Tug-of-war as a cooperative mechanism for bidirectional cargo transport by molecular motors

Intracellular transport is based on molecular motors that pull cargos along cytoskeletal filaments. One motor species always moves in one direction, e.g., conventional kinesin moves to the microtubule plus end, whereas cytoplasmic dynein moves to the microtubule minus end. However, many cellular cargoes are observed to move bidirectionally, involving both plus- and minus-end-directed motors. The presumably simplest mechanism for such bidirectional transport is provided by a tug-of-war between the two motor species. This mechanism is studied theoretically using the load-dependent transport properties of individual motors as measured in single-molecule experiments. In contrast to previous expectations, such a tug-of-war is found to be highly cooperative and to exhibit seven different motility regimes depending on the precise values of the single motor parameters. The sensitivity of the transport process to small parameter changes can be used by the cell to regulate its cargo traffic.     

Phospholipids as the molecular instruments of ion and solute transport in biological membranes.

Partition studies have established that phospholipids generally have the capabilities to mediate the transmembrane transport of the full range of ions and solutes that physiologically cross biological membranes. The list of transportable species includes cations, anions, amino acids, citric acid cycle intermediates, nucleotides, and sugars. Phospholipid-mediated transport can be readily modulated by altering the phospholipid mixture or by addition of detergents, nucleotides, divalent metals, proteins, peptides, or ring compounds. Containment of phospholipid within channels in protein appears to be the precondition for the formation of the micellar structure requisite for solute transport. Phospholipid-mediated transport is postulated to be a central feature of energy coupling, membrane-spanning systems, and membrane-bound, phospholipid-requiring enzymes.     

Physical mechanism for regulation of proton solute symport in Escherichia coli.

The activity of the Escherichia coli transport proteins for lactose and proline can be altered by changing the redox state of the dithiols in these carriers. A series of lipophilic oxidizing agents has been shown to inhibit and subsequent addition of dithiothreitol to restore full activity. Both systems are irreversibly inhibited by N-ethylmaleimide, but prior addition of oxidizing agents protects against this inhibition. These data, as well as studies on the inhibitory effect of the dithiol-specific reagent phenylarsine oxide, show that the redox-sensitive step is the conversion of a dithiol to a disulfide. Measurement of the initial rate as a function of the lactose and L-proline concentrations shows that the oxidation of a dithiol to a disulfide increases the Km of the carriers for lactose and L-proline. The reduced (dithiol) form of the carrier has a low Km and the oxidized (disulfide) form has a high Km for its substrate. The changes in Km brought about by reduction and oxidation are the same as those that accompany the generation and dissipation, respectively, of an electrochemical proton gradient (delta mu H+). These results support a mechanism in which an delta mu H+ or one of its components alters the ligand affinities of the carrier during a single transport cycle through conversion from the reduced to the oxidized form.     

Size-dependent control of colloid transport via solute gradients in dead-end channels

