Biomedical subjects

G Rivas

Publications and source records attributed to G Rivas.

At least 19 recordsLinked to original sources

PubMed2003

Tracer sedimentation equilibrium: a powerful tool for the quantitative characterization of macromolecular self- and hetero-associations in solution.

During the last two decades the measurement and analysis of sedimentation equilibrium has been used increasingly as a method for characterizing intermolecular interactions in solution. More recently, a variant of this technique has been developed, in which a trace amount of a single solute component is labelled so that the concentration gradient of that component at sedimentation equilibrium may be measured independently of the gradients of all other components. The dependence of equilibrium tracer gradients upon solution composition may be readily interpreted in the context of models for self- and hetero-association of the labelled component, and, in the case of concentrated solutions, repulsive as well as attractive solute-solute interactions. We present a summary of experimental and analytic methods, a brief review of some previously published applications, and a preview of new applications demonstrating capabilities beyond those afforded by other current techniques for characterizing macromolecular associations in solution.

Biophysics↗

PubMed2002

Analysis of radioactive pollution in the Gulf of Palermo.

PURPOSE: Sea water represents more than three quarters of the whole world. The radioactive fall out caused by nuclear experiments, radioactive drosses from nuclear power plants and other pollution sources, are able to contaminate the world ecosystems, and especially the sea water. The aim of this work is the evaluation of a possible radioactive pollution in the Gulf of Palermo. MATERIAL AND METHODS: The Gulf of Palermo was divided into 5 homogeneous hydrological parts, in which fish and water were collected. The dosage of 131I e del 137Cs was mainly assessed because they have a long half-life and their presence in the water can be due to nuclear accidents but also to a wrong removal of the waste materials of nuclear medicine procedures; the other radioisotopes assessed (144Ce, 106Rh, 140La) are due only to nuclear fall-out. Gamma spectrometry technique was used for the evaluation of the radioisotopes' concentration after an adequate treatment of the samples. RESULTS AND CONCLUSIONS: The concentration of the examined radionuclides was below the instrumental capability apart from the 137Cs, whose dose was increased, and can be due to nuclear accidents and explosions. The radionuclide levels detected do not determine a higher risk than normal for the population's health even regard the eatablity of ittical species.

Animals↗

PubMed2001

Direct observation of the enhancement of noncooperative protein self-assembly by macromolecular crowding: indefinite linear self-association of bacterial cell division protein FtsZ.

Recent measurements of sedimentation equilibrium and sedimentation velocity have shown that the bacterial cell division protein FtsZ self-associates to form indefinitely long rod-like linear aggregates in the presence of GDP and Mg(2+). In the present study, the newly developed technique of non-ideal tracer sedimentation equilibrium was used to measure the effect of high concentrations-up to 150 g/liter-of each of two inert "crowder" proteins, cyanmethemoglobin or BSA, on the thermodynamic activity and state of association of dilute FtsZ under conditions inhibiting (-Mg(2+)) and promoting (+Mg(2+)) FtsZ self-association. Analysis of equilibrium gradients of both FtsZ and crowder proteins indicates that, under the conditions of the present experiment, FtsZ interacts with each of the two crowder proteins essentially entirely via steric repulsion, which may be accounted for quantitatively by a simple model in which hemoglobin, albumin, and monomeric FtsZ are modeled as effective spherical hard particles, and each oligomeric species of FtsZ is modeled as an effective hard spherocylinder. The functional dependence of the sedimentation of FtsZ on the concentrations of FtsZ and either crowder indicates that, in the presence of high concentrations of crowder, both the weight-average degree of FtsZ self-association and the range of FtsZ oligomer sizes present in significant abundance are increased substantially.

Bacterial Proteins↗

PubMed2000

Interaction of the antitumor drug 9-aminoacridine with guanidinobenzoatase studied by spectroscopic methods: a possible tumor marker probe based on the fluorescence exciplex emission.

Fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), and analytical centrifugation are applied in this work to study the interaction of the antitumor drug 9-aminoacridine (9AA) with a trypsin-like protease, guanidinobenzoatase (GB), extracted from an Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission can be detected at a certain drug and enzyme concentration. The 9AA exciplex emission was also studied for 9AA interacting with others serin proteases: alpha-chymotrypsin, trypsin, and penicillin G-acylase (PGA), as well as with bovine serum albumin (BSA) in order to obtain information about the active center of GB. We have found that the exciplex 9AA emission may be induced by a ring-stacking interaction between the monomeric drug, under the amino form, and an aromatic residue placed in the catalytic site of the protein. The results derived from Raman spectroscopy corroborate this interaction mechanism, as demonstrated by the existence of typical protonated amino 9AA marker bands as well as an important modification of the ring vibrations, thus indicating the existence of an interaction through ring stacking. The analytical centrifugation technique was applied to study the GB association in aqueous solution, demonstrating that the 9AA/GB interaction depends on the enzyme quaternary structure. An interaction of 9AA with an associate form of GB, which may be the actual enzyme active form, is suggested.

Aminacrine↗

PubMed2000

Stripping analysis of nucleic acids at a heated carbon paste electrode.

A new electrically heated carbon paste electrode has been developed for performing adsorptive stripping measurements of trace nucleic acids. Such coupling of electrochemistry at electrically heated electrodes with adsorptive constant-current stripping chronopotentiometry offers distinct advantages for trace measurements of nucleic acids. The application of increased temperatures during the deposition step results in dramatic (4-34-fold, depending on temperature applied) enhancement of the stripping signal. Such improvement is attributed to the accumulation step at the heated electrode. Forced thermal convection near the electrode surface facilitates the use of quiescent solutions and hence of ultrasmall volumes. Using an electrode temperature of 32 degrees C and a quiescent solution during the 1-min accumulation, the response is linear over the 1-8 mg/L range tested, with a detection limit of 0.5 mg/L. Such electrode heating technology offers great promise for various applications involving thermal manipulations of nucleic acids.

Carbon↗

PubMed2000

Magnesium-induced linear self-association of the FtsZ bacterial cell division protein monomer. The primary steps for FtsZ assembly.

The bacterial cell division protein FtsZ from Escherichia coli has been purified with a new calcium precipitation method. The protein contains one GDP and one Mg(2+) bound, it shows GTPase activity, and requires GTP and Mg(2+) to polymerize into long thin filaments at pH 6.5. FtsZ, with moderate ionic strength and low Mg(2+) concentrations, at pH 7.5, is a compact and globular monomer. Mg(2+) induces FtsZ self-association into oligomers, which has been studied by sedimentation equilibrium over a wide range of Mg(2+) and FtsZ concentrations. The oligomer formation mechanism is best described as an indefinite self-association, with binding of an additional Mg(2+) for each FtsZ monomer added to the growing oligomer, and a slight gradual decrease of the affinity of addition of a protomer with increasing oligomer size. The sedimentation velocity of FtsZ oligomer populations is compatible with a linear single-stranded arrangement of FtsZ monomers and a spacing of 4 nm. It is proposed that these FtsZ oligomers and the polymers formed under assembly conditions share a similar axial interaction between monomers (like in the case of tubulin, the eukaryotic homolog of FtsZ). Similar mechanisms may apply to FtsZ assembly in vivo, but additional factors, such as macromolecular crowding, nucleoid occlusion, or specific interactions with other cellular components active in septation have to be invoked to explain FtsZ assembly into a division ring.

Bacterial Proteins↗

PubMed2000

The marine mites Hyadesia sp. and Copidognathus sp. Associated with the mussel Mytilus galloprovincialis.

Two species of marine mites belonging to the families Hyadesiidae and Halacaridae, Hyadesia sp. and Copidognathus sp., respectively, were found associated with the mussel Mytilus galloprovincialis from Baja California in NW México. The first species was found inside the mussel gut with an intensity ranging from one to six mites per mussel and their prevalence was from 20.0 to 46.7%; this species was also found living free in the sediment at a density of 0.7 mite/100 ml. The second species was found on the mantle and gills of the host with an intensity ranging from one to three mites per host and their prevalence was from 3.3 to 6.7%; this species was abundant (4.5 mites/100 ml) and living free in the sediment around mussel clumps. Hyadesia sp. was found alive and attached in the gut of the mussel. A histological analysis revealed this species in the lumen of intestine surrounded by mucus and attached to the epithelial cells of the intestine, where some disorder of epithelial cells was associated. Moreover, this mite may be encapsulated by hemocytes inside the digestive diverticulum, the reproductive follicle, or the connective tissue surrounding the diverticulum. No damages to branches or gills resulting from the presence of Copidognathus sp. were observed. The results suggest that these mites are occasional invaders of mussels; however, as a result of this infestation, Hyadesia sp. may produce damage in the host's tissues. This is the first record of marine mites inside the gut, reproductive follicles, branches, and mantle of a marine bivalve.

