Search PubMed⌕ Search

Biomedical subjects

R H Austin

Publications and source records attributed to R H Austin.

At least 37 records · Page 2Linked to original sources
PubMed1996

Biotechnology at low Reynolds numbers.

The shrinking of liquid handling systems to the micron and submicron size range entails moving into the area of small Reynolds numbers. The fluid dynamics in this regime are very different from the macroscale. We present an intuitive explanation of how the different physics of small Reynolds numbers flow, along with microscopic sizes, can influence device design, and give examples from our own work using fluid flow in microfabricated devices designed for biological processing.

Biotechnology↗
PubMed1995

Trapping of branched DNA in microfabricated structures.

We have observed electrostatic trapping of tribranched DNA molecules undergoing electrophoresis in a microfabricated pseudo-two-dimensional array of posts. Trapping occurs in a unique transport regimen in which the electrophoretic mobility is extremely sensitive to polymer topology. The arrest of branched polymers is explained by considering their center-of-mass motion; in certain conformations, owing to the constraints imposed by the obstacles a molecule cannot advance without the center of mass first moving a short distance backwards. The depth of the resulting local potential well can be much greater than the thermal energy so that escape of an immobilized molecule can be extremely slow. We summarize the expected behavior of the mobility as a function of field strength and topology and point out that the microfabricated arrays are highly suitable for detecting an extremely small number of branched molecules in a very large population of linear molecules.

Bacteriophage lambda↗
PubMed1995

Deformation and flow of red blood cells in a synthetic lattice: evidence for an active cytoskeleton.

We introduce the use of microfabrication techniques to construct on a silicon wafer a synthetic capillary bed with 2.5- to 4-micron (mu)-wide channels. Establishment of a fluid pressure gradient allowed us to observe simultaneously using optical microscopy hundreds of cells flowing through the bed at physiological speeds. We find a large distribution of mobilities among red cells flowing through the structure; smaller channels provide a greater impedance to flow than larger ones, indicating that kinetic drag variations provide the origin of the distribution. The mobility of a particular cell is not correlated with the cell diameter but appears to be inversely correlated with intracellular calcium concentration of the cell, as determined by fluorescence of the calcium-binding dye fluo-3 AM. Also, we are able to use the parallel processing nature of our arrays to observe isolated events where the rigidity of the red cell seems to change suddenly over several orders of magnitude as it blocks a channel in the array.

Capillaries↗
PubMed1995

Determination of the role of the 5-ht6 receptor in the rat brain: a study using antisense oligonucleotides.

The purpose of the present study was to determine possible physiological functions of the 5-ht6 receptor using antisense oligonucleotides (AOs) in male rats. Repeated intracerebroventricular treatment with AOs but not with a scrambled form of the antisense sequence (SO) gave rise to a specific behavioral syndrome of yawning, stretching and chewing and caused a 30% reduction in the number of [3H]-lysergic acid diethylamide binding sites (measured in the presence of 300 nM spiperone). Neither sequence, however, had any effect on other parameters measured (e.g., locomotor activity, body weight, food intake, body temperature and nociception). The specific behavioral syndrome did not appear to be caused by modulation of dopaminergic neurotransmission since no changes in the tissue levels of either dopamine or its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were seen. Furthermore, haloperidol (0.03 mg/kg s.c.) did not reduce the number of yawns or stretches. An increase in cholinergic neurotransmission did appear to be involved since the behavioral syndrome was dose-dependently antagonized by atropine. The present study suggests that 5-ht6 receptors are functionally expressed in the rat brain, where one of their functions appears to be the control of cholinergic neurotransmission.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

Thermodynamics and premelting conformational changes of phased (dA)5 tracts.

Using synthetic 45 bp long DNAs of known sequences, we have studied (i) the unusual structure that the phased A-tracts have at temperatures below 37 degrees C, (ii) the thermodynamics of the loss of that aberrant structure well below the duplex melting temperature, and (iii) the conformational changes that occur with temperature. Using temperature-dependent circular dichroism, we detect a low-temperature structural transition in a 45-mer duplex with four segments of phased (dA)5 tracks separated by five segments of five randomized G-C pairs, but none in the corresponding isomeric random sequence 45-mer duplex. Differential scanning calorimetry measurements reveal the enthalpy of this preglobal melting transition to be 3.5 kcal/mol.AT pair or 4.4 kcal/mol.AA step. The integrated enthalpy change for this helix-to-helix intramolecular event only is about 16% of the global duplex-to-single-strands melting enthalpy and is relatively broad compared to the global melting event (about 30 vs 15 degrees C for the full width at half maxima). Electric birefringence decay measurements show that the phased 45 bp duplex has a rotational time constant of 100 ns at 5 degrees C which increases to 220 ns above 40 degrees C. Simple modeling of the dynamics within the junction model for bending yields that the bend per A-tract is 42 degrees at 5 degrees C, decreasing to 0 degrees above 40 degrees C. We suggest that this sequence-dependent structure which "melts" at physiological temperatures may be relevant to DNA topology and function.

