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Biomedical subjects

C Bustamante

Publications and source records attributed to C Bustamante.

At least 37 records · Page 2Linked to original sources
PubMed2000

The mechanochemistry of molecular motors.

A theory of molecular motors is presented that explains how the energy released in single chemical reactions can generate mechanical motion and force. In the simplest case the fluctuating movements of a motor enzyme are well described by a diffusion process on a two-dimensional potential energy surface, where one dimension is a chemical reaction coordinate and the other is the spatial displacement of the motor. The coupling between chemistry and motion results from the shape of the surface, and motor velocities and forces result from diffusion currents on this surface. This microscopic description is shown to possess an equivalent kinetic mechanism in which the rate constants depend on externally applied forces. By using this equivalence we explore the characteristic properties of several broad classes of motor mechanisms and give general expressions for motor velocity versus load force for any member of each class. We show that in some cases simple plots of 1/velocity vs. 1/concentration can distinguish between classes of motor mechanisms and may be used to determine the step at which movement occurs.

Biomechanical Phenomena↗
PubMed2000

Stretching of single collapsed DNA molecules.

The elastic response of single plasmid and lambda phage DNA molecules was probed using optical tweezers at concentrations of trivalent cations that provoked DNA condensation in bulk. For uncondensed plasmids, the persistence length, P, decreased with increasing spermidine concentration before reaching a limiting value 40 nm. When condensed plasmids were stretched, two types of behavior were observed: a stick-release pattern and a plateau at approximately 20 pN. These behaviors are attributed to unpacking from a condensed structure, such as coiled DNA. Similarly, condensing concentrations of hexaammine cobalt(III) (CoHex) and spermidine induced extensive changes in the low and high force elasticity of lambda DNA. The high force (5-15 pN) entropic elasticity showed worm-like chain (WLC) behavior, with P two- to fivefold lower than in low monovalent salt. At lower forces, a 14-pN plateau abruptly appeared. This corresponds to an intramolecular attraction of 0.083-0.33 kT/bp, consistent with osmotic stress measurements in bulk condensed DNA. The intramolecular attractive force with CoHex is larger than with spermidine, consistent with the greater efficiency with which CoHex condenses DNA in bulk. The transition from WLC behavior to condensation occurs at an extension about 85% of the contour length, permitting looping and nucleation of condensation. Approximately half as many base pairs are required to nucleate collapse in a stretched chain when CoHex is the condensing agent.

Bacteriophage lambda↗
PubMed2000

Single-molecule studies of DNA mechanics.

During the past decade, physical techniques such as optical tweezers and atomic force microscopy were used to study the mechanical properties of DNA at the single-molecule level. Knowledge of DNA's stretching and twisting properties now permits these single-molecule techniques to be used in the study of biological processes such as DNA replication and transcription.

Animals↗
PubMed2000

Grabbing the cat by the tail: manipulating molecules one by one.

Methods for manipulating single molecules are yielding new information about both the forces that hold biomolecules together and the mechanics of molecular motors. We describe here the physical principles behind these methods, and discuss their capabilities and current limitations.

DNA↗
PubMed2000

[Impact of antiretroviral therapy in mortality of Chilean HIV (+) patients: a case control study].

