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R T Carr

Publications and source records attributed to R T Carr.

15 recordsLinked to original sources
PubMed2000

Nonlinear dynamics of microvascular blood flow.

Kiani et al. (Kiani, M. F., A. R. Pries, L. L. Hsu, I. H. Sarelius, and G. R. Cokelet, Am. J. Physics. 266: H1822-H1828, 1994) suggested that blood velocity, hematocrit, and nodal pressures can oscillate spontaneously in large microvascular networks in the absence of biological control. This paper presents a model of blood flow in microvascular networks that shows the possibility of sustained spontaneous oscillations in small networks (less than 15 vessel segments). The paper explores mechanisms which cause spontaneous oscillations under some conditions and steady states under others. The existence of sustained oscillations in the absence of biological control provides alternative interpretations of dynamic behavior in the microcirculation. The model includes the Fåhraeus-Lindqvist effect and plasma skimming but no biological control mechanisms. The model is a set of coupled nonlinear partial differential equations. The equations have been solved numerically with the direct marching numerical method of characteristics. The model shows steady-state fixed point and limit cycle dynamics (sometimes showing period doubling). Plasma skimming, and the Fåhraeus-Lindqvist effect, along with arcade type topology are necessary for oscillations to occur. Depending on network parameters, nonoscillating, damped oscillating and sustaining oscillating dynamics have been demonstrated. Oscillations are damped when the residence time one of the vessels is large compared to the residence times for the other vessels. When the residence times for all vessels are comparable, sustained oscillations are possible.

Blood Circulation↗
PubMed1995

Mechanism of metal-independent hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase.

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing a tetrahydrobiopterin cofactor. Several key mechanistic questions have yet to be resolved, specifically the identity of the hydroxylating species and the role of the non-heme iron which is present in all of the mammalian PAHs. Recently, we have demonstrated that a bacterial PAH from Chromobacterium violaceum does not require any redox active metal for activity [Carr, R. T., & Benkovic, S. J. (1993) Biochemistry 32, 14132-14138]. To identify the function of iron in the mammalian PAH's, we have undertaken a series of experiments to compare the mechanisms of this metal-independent PAH with the iron-dependent PAH from rat liver. Using [4-2H]phenylalanine as a substrate gave a kinetic isotope effect on hydroxylation of unity for CVPAH which is in agreement with previous values reported for RLPAH. The [4-2H]phenylalanine underwent an NIH shift upon hydroxylation by CVPAH. The extent of deuterium retention at the 3-position of the tyrosine product was identical within experimental error for both RLPAH and CVPAH using [4-2H]phenylalanine and [2,3,5,6-2H]phenylalanine as substrates. This suggests that PAH from either source probably does not directly mediate the NIH shift mechanism. No uncoupled pterin turnover was observed for CVPAH with either L-tyrosine or p-chloro-L-phenylalanine as substrate or tetrahydropterin as cofactor, each of which causes uncoupled turnover with RLPAH. CVPAH readily accepts 4-methylphenylalanine as a substrate giving 4-(hydroxymethyl)phenylalanine as the major product and 3-methyltyrosine as the only other minor product.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Separation surfaces for laminar flow in branching tubes--effect of Reynolds number and geometry.

Flow visualization experiments of Newtonian laminar flow through branching tubes have been performed to identify the shape of the separation surface or flow divider. The influences of Reynolds number, flow fraction into the side branch, and branch geometry on the separation surface shape have been considered. The shapes presented in this paper are formed by the intersection of the separation surface with the cross section of the parent tube. At low Re the separation surfaces are curved in a convex manner, bulging away from the opening of the side branch. Increasing Re causes the surface to become concave. At Re > 194 the surfaces can become closed for Q* > 0.3. The branch angle has no noticeable effect on the separation surface shape. The side to parent branch diameter ratio, Db/Dp, has a strong influence at low Re. As Re increases the diameter effect diminishes. Previous studies have shown no difference between the separation surfaces of T and Y type junctions at low Re. At Re = 194 there is a marked difference between the separation surfaces of T and Y bifurcations.