Adenine Nucleotides↗
PubMed1992

DNA electrophoresis in microlithographic arrays.

We have used optical microlithography to fabricate capped quasi-two-dimensional obstacle courses in SiO2. We report here observations using epifluorescence microscopy of the electrophoresis and length fractionation of large DNA molecules confined in arrays. Simple reptation theory, based on the work of deGennes, predicts that at low electric fields the electrophoretic mobility of a polymer of length L much greater than the persistence length p scales inversely with L (ref. 2). But elongation of the coil in the matrix at sufficiently strong electric fields results in a length-independent electrophoretic mobility. The application of suitably timed pulsed electric fields restores the fractionating power of gels for long molecules but the protocols of pulsed-field electrophoresis are semi-empirical because the complex and ill-understood gel matrix plays a critical role in fractionation. Microlithographically constructed obstacle arrays, with their low dimensionality, small volume and extremely reproducible topography, will make it possible to understand the motion and fractionation of large polymer molecules in complex but well characterized topologies.

DNA↗
PubMed1990

Physical studies of DNA premelting equilibria in duplexes with and without homo dA.dT tracts: correlations with DNA bending.

We have employed a variety of physical methods to study the equilibrium melting and temperature-dependent conformational dynamics of dA.dT tracts in fractionated synthetic DNA polymers and in well-defined fragments of kinetoplast DNA (kDNA). Using circular dichroism (CD), we have detected a temperature-dependent, "premelting" event in poly(dA).poly(dT) which exhibits a midpoint near 37 degrees C. Significantly, we also detect this CD "premelting" behavior in a fragment of kDNA. By contrast, we do not observe this "premelting" behavior in the temperature-dependent CD spectra of poly[d(AT)].poly[d(AT)], poly(dG).poly(dC), poly[d(GC)].poly[d(GC)], or calf thymus DNA. Thus, poly(dA).poly(dT) and kDNA exhibit a common CD-detected "premelting" event which is absent in the other duplex systems studied in this work. Furthermore, we find that the anomalous electrophoretic retardation of the kDNA fragments we have investigated disappears at temperatures above approximately 37 degrees C. We also observe that the rotational dynamics of poly(dA).poly(dT) and kDNA as assessed by singlet depletion anisotropy decay (SDAD) and electric birefringence decay (EBD) also display a discontinuity near 37 degrees C, which is not observed for the other duplex systems studied. Thus, in the aggregate, our static and dynamic measurements suggest that the homo dA.dT sequence element [common to both poly(dA).poly(dT) and kDNA] is capable of a temperature-dependent equilibrium between at least two helical states in a temperature range well below that required to induce global melting of the host duplex. We suggest that this "preglobal" melting event may correspond to the thermally induced "disruption" of "bent" DNA.

Animals↗
PubMed1990

Ligand dynamics in the photodissociation of carboxyhemoglobin by subpicosecond transient infrared spectroscopy.

Time-resolved infrared spectroscopy with 0.5-ps resolution is used to track the evolution of the CO stretching vibration after visible photoexcitation of carboxyhemoglobin in water at room temperature. Polarization measurements determine that the iron-complexed CO is oriented nearly perpendicular to the porphyrin plane. The dissociation appears to proceed via a metastable excited state with 2 +/- 1 ps lifetime. The dissociated CO binds weakly in the heme pocket for at least 500 ps. This state correlates with the internally bound state observed by Frauenfelder et al. at low temperatures in myoglobin.

Carboxyhemoglobin↗
PubMed1989

DNA flexibility variation may dominate DNase I cleavage.

In a previous experimental study, we proposed that the bending and torsional stiffness of DNA display a systematic sequence dependence. Subsequently, we developed an elastic strain model to quantify the sequence dependence of the bending and torsional rigidity in terms of nearest neighbor interactions and used that model to analyze the sequence dependence of the 434 repressor binding to its operator. The analysis presented here shows that, in the absence of significant local variation of DNA secondary structure, DNase I cleavage is strongly correlated with local variation in the bending flexibility as calculated from our elastic strain model and that the agreement is also quantitatively significant. It is proposed that analysis using elastic strain models will provide a preliminary set of biochemical and chemical tools to explore the relation between DNA flexibility and the binding of other proteins.

Base Sequence↗
PubMed1987

Terbium luminescence-lifetime heterogeneity and protein equilibrium conformational dynamics.

The fluorescence decay of the rare earth terbium when bound to the protein calmodulin changes from a simple exponential decay to a complex nonexponential decay as the temperature is lowered below 200 K. We have fit the observed decay curves by assuming that the terbium emission is a forced electric dipole transition and proteins have a distribution of continuous conformational states. Quantitative fits to the data indicate that the root-mean-square configurational deviation of the atoms surrounding the terbium ion is 0.2 A, in good agreement with other measurements. We further point out that because the protein seems to undergo a glass transition yet retains configurational order at room temperature, the proper name for the physical state of a protein at room temperature is the rubber-like state.