BACKGROUND: Combined antiretroviral therapy (AVR) has shown a protective effect (PE) on morbidity and survival in HIV (+) patients of industrialised countries where triple-drug therapy (ARV-3) is standard. In Chile the public health system began providing double-drug therapy in 1997 (ARV-2) with 2 reverse transcriptase inhibitors. AIM: To assess the impact of ARV in morbimortality of HIV (+) patients in Chile after a year of follow up. PATIENTS AND METHODS: Retrospective case-control (1:1) study. Cases were 97 patients followed during 1997 for 6 or more months and dying during that period. Each case had a control of the same gender and CDC stage, similar age and CD4 count, but surviving a same period of follow up. A comparison of ARV before and during follow up (rate and type) was done. P carinii prophylaxis, pneumococcal immunization at baseline or follow up, frequency of hospital admissions and occurrence of opportunistic infections in both groups were assessed. Odds ratio (OR) for mortality, hospitalization and opportunistic infections in ARV user, as well as treatment PE were calculated. RESULTS: Twenty four (24.7%) cases and sixty six (68%) controls received ARV during follow up (p < 0.001), OR was 0.15 (CI 95% 0.08-0.3), p < 0.001, the PE was 6.6 for ARV users versus non users, among cases 19 patients received ARV-2 and five received ARV-3. Among controls, 41 patients received ARV-2 and 25 received ARV-3. These differences established an OR of 0.20 (CI 95% 0.09-0.04) and a PE of 5 for ARV-2 versus no ARV. For ARV-3 compared with no ARV the OR was 0.08 (CI 95% 0.003-0.26), and the PE 12.5. Fifty three (54.6%) cases and 13 (13.4%) controls required hospital admission, OR 0.49 (CI 95% 0.25-0.94), p = 0.03, and PE of 2.04 of ARV versus no ARV; 82 (85.3%) cases and 50 (51%) controls had opportunistic infections, OR 0.5 (CI 95% 0.26-0.96), p = 0.03 and PE of 2 for ARV versus no ARV. There were no significant differences in prior ARV, prophylaxis and immunisation between cases and controls. CONCLUSIONS: This study showed the high impact of ARV in short term morbimortality of HIV(+) patients and the need to implement antiretroviral therapy to all patients as an official health policy. This study did not answer the question of the role, if any, of weaker-than standard antiretroviral therapy.

AIDS-Related Opportunistic Infections↗
PubMed1999

How RNA folds.

We describe the RNA folding problem and contrast it with the much more difficult protein folding problem. RNA has four similar monomer units, whereas proteins have 20 very different residues. The folding of RNA is hierarchical in that secondary structure is much more stable than tertiary folding. In RNA the two levels of folding (secondary and tertiary) can be experimentally separated by the presence or absence of Mg2+. Secondary structure can be predicted successfully from experimental thermodynamic data on secondary structure elements: helices, loops, and bulges. Tertiary interactions can then be added without much distortion of the secondary structure. These observations suggest a folding algorithm to predict the structure of an RNA from its sequence. However, to solve the RNA folding problem one needs thermodynamic data on tertiary structure interactions, and identification and characterization of metal-ion binding sites. These data, together with force versus extension measurements on single RNA molecules, should provide the information necessary to test and refine the proposed algorithm.

Algorithms↗
PubMed1999

Polymerization and mechanical properties of single RecA-DNA filaments.

The polymerization of individual RecA-DNA filaments, containing either single-stranded or double-stranded DNA, was followed in real time, and their mechanical properties were characterized with force-measuring laser tweezers. It was found that the stretch modulus of a filament is dominated by its (central) DNA component, while its bending rigidity is controlled by its (eccentric) protein component. The longitudinal stiffness of DNA increases 6- to 12-fold when the DNA is contained in the protein helix. Both the stretch modulus and the bending rigidity of a fiber change in the presence of various nucleotide cofactors-e.g., [gamma-thio]ATP, ATP, and ADP-indicating a substantial re-arrangement of spatial relationships between the nucleic acid and the protein scaffold. In particular, when complexed with ATP, a fiber becomes twice as extensible as a [gamma-thio]ATP fiber, suggesting that 32% of the DNA-binding sites have been released in its core. Such release may enable easy rotation of the DNA within the protein helix or slippage of the DNA through the center of the protein helix.

Adenosine Triphosphate↗
PubMed1999

Wrapping of DNA around the E.coli RNA polymerase open promoter complex.

High-resolution atomic force microscopy (AFM) and biochemical methods were used to analyze the structure of Escherichia coli RNA polymerase.sigma(70) (RNAP) open promoter complex (RP(o)). A detailed analysis of a large number of molecules shows that the DNA contour length of RP(o) is reduced by approximately 30 nm (approximately 90 bp) relative to the free DNA. The DNA bend angle measured with different methods varied from 55 to 88 degrees. The contour length reduction and the DNA bend angle were much less in inactive RNAP-DNA complexes. These results, together with previously published observations, strongly support the notion that during transcription initiation, the promoter DNA wraps nearly 300 degrees around the polymerase. This amount of DNA bending requires an energy of 60 kJ/mol. The structural analysis of the open promoter complexes revealed that two-thirds of the DNA wrapped around the RNAP is part of a region upstream of the transcription start site, whereas the remaining one-third is part of the downstream region. Based on these data, a model of the sigma(70).RP(o) conformation is proposed.