Biomechanical Phenomena↗
PubMed1995

Plasma skimming in vascular trees: numerical estimates of symmetry recovery lengths.

A new model of plasma skimming in vascular trees that includes the history of disturbances at previous bifurcations is developed. The model calculates the shift in the red blood cell concentration profile by mapping streamlines through the junction; it then computes the dissipation of that disturbance through random collisions among the cells. A numerical solution of this model is compared to experimental data on plasma skimming in serial bifurcations. Numerical values of symmetry recovery lengths downstream of the bifurcation are calculated using the model. Comparison of the computed symmetry recovery lengths with anatomical measurements in hamster cremaster muscle indicate that asymmetries in red blood cell concentration profiles are important in vessels with diameters below 55 microns.

Animals↗
PubMed1994

Identification of metal ligands in Cu(II)-inhibited Chromobacterium violaceum phenylalanine hydroxylase by electron spin echo envelope modulation analysis of histidine to serine mutations.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) is known to bind an equivalent of divalent copper. The "metal-free" form of the protein is fully active, and Cu(II) is now shown to be an inhibitor of CVPAH rather than an activator of the enzyme [Carr, R. T., & Benkovic, S. J. (1994) Biochemistry 32, 14132-14138]. On the basis of amino acid sequence homology, the metal binding site may be related to those of rat liver PAH and other eukaryotic pterin-dependent hydroxylases, which require Fe(II) for activity. The conserved histidines at that site in CVPAH, histidines 138 and 143, were each mutated to serines. The mutant enzymes H138S and H143S were both catalytically inactive, but still able to bind Cu(II). Binding studies further demonstrated that both mutant enzymes still bind L-phenylalanine. Electron spin echo envelope modulation (ESEEM) studies on each of the mutants showed the presence of only a single copper-coordinating histidine, rather than the two histidine ligands suggested for the wild-type protein. This result supports a model in which Cu(II) is equatorially ligated to only two histidines in the Cu(II)-inhibited protein and allows us to unambiguously assign histidines 138 and 143 as these ligands. That the enzyme is inactive when these histidines are either bound with copper or when replaced with serines suggests that these histidines perform a catalytic function. Possible catalytic roles for these histidines in the hydroxylation mechanism of pterin-dependent monooxygenases are discussed along with potential future applications of the combination of ESEEM with site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1993

An examination of the copper requirement of phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) was classified as a copper metalloenzyme by virtue of a 1/1 Cu/enzyme stoichiometry and its inhibition with various chelators [Pember, S. O., Villafranca, J. J., & Benkovic, S. J. (1986) Biochemistry 25, 6611]. We have prepared "copper-free" CVPAH by extraction with DTT. These preparations retained full activity though the Cu/enzyme ratio averaged 0.015. Reconstitution by extraneous copper was disproved by measuring a Cu/enzyme ratio of 0.09 in assay mixtures after the specific activity was determined to be within 85% of a fully copper-complexed control. Several copper chelators were examined and were not inhibitory. The "copper-free" enzyme had significant activity without a thiol or other reducing agent capable of reducing the copper center whereas copper-complexed CVPAH had minimal activity under these conditions. Copper-complexed CVPAH can be activated, however, by nonreducing copper ligands such as imidazole. From these results, we conclude that copper is not a requirement of activity. Iron, cobalt, nickel, manganese, molybdenum, and chromium were not found in the "copper-free" preparation, indicating that the active hydroxylating species may not require a redox-active metal. The KdS for binding Cu2+ and Zn2+ were measured to be 0.48 and 0.85 microM, respectively. Both copper and zinc were found to be potent inhibitors of "copper-free" CVPAH in the absence of thiols. DTT reverses inhibition due to Cu2+ but not inhibition caused by Zn2+. The product stoichiometry indicates the same tightly coupled turnover found with all other pterin-dependent hydroxylases when using natural substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1992

X-ray absorption studies of the Cu-dependent phenylalanine hydroxylase from Chromobacterium violaceum. Comparison of the copper coordination in oxidized and dithionite-reduced enzymes.

