PubMed Health⌕ Search

Biomedical subjects

R W Briehl

Publications and source records attributed to R W Briehl.

At least 19 recordsLinked to original sources
PubMed2006

Fiber depolymerization.

Depolymerization is, by definition, a crucial process in the reversible assembly of various biopolymers. It may also be an important factor in the pathology of sickle cell disease. If sickle hemoglobin fibers fail to depolymerize fully during passage through the lungs then they will reintroduce aggregates into the systemic circulation and eliminate or shorten the protective delay (nucleation) time for the subsequent growth of fibers. We study how depolymerization depends on the rates of end- and side-depolymerization, k(end) and k(side), which are, respectively, the rates at which fiber length is lost at each end and the rate at which new breaks appear per unit fiber length. We present both an analytic mean field theory and supporting simulations showing that the characteristic fiber depolymerization time tau= square root 1/k(end)k(side) depends on both rates, but not on the fiber length L, in a large intermediate regime 1 << k(side)L(2)/k(end) << (L/d)(2), with d the fiber diameter. We present new experimental data which confirms that both mechanisms are important and shows how the rate of side depolymerization depends strongly on the concentration of CO, acting as a proxy for oxygen. Our theory remains rather general and could be applied to the depolymerization of an entire class of linear aggregates, not just sickle hemoglobin fibers.

Carbon Monoxide↗
PubMed2006

Anisotropy in sickle hemoglobin fibers from variations in bending and twist.

We have studied the variations of twist and bend in sickle hemoglobin fibers. We find that these variations are consistent with an origin in equilibrium thermal fluctuations, which allows us to estimate the bending and torsional rigidities and effective corresponding material moduli. We measure bending by electron microscopy of frozen hydrated fibers and find that the bending persistence length, a measure of the length of fiber required before it starts to be significantly bent due to thermal fluctuations, is 130microm, somewhat shorter than that previously reported using light microscopy. The torsional persistence length, obtained by re-analysis of previously published experiments, is found to be only 2.5microm. Strikingly this means that the corresponding torsional rigidity of the fibers is only 6x10(-27)Jm, much less than their bending rigidity of 5x10(-25)Jm. For (normal) isotropic materials, one would instead expect these to be similar. Thus, we present the first quantitative evidence of a very significant material anisotropy in sickle hemoglobin fibers, as might arise from the difference between axial and lateral contacts within the fiber. We suggest that the relative softness of the fiber with respect to twist deformation contributes to the metastability of HbS fibers: HbS double strands are twisted in the fiber but not in the equilibrium crystalline state. Our measurements inform a theoretical model of the thermodynamic stability of fibers that takes account of both bending and extension/compression of hemoglobin (double) strands within the fiber.

Anisotropy↗
PubMed2006

Deforming biological membranes: how the cytoskeleton affects a polymerizing fiber.

We give a theoretical treatment of the force exerted by a fluctuating membrane on a polymer rod tip, taking into account the effects of an underlying biological cytoskeleton by way of a simple harmonic dependence on displacement. We also consider theoretically and experimentally the dynamics of a growing fiber tip under the influence of such a fluctuation-induced membrane force, including the effects of an underlying cytoskeletal network. We compare our model with new experimental data for the growth of hemoglobin fibers within red blood cells, revealing a good agreement. We are also able to estimate the force and membrane/cytoskeletal displacement required to stall growth of, or buckle, a growing fiber. We discuss the significance of our results in a biological context, including how the properties of the membrane and cytoskeleton relate to the thermodynamics of rod polymerization.

Biophysics↗
PubMed2005

Measuring forces between protein fibers by microscopy.

We propose a general scheme for measuring the attraction between mechanically frustrated semiflexible fibers by measuring their thermal fluctuations and shape. We apply this analysis to a system of sickle hemoglobin (HbS) fibers that laterally attract one another. These fibers appear to "zip" together before reaching mechanical equilibrium due to the existence of cross-links into a dilute fiber network. We are also able to estimate the rigidities of the fibers. These rigidities are found to be consistent with sickle hemoglobin "single" fibers 20 nm in diameter, despite recent experiments indicating that fiber bundling sometimes occurs. Our estimate of the magnitude of the interfiber attraction for HbS fibers is in the range 8 +/- 7 kBT/microm, or 4 +/- 3 k(B)T/microm if the fibers are assumed, a priori to be single fibers (such an assumption is fully consistent with the data). This value is sufficient to bind the fibers, overcoming entropic effects, although extremely chemically weak. Our results are compared to models for the interfiber attraction that include depletion and van der Waals forces. This technique should also facilitate a similar analysis of other filamentous protein assembles in the future, including beta-amyloid, actin, and tubulin.