Animals↗

PubMed1999

Direct observation of the self-association of dilute proteins in the presence of inert macromolecules at high concentration via tracer sedimentation equilibrium: theory, experiment, and biological significance.

The technique of tracer sedimentation equilibrium [Rivas, G., et al. (1994) Biochemistry, 2341-2348 (1); Rivas, G., et al. (1996) J. Mol. Recognit. 9, 31-38 (2)] is utilized, together with an extension of the theory of sedimentation equilibrium of highly nonideal solutions [Chatelier and Minton, (1987) Biopolymers 26, 1097-1113 (3)], to characterize the thermodynamic activity and/or the state of association of a dilute, labeled macromolecular solute in the presence of an arbitary concentration of a second, unlabeled macromolecular solute. Experiments are performed on solutions of labeled fibrinogen (0.25-1 g/L) in bovine serum albumin (0-100 g/L) in the presence and absence of divalent cations (Ca(2+), Mg(2+)), and on solutions of labeled tubulin (0.2-0.6 g/L) in dextran (0-100 g/L). It is found that in the absence of the divalent cations, the large dependence of the thermodynamic activity of fibrinogen on BSA concentration is well accounted for by a simple model for steric repulsion. In the presence of the cations and sufficiently large concentrations of BSA (>30 g/L), fibrinogen appears to self-associate to a weight-average molar mass approximately twice that of monomeric fibrinogen. Tubulin appears to self-associate to an extent that increases monotonically with increasing dextran concentration, reaching a weight-average molar mass almost 3 times that of the alphabeta dimer in the presence of 100 g/L dextran. Possible biological ramifications are discussed.

Buffers↗

PubMed1999

Sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus, is a monomer-tetramer associating protein.

Sticholysin II (Stn-II) is a pore-forming cytolysin. Stn-II interacts with several supports for size exclusion chromatography, which results in an abnormal retardation precluding molecular mass calculations. Sedimentation equilibrium analysis has revealed that the protein is an associating system at neutral pH. The obtained data fit a monomer-tetramer equilibrium with an association constant K4c of 10(9) M(-3). The electrophoretic pattern of Stn-II treated with different cross-linking reagents, in a wide range of protein concentrations, corroborates the existence of tetrameric forms in solution. A planar configuration of the four monomers, C4 or D2 symmetry, is proposed from modelling of the cross-linking data.

Animals↗

PubMed1999

Characterization of heterologous protein-protein interactions using analytical ultracentrifugation.

Methods for quantitative characterization of heterologous protein-protein interactions by means of analytical ultracentrifugation (AUC) include sedimentation equilibrium, tracer sedimentation equilibrium, sedimentation velocity, and analytical band sedimentation. Fundamental principles governing the behavior of macromolecules in a centrifugal field are summarized, and the application of these principles to the interpretation of data obtained from each type of experiment is reviewed. Instrumentation and software for the acquisition and analysis of data obtained from different types of AUC experiments are described.

Kinetics↗

PubMed1998

Structural features of the plasmid pMV158-encoded transcriptional repressor CopG, a protein sharing similarities with both helix-turn-helix and beta-sheet DNA binding proteins.

The small transcriptional repressor CopG protein (45 amino acids) encoded by the streptococcal plasmid pMV158 was purified to near homogeneity. Gel filtration chromatography and analytical ultracentrifugation showed that the native protein is a spherical dimer of identical subunits. Circular dichroism measurements of CopG indicated a consensus average content of more than 50% alpha-helix and 10-35% beta-strand and turns, which is compatible with the predicted secondary structure of the protein. CopG exhibited a prolonged intracellular half-life, but deletions in regions other than the C-terminal affected the global structure of the protein, severely reducing the half-lives of the CopG variants. This indicates that CopG has a compact structure, perhaps constituted by a single domain. Molecular modeling of CopG showed a good fitting between the helix-turn-helix motifs of well-known repressor proteins and a bihelical unit of CopG. However, modeling of CopG with ribbon-helix-helix class of DNA binding proteins also exhibited an excellent fit. Eleven out of the 12 replicons belonging to the pMV158 plasmid family could also encode Cop proteins, which share features with both helix-turn-helix and beta-sheet DNA binding proteins.