Transport of colloids in dead-end channels is involved in widespread applications including drug delivery and underground oil and gas recovery. In such geometries, Brownian motion may be considered as the sole mechanism that enables transport of colloidal particles into or out of the channels, but it is, unfortunately, an extremely inefficient transport mechanism for microscale particles. Here, we explore the possibility of diffusiophoresis as a means to control the colloid transport in dead-end channels by introducing a solute gradient. We demonstrate that the transport of colloidal particles into the dead-end channels can be either enhanced or completely prevented via diffusiophoresis. In addition, we show that size-dependent diffusiophoretic transport of particles can be achieved by considering a finite Debye layer thickness effect, which is commonly ignored. A combination of diffusiophoresis and Brownian motion leads to a strong size-dependent focusing effect such that the larger particles tend to concentrate more and reside deeper in the channel. Our findings have implications for all manners of controlled release processes, especially for site-specific delivery systems where localized targeting of particles with minimal dispersion to the nontarget area is essential.The ability of a particle to migrate along a local solute concentration gradient, which is referred to as diffusiophoresis, has been exploited to direct transport in a variety of systems, e.g., artificial swimmers (1, 2) and collective behaviors of active colloids (3, 4). One physical mechanism for diffusiophoresis originates from surface–solute interactions, where the solute gradient sets up an osmotic pressure gradient within a narrow interaction region. This gradient leads to fluid flow along the surface of a particle, in which case propulsion occurs in the opposite direction and is referred to as chemiphoresis (5, 6). In addition, differences in diffusivities between anions and cations lead to spontaneous electrophoresis of a particle, giving an additional propulsion mechanism. A particular feature of diffusiophoresis is that the diffusiophoretic mobility, or the phoretic velocity, of a particle is independent of its size, as long as the thickness of the interaction region, e.g., the Debye screening layer when the interaction is electrostatic, is much thinner than the size of the particle (6). This feature allows the utilization of diffusiophoresis for enhancing transport of microscale particles, leading to orders of magnitude higher transport rates compared with pure diffusion (7). However, this size independence could also be a source of frustration because it precludes useful effects such as sorting or controlling transport by particle size.We anticipate that size-independent particle mobility breaks down when the thickness of the surface–solute interaction region becomes comparable to the size of the particle. Already more than a century ago this feature has been well appreciated in the field of electrokinetics as the Hückel limit (8) where the electrophoretic mobility of a particle is 2/3 of the Smoluchowski mobility if the thickness of Debye screening layer κ^?1 is larger than the particle radius a, i.e., κa < 1. Likewise, there are a number of investigations, mostly theoretical, on the effect of finite Debye layer thickness on the diffusiophoretic mobility, which have shown that the influence of finite κa is much stronger than for electrophoresis due to the presence of chemiphoresis (9–11). Because a finite Debye layer effect can lead to size-dependent particle mobility, we revisit the influence of finiteness of κa on the diffusiophoretic mobility, and exploit it in a useful way.One application of size-dependent diffusiophoresis is particle transport into dead-end geometries, which is important to many industrial applications including drug delivery and disinfection. When characterizing transport processes, transferring fluid and/or particles into dead-end geometries or pores is a significant challenge as such geometries do not allow any net fluid flow within the system. Recently, it has been shown that diffusiophoresis can be used to pump colloidal particles and oil emulsions in and out of dead-end capillaries, which have significant implications for oil recovery systems (12).Here, we use size-dependent diffusiophoresis to control colloid transport in a dead-end channel. The observed size dependence is attributed to the abovementioned finite Debye layer effect. This key insight can be exploited pragmatically, to achieve useful end points such as size-dependent particle sorting and separation. We show that theory accounting for finite κa is in a good agreement with the experimental observations.Further, we demonstrate that a judicious choice of ions for multicomponent solutes can also be used to gain exquisite control of the phenomenon, importantly at constant osmolarity, which is critical for in vivo applications, either preventing colloid particles from entering a dead-end channel, or promoting entry as for a single binary ionic solute such as NaCl. These observations suggest future applications in drug delivery (13, 14).To impose a solute gradient along a dead-end channel, we have used a microfluidic approach (Fig. 1A), where the entrance of the dead-end channel is connected to the main flow channel so that the solute concentration (c_o) can be regulated at the entrance. Additionally, the height of the dead-end channel is much thinner ( ≈ 10 μm) than the main channel ( ≈ 100 μm), such that the disturbance from the main channel can be minimized (15).Open in a separate windowFig. 