The coordination chemistry of the Cu sites of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum has been studied by X-ray absorption spectroscopy (XAS). The EXAFS of the Cu(II) form of the enzyme resembles that of other non-blue copper proteins such as plasma amine oxidases and dopamine-beta-hydroxylase and is characteristic of a mixed N/O coordination shell containing histidine ligation. Detailed simulations of the raw EXAFS data have been carried out using full curved-wave restrained refinement methodologies which allow imidazole ligands to be treated as structural units. The results suggest a Cu(II) coordination of two histidines and two additional O/N-donor groups. A reasonable fit to both data sets can be obtained by assuming that the non-imidazole first-shell donor atoms are derived from solvent (H2O or OH-). The EXAFS of the reduced enzyme shows major differences. The amplitude of the first shell in the Fourier transform is only 50% of that of the oxidized enzyme, indicative of a substantial reduction in coordination number. In addition, the first shell of the transform is split into two components. Simulations of the reduced data can be obtained by either two histidines at a long distance of 2.08 A and an O ligand at a short distance of 1.88 A or two histidines at a short distance of 1.90 A and one second-row scatterer such as S or Cl at 2.20 A. Comparison of absorption edge data on the reduced enzyme with data from Cu(I) bis- and tris(1,2-dimethylimidazole) complexes suggests a pseudo-three-coordinate structure.

Chromobacterium↗
PubMed1991

Influence of vessel diameter on red cell distribution at microvascular bifurcations.

The uneven distribution of red blood cells (RBCs) to daughter branches at microvascular bifurcations, often referred to as "plasma skimming," has been characterized in previous studies by comparisons of flux-flow plots of red cell flow fraction (F*) versus volumetric flow fraction (Q*) at single junctions. Comparisons of flux-flow plots for single bifurcations with differing daughter/parent vessel diameter ratios (Dd/Dp = 1.0 vs 0.5) have been made to reveal the influence of vessel size on red cell distribution at vessel junctions of four different geometries (100 x 100, 50 x 50, 100 x 50, 50 x 25 microns). Experimental data determined by effluent collection were compared to those from video fluorescence microscopy experiments, as well as to numerical computations from theoretical models. Statistical comparisons using a likelihood ratio test indicate that vessel diameter ratio, Dd/Dp, has no detectable influence on RBC distribution in these microvascular bifurcations.

Blood Flow Velocity↗
PubMed1990

Plasma skimming in serial microvascular bifurcations.

Red cell distribution in simple two bifurcation networks has been studied experimentally. The results indicate that fractional red cell flux/fractional volumetric flow curves can be asymmetric at the downstream bifurcation. The important parameters affecting this asymmetry are the fractional flow into the upstream branch Q1*, and the ratio of the distance between junctions to the volumetric flow rate, z/Q. The asymmetry is attenuated as z/Q increases. In 50-micron tubes with Q1* of 0.5, symmetric phase separation behavior is regained when z/Q is greater than 200 sec/mm2. In 25-micron tubes symmetry is recovered before z/Q reaches the value of 50 sec/mm2. These results agree with in vivo data of previous studies and provide additional evidence that flow history can be important in microvascular networks if junctions are close together or flow rates are sufficiently high.

Blood Flow Velocity↗
PubMed1990

Dye studies on flow through branching tubes.