Amino Acid Sequence↗

PubMed1998

Control of the structural stability of the tubulin dimer by one high affinity bound magnesium ion at nucleotide N-site.

Tubulin liganded with GTP at the N-site in the alpha-subunit and with GDP at the E-site in the beta-subunit (GDP-tubulin) reversibly binds one high affinity Mg2+ cation (Kb = 1.1 x 10(7) M-1), whereas tubulin liganded with GTP at both subunits (GTP-tubulin) binds one more high affinity Mg2+. The two cation binding loci are identified as nucleotide sites N and E, respectively. Mg2+ at the N-site controls the stability and structure of the alphabeta-tubulin dimer. Mg2+ dissociation is followed by the slow release of bound nucleotide and functional inactivation. Mg2+ bound to the N-site significantly increases the thermal stability of the GDP-tubulin dimer (by 10 degrees C and approximately 50 kcal mol-1 of experimental enthalpy change). However, the thermal stability of Mg2+-liganded GDP- and GTP-tubulin is the same. Mg2+ binding to the N-site is linked to the alphabeta-dimer formation. The binding of Mg2+ to the alpha-subunit communicates a marked enhancement of fluorescence to a colchicine analogue bound to the beta-subunit. Colchicine, in turn, thermally stabilizes Mg2+-depleted tubulin. The tubulin properties described would be simply explained if the N-site and the colchicine site are at the alpha-beta dimerization interface. It follows that the E-site would be at the beta-end of the tubulin dimer, consistent with the known functional role of the E nucleotide gamma-phosphate and coordinated cation controlling microtubule stability.

Animals↗

PubMed1998

The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple coiled-coil structure and interacts with the 21-kilodalton (A17L) virus membrane protein through a C-terminal alpha-helix.

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third alpha-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.

Amino Acid Sequence↗

PubMed1997

Phage phi29 protein p6 is in a monomer-dimer equilibrium that shifts to higher association states at the millimolar concentrations found in vivo.

Protein p6 from Bacillus subtilis phage phi29 (Mr = 11 800) binds in vitro to DNA forming a large nucleoprotein complex in which the DNA wraps a multimeric protein core. The high intracellular abundance of protein p6 together with its ability to bind the whole phi29 DNA in vitro strongly suggests that it plays a role in viral genome organization. We have determined by sedimentation equilibrium analysis that protein p6 (1-100 microM range), in the absence of DNA, is in a monomer-dimer equilibrium, with an association constant (K2) of approximately 2 x 10(5) M-1. The intracellular concentration of protein p6 (approximately 1 mM) was estimated measuring the number of copies per cell (7 x 10(5)) and the cell volume (1 x 10(-15) L). At concentrations around 1 mM, protein p6 associates into oligomers. This self-association behavior is compatible with a dimer-hexamer model (K2,6 = 3.2 x 10(8) M-2) or with an isodesmic association of the dimer (K = 950 M-1), because the apparent weight-average molecular mass (Mw,a) does not reach saturation at the highest protein concentrations. The sedimentation coefficients of protein p6 monomer and dimer were 1.4 and 2.0, respectively, compatible with translational frictional ratios (f/fo) of 1.15 and 1.30, which slightly deviate from the hydrodynamics of a rigid globular protein. Taking together these results and considering the structure of the nucleoprotein complex, we speculate that the observed oligomers of protein p6 could mimic a scaffold on which DNA folds to form the nucleoprotein complex in vivo.

Bacillus Phages↗

PubMed1997

Destabilization, oligomerization and inhibition of the mitogenic activity of acidic fibroblast-growth factor by aurintricarboxylic acid.

