-5HTP) (with and without carbidopa) on plasma beta-endorphin (beta-EP) levels and subjective pain threshold and tolerance were investigated in seven healthy volunteers. To measure also an objective indicator of pain, the nociceptive flexion reflex threshold was studied. L-5HTP treatment with and without carbidopa administration increased beta-EP levels significantly (p less than 0.05). L-5HTP plus carbidopa induced an increase in beta-EP significantly (p less than 0.05) greater than that induced by L-5HTP alone. Neither the subjective pain threshold and tolerance nor the RIII threshold was modified by either treatment. Our data seem to point to the existence of a complex linkage between plasma opioid levels and pain perception.

Adult↗
PubMed1986

Effects of L-5HTP with and without carbidopa on plasma beta-endorphin and pain perception. Possible implications in migraine prophylaxis.

L-Tryptophan (L-TP) has been used in migraine and other pain conditions. The mechanism underlying the analgesic effect is still partly undefined. In this study the effects of subchronic administration of L-5-hydroxy-tryptophan (L-5HTP) (with and without carbidopa) on plasma beta-endorphin (beta-EP) levels and subjective pain threshold and tolerance were investigated in seven healthy volunteers. To measure also an objective indicator for pain, the nociceptive flexion reflex threshold was studied. L-5HTP treatment with and without carbidopa administration increased beta-EP levels significantly (p less than 0.05). L-5HTP plus carbidopa induced an increase in beta-EP significantly (p less than 0.05) higher than that after L-5HTP alone. Neither subjective pain threshold and tolerance nor RIII threshold was modified by either treatment. Our data seem to point to the existence of a complex linkage between plasma opioid levels and pain perception.

5-Hydroxytryptophan↗
PubMed1985

Molecular shape and dipole moment of alamethicin-like synthetic peptides.

The peptides Boc-(L-Ala-Aib-L-Ala-Aib-L-Ala)n-OMe, with n = 2 (P10) and n = 4 (P20), have been synthesized as purely hydrophobic models of the antibiotic alamethicin, which is known to be a voltage-dependent pore former in membranes and is apparently alpha-helical in lipophilic media. These peptides were investigated in 1-octanol, a solvent which resembles the membrane environment. From dielectric dispersion studies quantitative information on the molecular shape and dipole moments could be derived. Further independent data concerning conformation and extent of aggregation of the peptides were obtained by circular dichroism and ultracentrifuge measurements. The results suggest that the peptides assume the form of elongated particles having a significant amount of ordered secondary structure and carrying a dipole parallel to the long axis. Apparently the monomeric peptide molecules undergo, to some extent, a head-to-tail aggregation which is slightly enhanced at lower temperatures. Based on the high-frequency parts of the dielectric dispersion curves the lengths, diameters, and dipole moments of the monomer particles have been determined as 22.5A, 10A, 36 D (P10) and 28.5A, 12A, 64D (P20).

Alamethicin↗
PubMed1976

Co-oligopeptides of glycine and aromatic amino acids with variable distance between the aromatic residues. IV. Spectroscopic properties of tryptophan-containing peptides in a cyclohexane phase.

L-Tryptophan, L-tryptophanylglycine, glycyl-L-tryptophan, glycyl-L-tryptophanylglycine and glycyl-L-tryptophanylglycylglycyl-L-tryptophanylglycine have been transferred from an aqueous solution (generally 0.1 M NaOH) to cyclohexane, using the quaternary ammonium salt trioctylmethyl ammonium chloride (NR+4Cl-, soluble in cyclohexane but not in water) as the transporting agent. The spectroscopic properties of L-tryptophan and tryptophan-containing peptides have been studied in the cyclohexane phase. With respect to the aqueous solutions, ultraviolet absorption spectra are characterized by a considerable red shift of the absorption maxima and by a hypochromicity of up to 10%. Fluorescence spectra generally show emission maxima which are characteristic of polar environments, accompanied by a significant enhancement of the quantum yield. CD spectra have also been investigated for all peptides and compared with those for aqueous systems reported in preceding publications. All these spectral changes cannot be attributed solely to the cyclohexane solvent effect. It is suggested that these anomalous spectral properties of the tryptophan-containing compounds in the cyclohexane-NR+4 solution are due to the influence the electrostatic field of the ion pair has on the indole chromophore. The possible implications of this finding for the spectroscopic properties of aromatic residues buried in the polar interior of proteins are discussed.

Circular Dichroism↗