Bacterial Proteins↗
PubMed1999

Presence of dinucleotide and ATP receptors in human cerebrocortical synaptic terminals.

Human cerebrocortical synaptic terminals elicited concentration-dependent Ca2+ transients after Ap5A (diadenosine pentaphosphate) and ATP stimulation, with EC50 values of 23.44 +/- 3.70 microM and 11.48 +/- 2.12 microM, respectively. The lack of cross-desensitisation and the selective antagonism by Ip5I (diinosine pentaphosphate), suggests the activation of a dinucleotide receptor by Ap5A, and a P2X receptor by ATP. Ap5A Ca2+ transients were partially abolished by omega-conotoxin GVI-A (53%), suggesting the participation of a N-type Ca2+ channel in the dinucleotide response. ATP effect on Ca2+ entry was abolished by nicardipine (44%) and by omega-conotoxin GVI-A (52%), suggesting the participation of L- and N-type Ca2+ channels. These data suggest that Ap5A and ATP activate dinucleotide and P2X receptors, respectively, in human brain synaptic terminals.

Adenosine Triphosphate↗
PubMed1999

Trapping of megabase-sized DNA molecules during agarose gel electrophoresis.

Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated lambda-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed.

Bacteriophage lambda↗
PubMed1999

Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase.

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

Adsorption↗
PubMed1999

Palatability, reactogenicity and immunogenicity of engineered live oral cholera vaccine CVD 103-HgR in Chilean infants and toddlers.

BACKGROUND: Live oral cholera vaccine CVD 103-HgR is well-tolerated and immunogenic when administered to adults, school age children and preschool children in a single 5 x 10(9) colony-forming unit dose. Because elicitation of immune responses after administration of a single dose is exceptional for any oral vaccine in any age group, CVD 103-HgR was used as a probe to investigate the clinical acceptability, practicality and immunogenicity of this vaccine in infants and toddlers 3 to 17 months of age. METHODS: The study was undertaken successively in 12- to 17-month-olds (n = 104), 7- to 11-month-olds (n = 106) and 3- to 5-month-olds (n = 102). One-half of the subjects were randomly allocated to receive vaccine and the other one-half to receive placebo, in double blind fashion. After 2 weeks of double blind follow-up, all subjects received a dose of vaccine. Vibriocidal antibody titers were measured on coded sera collected at baseline and 2 weeks after each dosing. The buffered vaccine "cocktail" had a volume of 100 ml; subjects who ingested > or =70 ml were considered fully vaccinated. FINDINGS: Only 37% of subjects overall (25% of 3- to 5-month-olds) ingested > or =70 ml of the cocktail. The vaccine was well-tolerated with no significant differences in the rate or severity of adverse reactions after ingestion of vaccine vs. placebo. Seroconversion after ingestion of a single dose of CVD 103-HgR was similar in fully vaccinated subjects (66%) and in those who ingested a smaller fraction of the vaccine cocktail (63%). Of subjects who ingested two doses, 5 of 118 excreted vaccine organisms on Day 7 after the first dose vs. 0 of 118 after the second dose. INTERPRETATION: Single dose oral CVD 103-HgR is well-tolerated and immunogenic in infants even when a partial dose is ingested. The buffered vaccine cocktail that is readily imbibed by older children is not appealing to young infants, and improved vaccine formulations and delivery vehicles for immunizing infants must be sought.

Administration, Oral↗
PubMed1999

[Serum antibodies against hepatitis A virus among subjects of middle and low socioeconomic levels in urban area of Santiago, Chile].