The triphenylmethane derivative aurintricarboxylic acid has been used to inhibit angiogenesis, vascular smooth muscle cell proliferation and cell transformation, an effect that has been attributed to its relatively nonspecific inhibitory activity of protein-nucleic acid interactions. Here, we show that this compound binds to acidic fibroblast growth factor, a prototypic member of a family of protein mitogens activated by heparin, altering its physicochemical properties and decreasing its mitogenic activity. Counteraction of the effects of aurintricarboxylic acid by heparin shows that the two compounds have opposite and reversible effects on acidic fibroblast growth factor structure and biological activity. The studies reported here may contribute to a deeper understanding of the inhibition of fibroblast-growth-factor-dependent mitogenesis of relevance to future pharmacologic developments.

3T3 Cells↗

PubMed1997

Structural features of phi29 single-stranded DNA-binding protein. II. Global conformation of phi29 single-stranded DNA-binding protein and the effects of complex formation on the protein and the single-stranded DNA.

The strand-displacement mechanism of Bacillus subtilis phage phi29 DNA replication occurs through replicative intermediates with high amounts of single-stranded DNA (ssDNA). These ssDNA must be covered by the viral ssDNA-binding protein, phi29 SSB, to be replicated in vivo. To understand the characteristics of phi29 SSB-ssDNA complex that could explain the requirement of phi29 SSB, we have (i) determined the hydrodynamic behavior of phi29 SSB in solution and (ii) monitored the effect of complex formation on phi29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 +/- 0.1) x 10(8) gs(-1), and its rotational correlation time, 7.0 +/- 0.5 ns, phi29 SSB was modeled as a nearly spherical ellipsoid of revolution. The axial ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) or 1.0 to 3.2 (prolate model, a > b). Far-UV CD spectra, indicated that phi29 SSB is highly organized within a wide range of temperatures (15 to 50 degrees C), being mainly constituted by beta-sheet elements (approximately 50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of phi29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, phi29 SSB binding leads to non-conservative changes of the near-UV CD spectra of ssDNA, which are consistent with different nearest-neighbor interactions of the nucleotide bases upon complex formation. The above results will be compared to those reported for other SSBs and discussed in terms of the functional roles of phi29 SSB.

Bacillus Phages↗

PubMed1997

Nucleic-acid immobilization, recognition and detection at chronopotentiometric DNA chips.

Wide-scale DNA testing requires the development of fast, small, easy-to-use biosensing devices. Various synthetic oligonucleotides and DNA have thus been immobilized onto microfabricated thick-film carbon transducers for performing several new nucleic-acid assay protocols. These include hybridization detection of nucleic acid sequences, determination of small molecules (drugs, pollutants) based on their collection into the dsDNA layer or via monitoring their effect upon the intrinsic DNA oxidation signal, and direct adsorptive stripping measurements of ultratrace levels of nucleic acids. Transduction of these DNA recognition processes is accomplished by a new highly-sensitive constant-current stripping chronopotentiometric operation. Comparison to traditional electrodes indicates that the biosensing performance is not compromised by the use of mass-producible disposable transducers. Such thick-film DNA biosensors have been coupled to a compact, user-friendly, hand-held analyzer. Applicability for the detection of sequences from M. tuberculosis and HIV-1 DNAs is illustrated. Such activity in the author's laboratory, aimed at developing DNA-coated screen-printed electrodes, is reviewed.

Biosensing Techniques↗

PubMed1996

DNA electrochemical biosensor for the detection of short DNA sequences related to the human immunodeficiency virus.

An electrochemical biosensor for the detection of short DNA sequences related to the human immunodeficiency virus type 1 (HIV-1) is described. The sensor relies on the immobilization and hybridization of the 21- or 42-mer single-stranded oligonucleotide from the HIV-1 U5 long terminal repeat (LTR) sequence at carbon paste or strip electrodes. The extent of hybridization between the complementary sequences is determined by the enhancement of the chronopotentiometric peak of the Co(phen)3(3+) indicator. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions are optimized to maximize the sensitivity and speed the assay time. A detection limit of 4 x 10(-9) M HIV-1 U5 LTR segment is reported following a 30 min hybridization. The hybridization biosensor format obviates the use of radioisotopes common in radioactive methods for the detection of HIV-1 DNA. We also report on the direct adsorptive chronopotentiometric stripping measurements of trace levels of various HIV-1 DNAs.

Base Sequence↗