This study was aimed at identifying the shape of the separating surface at the junction of two vessels and determining the shift in concentration profiles due to streamline bending at the junction. These data are useful in modeling phase separation during plasma skimming in serial microvascular bifurcations. It was hypothesized that the separating surface shape should be a function of the ratio of the branch diameters and the fractional flow split at the junction. Streamlines bend as blood flows through a junction thereby disturbing the concentration profile of several blood components downstream from the branch point. Model experiments have been conducted to test these ideas. Scaled-up dye studies have shown that the separating surface is indeed a function of the branch diameter ratio. When all branches have the same diameter the separating surface is virtually flat. When the side branch diameter is half that of the parent branch, the surface is curved, bulging away from the opening of the side branch. At Reynold's numbers greater than 20, when vortices form in the daughter branches, the shape of the separating surface is complicated and may even be discontinuous. Other dye experiments have been used to illustrate the magnitude of streamline bending. The data are then used to estimate the shift in concentration profiles due to flow through junctions. A technique for mapping upstream profiles to their corresponding downstream location has been developed. This mapping technique provides the necessary initial condition for the equation which describes the dispersion of red blood cells as they flow between the junctions.

Animals↗
PubMed1989

Enhancement of heat transfer in red cell suspensions in vitro experiments.

New data on laminar heat convection with red cell suspensions have been gathered for both heating and cooling. When compared to data for the suspending medium alone, it is apparent that the red cells enhance laminar heat transfer when Pe greater than 4. This is probably due to particle movements. These new data disagree with earlier studies which indicated no enhancement of heat transfer for blood cell suspensions. The data do agree with previous correlations for enhanced thermal transport in sheared suspensions.

Erythrocytes↗
PubMed1989

Estimation of hematocrit profile symmetry recovery length downstream from a bifurcation.

Downstream from a microvascular bifurcation the distribution of blood cells in the vessel lumen is not symmetric. A diffusion process is used to model the rearrangement of red cells as blood flows between junctions in the microcirculation. A Fourier series approach is used to solve the model diffusion convection equation in slit geometry. Both flat and parabolic velocity profiles are considered. The eigenvalues, found using the Rayleigh-Ritz method, are used to find an upper bound on distance required for a symmetric red cell distribution to be obtained. The method has also been applied to cylindrical geometry and the computed symmetry recovery lengths are compared to distances between bifurcations measured in vivo. These estimates indicate that red cell distributions are frequently asymmetric in the microcirculation. Such asymmetries can have a strong effect on plasma skimming and material balance calculations.

Animals↗
PubMed1985

Nonuniform red cell distribution in 20 to 100 micrometers bifurcations.

Several potentially important parameters influencing the disproportionate distribution of red cell flux and blood flow at a bifurcation are examined. These include parent vessel hematocrit, vessel diameter, suspending medium, cell distensibility, parent vessel blood flow rate, and local geometry. Measurements were performed on 20 to 100 micrometers bifurcations, fabricated such that all vessels of a given bifurcations have the same diameter. Suspensions of human red cells, hardened red cells, and mixtures of each in albumin-saline, Dextran 75, or plasma were flowed through the bifurcations and determinations of flow rates and discharge hematocrits were made for each of the channels. For the 20-micrometers channels, hematocrits were found using videophotometric techniques, and for the larger channels, hematocrits were measured directly from the exit streams. Flow rates for both were measured by meniscus travel downstream in small-diameter glass tubes. Within the limits of the present experiments, three of the variables proved to be of major importance: feed hematocrit, tube diameter, and flow-rate distribution. It was clearly demonstrated that red cell flux varies nonlinearly with fractional flow rate. Critical flow rates, at which all or none of the cells entered one of the branches, were found to vary with diameter and hematocrit as has been reported in other studies. The data were analyzed with a theoretical model which assumes that the parent vessel contains a core of uniformly distributed red cells surrounded by a marginal gap of suspending medium; in the parent vessel lumen, the flows to the two daughter branches were assumed to be separated by a chord. The marginal gap widths and tube hematocrits deduced from the data with this model are of reasonable magnitudes.

Albumins↗