Biophysics↗
PubMed2003

Twisted protein aggregates and disease: the stability of sickle hemoglobin fibers.

We describe how twist could play an essential role in stabilizing 20 nm diameter sickle hemoglobin fibers. Our theory successfully reproduces the observed variation of helical pitch length with fiber diameter. With no remaining adjustable parameters it also yields a prediction for the torsional rigidity of sickle hemoglobin fibers that is in good agreement with experiment and hence retains the striking feature that such fibers can be highly mechanically anisotropic, even with a ratio of bending to torsional rigidity of about 50. We discuss how our study might be relevant to the development of treatment strategies.

Hemoglobin, Sickle↗
PubMed2000

Nonideality and the nucleation of sickle hemoglobin.

The homogeneous and heterogeneous nucleation kinetics of sickle hemoglobin (HbS) have been studied for various degrees of solution crowding by substitution of cross-linked hemoglobin A, amounting to 50% of the total hemoglobin. By cross-linking hemoglobin A, hybrid formation between hemoglobin A and hemoglobin S was prevented, thus simplifying the analysis of the results. Polymerization was induced by laser photolysis, and homogeneous nucleation kinetics were determined by observation of the stochastic behavior of the onset of light scattering. Heterogeneous nucleation was determined by observing the exponential growth of the progress curves, monitored by light scattering. At concentrations between 4 and 5 mM tetramer (i.e., approximately 30 g/dl), the substitution of 50% HbA for HbS slows the reaction by a factor of 10(3) to 10(4). Using scaled particle theory to account for the crowding of HbA, the observed decrease in the homogeneous nucleation rate was accurately predicted, with no variation of parameters required. Heterogeneous nucleation, on the other hand, is not well described in the present formulation, and the theory for this process appears to require modification of the way in which nonideality is introduced. Nonetheless, the accuracy of the homogeneous nucleation description suggests that such an approach may be useful for other assembly processes that occur in a crowded intracellular milieu.

Cross-Linking Reagents↗
PubMed1995

Nucleation, fiber growth and melting, and domain formation and structure in sickle cell hemoglobin gels.

Pathogenesis in sickle cell disease depends on polymerization and gelation of deoxyhemoglobin S. Under the double nucleation model, polymerization is initiated by homogeneous nucleation, followed by heterogeneous nucleation on pre-existing fibers. Fibers grow by non-cooperative addition of hemoglobin. The model derives from macroscopic results rather than direct observation of individual events. We observe individual events and structures by differential interference contrast (DIC) microscopy to show consistency with the model, to define structure and development of gel domains and their relation to kinetics, and to demonstrate the mechanism of fiber melting. Kinetics were controlled by producing deoxyhemoglobin by photolysis of CO hemoglobin under DIC observation. The first visible polymers appeared randomly and were usually linear aggregates less than 1 micron long, consistent with homogeneous nucleation and immediate post-nucleation aggregates. Aggregates then branched extensively, consistent with heterogeneous nucleation. This branching of new fibers was also induced at countable rates on isolated single fibers. Branching and fiber growth rapidly produced dense domains. Changes in photolytic intensity altered domain growth rates and domain structure. At low intensity and slow growth, fibers grew radially without branching. Domains lacked cross-links and polymer density was low. High intensity produced faster growth, much heterogeneous nucleation and highly cross-linked, dense, domains. At still higher intensity, homogeneous nucleation was very rapid, producing many small domains. These results show a hierarchy of processes: as deoxyhemoglobin concentration increases, growth occurs without observable nucleations, and then heterogeneous and finally homogeneous nucleation become dominant. This is consistent with the double nucleation model under which the concentration dependence of growth is low, and that of heterogeneous and homogeneous nucleation successively higher. Under decreased photolysis, fiber ends melted continuously without fiber breakage; increased photolysis reversed this, producing growth. Isolated fibers melted and grew at both ends. The results are consistent with a fiber melting mechanism that is the reverse of growth.