Calmodulin↗
PubMed1987

Political risk assessment, from an elected safety belt law advocate's point of view and experience.

Voters of Michigan have elected Secretary Austin, prominent safety belt law advocate, to four successive four year terms, by substantial margins. He is now the longest serving black statewide official in the USA. Michigan's Secretary of State, in addition to being the State's official record keeper and elections supervisor, is responsible for motor vehicle administration and driver licensing. He serves as chairman of the state's traffic safety commission. In his first campaign for election in 1970, Austin included in his traffic safety program a proposal for safety belt legislation, as the best way to encourage usage. His opponent was opposed. In each of three campaigns for re-election and at every opportunity, Austin urged usage and advocated mandatory safety belt legislation. Michigan enacted a safety belt law in 1985, culminating a 14-year struggle for Austin. In this article he shares some of his experience and views.

Accidents, Traffic↗
PubMed1986

Ion-induced DNA structure change in nucleosomes.

Physical methods have been used to study calcium binding to the nucleosome core particle. Equilibrium dialysis of Ca2+ and spectroscopic analysis of a Ca2+ analogue show that the ion binds tightly to the particles, resulting in a significant change of DNA circular dichroism. This suggests that base stacking may be altered as a result of Ca2+ binding. In the presence of Ca2+, the absorbance and fluorescence properties of methylene blue (MB), a DNA-specific intercalator, confirm that the dye binds tightly to nucleosomes by intercalation. However, secondary changes occur which suggest that the MB binding site is altered as a result of Ca2+ binding. Triplet state anisotropy decay and triplet lifetime quenching both show that in the Ca2+-nucleosome complex, methylene blue is capable of wobbling over a substantial angular range at its binding site. To explain these data, it is proposed that Ca2+ binding to nucleosomes causes DNA to fold by means of a series of sharp bends (kinks). The properties of bound MB are best explained if it is presumed that the intercalator binds tightly to such kinked sites in the nucleosome. On the basis of these observations, we discuss the possibility that multivalent ion concentration in the nucleus is high enough that the smooth to kinked helix equilibrium may be near to its midpoint. Near such a midpoint, the secondary structure of DNA in the nucleosome might prove to be sensitive to effector molecule binding and to site-specific variation of DNA or histone composition within genes.

Animals↗
PubMed1985

Kinetic differences at low temperatures between R and T state carbon monoxide-carp hemoglobin.

We use the low-temperature recombination kinetics of carbon monoxide with carp hemoglobin to determine that the R and T states of hemoglobin exhibit different low-temperature geminate recombination kinetics. The peak of the fitted Gaussian activation energy spectrum is at 1.5 kcal/mol for R state and 1.8 kcal/mol for T state. The distribution in activation energies is fit well by the Agmon-Hopfield linear strain model. The T state is fit with a stronger elastic constant than R, and has a larger displacement of the protein conformation coordinate than does the R state, indicating that the T state does have a significantly greater rigidity and also stores more strain energy in its conformational states than does R hemoglobin. The pre-exponential in the activation energy spectrum is only a factor of two greater in the R than the T state and the low-temperature activation energy spectrum does not correctly predict the difference in the on rates for R and T states at 300 degrees K, indicating that processes removed from the binding site are important in cooperativity.

Animals↗
PubMed1984

Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence.

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

On the role of Fe2+ in bacterial photosynthesis. The effect of biosynthetic substitution of Fe2+ by Mn2+ on the electron transfer step Q-1Q2----Q1Q-2 in reaction centers.

A test of the 'iron-wire' hypothesis for the role of Fe2+ in promoting the electron transfer between the primary (Q1) and secondary (Q2) quinones in bacterial reaction centers of Rhodopseudomonas sphaeroides strain R-26.1 has been conducted. Kinetics of this step, P+Q-1Q2----P+Q1Q-2, and of recombination with the oxidized donor, P+Q-1----PQ1 and P+Q-2----PQ2, were followed optically at 4 degrees C in normal iron-containing reaction centers and in reaction centers having 58% Mn2+, replacing Fe2+. This significant replacement is accomplished biosynthetically by control of the growth conditions, and so should preserve the native interactions between the cofactors. There are no significant differences observed in the recombination kinetics of the two types of reaction centers. The electron transfer between the quinones was observed to show apparent biphasic kinetics with major components of approx. 170 microseconds and 1.5 ms at 4 degrees C and pH = 7.5. There is no statistically significant difference observed between the two types of reaction centers. This major change in the electronic structure of the metal and the unaltered kinetics discount the likelihood of any direct orbital participation of the metal in the electron transfer between the quinones.

Darkness↗