BACKGROUND: The epidemiology of hepatitis A virus (HAV) infection is closely associated to the level of hygiene and sanitation of the population. Newly industrialising areas experience a transition from high to intermediate endemicity, which is characterized by a shift in the exposure age to HAV, from early childhood to school ages or adolescence. AIM: To measure the prevalence of HAV antibodies in subjects living in urban Santiago. SUBJECTS AND METHODS: A HAV antibody survey in five medium and low socioeconomic level urban districts of northern Santiago, was conducted in 1996. Healthy subjects aged 1 to 39 years old were recruited from randomly selected households. RESULTS: Five hundred three subjects were studied. Anti HAV antibodies were found in 13.2, 29.5, 59.6, 78.1, 95.6 and 98.2% of individuals aged 1 to 4, 5 to 9, 10 to 14, 15 to 19, 20 to 29 and 30 to 39 years old, respectively. CONCLUSIONS: The profile of positive antibodies is compatible with an intermediate pattern of transmission of HAV. The current data supports the idea that infection is shifting towards older ages. A progressive increase in the number of susceptible school age children and teenagers is propitious for the occurrence of common source hepatitis A outbreaks.

Adolescent↗
PubMed1998

Polymer chain statistics and conformational analysis of DNA molecules with bends or sections of different flexibility.

The worm-like chain model has often been employed to describe the average conformation of long, intrinsically straight polymer molecules, including DNA. The present study extends the applicability of the worm-like chain model to polymers containing bends or sections of different flexibility. Several cases have been explicitly considered: (i) polymers with a single bend; (ii) polymers with multiple coplanar bends; (iii) polymers with two non-coplanar bends; and (iv) polymers comprised of sections with different persistence lengths. Expressions describing the average conformation of such polymers in terms of the mean-square end-to-end distance have been derived for each case. For cases (i) and (iv), expressions for the projection of the end-to-end vector onto the initial orientation of the chain are presented. The expressions derived here have been used to investigate DNA molecules with sequence-induced bending (A-tracts). Mean-square end-to-end distance values determined from a large number of A-tract containing DNA molecules visualized by scanning force microscopy resulted in an average bend angle of 13.5 degrees per A-tract. A similar study was performed to characterize the flexibility of double-strandedDNA molecules containing a single-stranded region. Analysis of their mean-square end-to-end distance yielded a persistence length of 1.3 nm for single-stranded DNA.

DNA↗
PubMed1998

Complete unfolding of the titin molecule under external force.

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several earlier studies have implicated titin as playing a fundamental role in maintaining sarcomeric structural integrity and generating the passive force of muscle. The elastic properties of titin were characterized in recent single-molecule mechanical works that described the molecule as an entropic spring in which partial unfolding may take place at high forces during stretch and refolding at low forces during release. In the present work titin molecules were stretched using a laser tweezer with forces above 400 pN. The high external forces resulted in complete mechanical unfolding of the molecule, characterized by the disappearance of force hysteresis at high forces. Titin refolded following complete denaturation, as the hysteresis at low forces reappeared in subsequent stretch-release cycles. The broad force range throughout which unfolding occurred indicates that the various globular domains in titin require different unfolding forces due to differences in the activation energies for their unfolding.

Antibodies↗
PubMed1998

Theory for the hydrodynamic and electrophoretic stretch of tethered B-DNA.

We have developed a theory for the extension and force of B-DNA tethered at a fixed point in a uniform hydrodynamic flow or in a uniform applied electric field. The chain tethered in an electric field is considered to be subject to free electrophoresis compensated by free sedimentation in the opposite direction. This allows the use of results of free electrophoresis for including the effects of small ions. The force on the chain is derived for a sequence of ellipsoidal segments, each twice the persistence length of the wormlike chain. Hydrodynamic interaction between these segments is based on the long-range limit of flow around the prolate ellipsoids, as derived from equivalent Stokes spheres. The chain extension is derived by applying the entropic elasticity relation of Marko and Siggia (1995 Macromolecules. 28:8759-8770) to each segment for polymer chains under constant tension. We justify this procedure by comparing with extension results based on the Boltzmann averaged orientation of straight, freely jointed segments. Predicted results agree well with recent extension-flow experiments by Perkins et al., 1995. Science. 258:83-87, and with electrophoretic stretch experiments by Smith and Bendich (1990 Biopolymers. 29:1167-1173) on fluorescently stained B-DNA. We find that the equivalence of hydrodynamic and electrophoretic stretch, proposed by Long et al. (1996 Phys. Rev. Lett. 76:3858-3861; 1996 Biopolymers 39:755-759), is valid only for very small chain deformations, but not in general.

Biophysical Phenomena↗