Biopolymers↗
PubMed1994

Fragility and structure of hemoglobin S fibers and gels and their consequences for gelation kinetics and rheology.

Pathogenesis in sickle cell disease depends on whether red blood cells can pass the microvasculature during the delay time before hemoglobin S gelation and cell rigidification occur. Here we observe individual hemoglobin S fibers by differential interference contrast (DIC) microscopy and show that hemoglobin S gels and fibers are fragile and easily broken by mechanical perturbation, and that breakage results in vast acceleration of gelation kinetics due to the creation of new, growing fiber-ends. Hence, in vivo this may be an important factor, in addition to hemoglobin concentration and degree of deoxygenation, that governs delay time and pathogenesis. Pathogenesis also depends on gel rheology and cell rigidification, which depend on fiber cross-linking. We show different mechanisms by which X-shaped, Y-shaped, and "zippering" cross-links form. Finally, we estimate the "on" rate constant for fiber growth to be about 200 mmol/(L.s) and obtain a value for the heterogeneous nucleation rate at 13.5 mmol/L heme.

Gels↗
PubMed1993

Hemoglobin S polymerization and gelation under shear II. The joint concentration and shear dependence of kinetics.

The kinetics of hemoglobin S gelation are critical in sickle disease because microvascular obstruction can be avoided if red blood cells pass these vessels during the delay time, before polymerization and gelation occur in sufficient degree to rigidify the cells. Kinetics, including the delay time and the closely related exponential progress rate, are highly sensitive to hemoglobin concentration and degree of deoxygenation. Kinetics are also greatly accelerated by shear, an effect that may contribute to pathogenesis, since red blood cells deform and can undergo shear in vivo. Here we examine the joint dependence of kinetics on shear and hemoglobin concentration. As shear rate increases, the concentration dependence of the exponential progress rate decreases. The large decrease in concentration dependence supports the conclusion that acceleration of gelation by shear is due to breakage and not to enhancement of heterogeneous nucleation. Under shear, new fibers are created by breakage of existing ones, as well as by heterogeneous nucleation. At high shear, the rate of new fiber creation by breakage is very great and dominates that by heterogeneous nucleation. Therefore, if breakage depended only on shear rate and were independent of the concentration of hemoglobin in solution, the concentration dependence of kinetics should vanish. Although it decreases, it does not disappear. The concentration dependence that remains at high shear arises from (1) the direct contribution of fiber growth rate to the exponential progress rate, (2) the dependence of breakage rate on fiber growth rate, and (3) the dependence of solution viscosity on hemoglobin concentration.

Dose-Response Relationship, Drug↗
PubMed1993

Kinetics of hemoglobin S polymerization and gelation under shear: I. Shape of the viscosity progress curve and dependence of delay time and reaction rate on shear rate and temperature.

Polymerization and gelation of deoxyhemoglobin S makes red blood cells (RBCs) rigid and is the immediate basis of pathogenesis in sickle cell disease. Hence, characterization of hemoglobin S viscosity and its time-dependent development as RBCs pass through the microvasculature is important in understanding pathogenesis. Because RBCs and the intraerythrocytic milieu in vivo are subject to shear, the shear dependence of polymerization kinetics is also important. In steady-state cone-plate viscometry we find: (1) gelation under shear progresses exponentially with time; (2) shear markedly increases exponential rate and (3) shortens delay time independent of when in the delay time it is applied; (4) shear greatly decreases the temperature dependence of the exponential rate and delay time; (5) simultaneous with its acceleratory effect on polymerization, shear breaks down gel structure. We conclude that shear acts to accelerate gelation by breaking fibers and creating new growing ends, a process that occurs in addition to the homogeneous and heterogeneous nucleation of new fibers that occurs in the absence of shear. Fibers that break are part of a gel network rather than in free solution. The shear dependence of gelation rates means that the critical clinical issue, whether the delay time is long enough and gelation slow enough to permit deoxygenated cells to pass through the microvasculature before they rigidify, depends on in vivo shear rates as well as on degree of unsaturation and hemoglobin concentration.

Gels↗
PubMed1990

Nucleation and growth of fibres and gel formation in sickle cell haemoglobin.

Deoxygenated sickle haemoglobin polymerizes into long 210-A diameter fibres that distort and decrease the deformability of red blood cells, and cause sickle cell disease. The fibres consist of seven intertwined double strands. They can form birefringent nematic liquid crystals (tactoids) and spherulites. Rheologically, the system behaves as a gel. The equilibria show a phase separation and a solubility. The reaction kinetics show a delay time, are then roughly exponential and are highly dependent on concentration and temperature, and accord with the double nucleation model. But these conclusions are derived from macroscopic data, without direct observation of individual fibres. We have now used non-invasive video-enhanced differential interference contrast (DIC) and dark-field microscopy to observe nucleation, growth and interaction of sickle deoxyhaemoglobin fibres in real time. The fibres originate both from centres that produce many radially distributed fibres and on the surface of pre-existing fibres, from which they then branch. The resulting network is cross-linked and dynamic in that it is flexible and continues to grow and cross-link. Our results support most aspects of the double nucleation model.

Gels↗
PubMed1990

Length distributions of hemoglobin S fibers.

Electron microscopy of sickle cell hemoglobin fibers fixed at different times during gelation shows an exponential distribution of fiber lengths, with many short fibers and few long ones. The distribution does not change significantly with time as polymerization progresses. If this distribution of lengths reflects kinetic mechanism of fiber assembly, it complements information from studies of the progress of average properties of the polymers and, as has been done for other rod-like polymerizing systems, permits testing of models for the mechanism of fiber assembly. In this case, the results are consistent with the double nucleation model of Ferrone et al. or with a related alternative model based on fiber breakage. However, other possible causes of this microheterogeneity exist, including: breakage due to solution shearing of the long, rod-like, fibers; the presence of residual nuclei; equilibrium relations governing polymerization; and breakage of solid-like but weak gels that develop early and adhere to the grid. The arguments against the first three of these possibilities suggest that they are not responsible. However, breakage of entanglements or cross-links in a solid-like and adherent gel is consistent with the distributions.

Hemoglobin, Sickle↗
PubMed1983

Rheology of hemoglobin S gels: possible correlation with impaired microvascular circulation.

The sequence of pathogenic events in sickle cell disease begins with the genetic abnormality and proceeds through molecular and red cell abnormalities to clinical events of vascular obstruction, hemolysis, and crisis. The least studied event, central in the sequence, is altered viscosity and rheology of the gelled deoxyhemoglobin S. In this work, shear is shown not only to measure the formation of gels, but to alter the progress of gelation. Thus, intraerythrocytic shear may be an important factor in pathogenesis. Increasing shear decreases the delay time for gelation as measured directly and by experiments in which shear rate is altered during the delay period. After the delay time, during the growth stage, characterized by a large increase in viscosity, shearing increases the rate of viscosity increase. On the other hand, as previously shown, shearing breaks down solid-like gels. These two effects of shear, one detrimental and the other possibly beneficial, may contribute to the variations known to exist in the clinical picture of sickle cell disease. The growth stage progress curve of gelation is here shown to be exponential in shape. This suggests that fiber breakage occurs under shear and/or that new fibers nucleate on the surface of existing fibers (i.e. heterogeneous nucleation). Finally, the progress curve is shown to be composed of plastic (i.e. solid-like) as well as viscous components early in gel development.

Anemia, Sickle Cell↗
PubMed1982

The absence of volume change in the gelation of hemoglobin-S.

The volume change for the gelation of deoxygenated sickle cell hemoglobin has been measured by dilatometry at 22.0 degrees C and found to be zero. The precision of the result is 0 +/- 1.4 ml/mol of protein present in the sample. When the solubility of the protein is taken into account, the precision is 0 +/- 5.1 ml/mol of gelled hemoglobin. The participation of "hydrophobic interactions" in sickle cell hemoglobin gelation and model compound studies of the volume change associated with transferring hydrophobic solutes from an aqueous to a hydrophobic milieu, as well as the volume changes of other globular protein polymerizations, led us, initially, to expect a large positive delta V. The results are discussed in the context of concentration effects in sickle cell hemoglobin solutions and of recent work on the pressure-induced denaturation of globular proteins, which also gives smaller volume effects than had been anticipated.

Chemical Phenomena↗