1.Colloid transport in a dead-end channel induced by a solute gradient. (A) Setup for enabling transport experiments in a dead-end channel with minimum disturbance from a continuous flow. (B) Steplike initial solute and colloid concentrations were realized by inserting an oil droplet or an air bubble in between two liquids with different solute concentrations. (C) Otherwise, a gradual concentration was observed due to mixing. Insets are the fluorescent intensity distributions of colloidal particles (polystyrene latex beads, diameter 0.19 μm) along the dead-end channel. (D) Sequential images and (E) intensity distributions of colloids (particle diameter 0.19 μm) migrating along a dead-end channel in the presence of a solute gradient (NaCl: c_i = 2 mM, c_o = 0.02 mM). Channelwise direction, x, is normalized by the length of the dead-end channel, L (=400 μm). (F) Time taken for the colloidal particles to reach the middle of the dead-end channel with 50% of the inlet fluorescent intensity (0.5I_i) under different solute gradients. (Scale bars: 50 μm.)Moreover, unlike electrophoresis or thermophoresis, where the field gradient is generally constant, diffusion in a dead-end channel is time dependent. Therefore, to observe the transient dynamics, especially at the early stages of the solute and colloid transport, a steplike initial concentration profile is desired. To achieve this condition, we inserted in the main channel an immiscible fluid such as an oil droplet or an air bubble to separate the leading solution, which fills in the dead-end channel first, with the trailing solution that has different solute conditions and contains the colloidal particles (Fig. 1B). This sequential approach works as an effective gate leading to a repeatable constant inlet concentration at the start of the experiment. In the absence of this flow design, the trailing fluid that contains the colloidal particles will be mixed with the leading fluid during the delivery to the inlet of the dead-end channel. This results in  > 10 min of dead time before the steady-state inlet condition is reached (Fig. 1C); such a delay would obscure and compromise the results we report here.Using this setup, we study the colloid transport in a dead-end channel induced by a solute gradient. Because the colloidal particles migrate toward higher solute concentrations with our current choice of solutes, we keep the initial inner solute concentration (c_i) high and the outer solute concentration (c_o) low, unless otherwise noted. Typical transport dynamics of colloids in the presence of a solute gradient are shown in Fig. 1 D and E and Movie S1. Owing to the solute (NaCl) gradient, the colloid transport into the dead-end channel is accelerated by nearly two orders of magnitude compared with the pure diffusion case (i.e., no solute gradient) as shown in Fig. 1F.The enhanced colloid transport is not surprising as it has already been reported in a number of studies (7, 16, 17). However, we have also observed that the density profile of the leading colloids is nonuniform in the lateral direction, and the colloidal particles tend to concentrate as they transport along the channel (Fig. 1 D and E). These features stem from the intrinsic geometrical confinement of the dead-end channel. In the presence of a solute gradient along a channel, a diffusioosmotic flow is induced, which has a pluglike flow profile. Because a dead-end geometry does not allow any net flux, a pressure gradient is established that opposes the osmotic flow. In consequence, as the pressure-driven flow has an essentially parabolic profile, summing up these two components results in a circulating flow where the velocity at the center of the channel is opposite to the wall slip velocity. This flow is analogous to an electrokinetic pump where an electroosmotic flow that is induced by an external electric field is balanced by a Poiseuille flow in a dead-end channel and leads to a circulating flow developed nearly instantaneously within the entire channel (18).In our system, because the solute concentration is diffusing out over time (c_i > c_o), the circulating flow slowly propagates toward the closed end of the channel (Supporting Information, Movie S2). Along with this flow, particles experience phoretic motion, and thus the net motion of the particles is the sum of the fluid flow and the phoretic movement, which leads to the lateral curvature of the density profile, as also observed in a recent study by Kar et al. (12).The transient nature of solute diffusion in a dead-end channel also leads to another significant effect: particle focusing. Because the gradient of the solute concentration is always decreasing along the channel due to diffusion, the particle phoretic velocity, which is proportional to ?lnc, decreases as the particles move deeper into the channel (Fig. S1). Thus, particles tend to accumulate near the leading edge of the migrating colloidal front (17, 19), which creates a pluglike colloidal “wave” that can be quantitatively identified from the time-dependent fluorescence intensity distribution along the channel (Fig. 1 D and E).Open in a separate windowFig. S1.Measurement of the particle (diameter 1.01??μm) speed along the dead-end channel driven by a solute gradient (NaCl: c_i = 2?mM, c_o = 0.02?mM). Solid curves represent theoretical results.This colloidal wave leads to preconcentration, separation, and sorting of particles, which could be useful to many applications. The quantitative factors that define the colloidal wave are the location of the peak, the amplitude, and the width of the wave. In theory, these factors are set by the transport properties of the particle and the solute, namely the diffusiophoretic mobility of the particle (Γ_p) and diffusivity of the particle (D_p) and the solute (D_s). In detail, the transient distribution of colloids (concentration n) in the presence of a solute gradient can be described by an advection–diffusion equation that is coupled to the solute diffusion, which drives the advection of colloids. The velocity of the particles is defined as v = v_p + v_f where v_p = Γ_p?lnc is the particle velocity driven by diffusiophoresis and v_f is the flow velocity driven by solute gradient (see Supporting Information for full derivation of v_f). Using the conservation law ??n/??t? + ??? ? ?j? = ?0, where j? = ??D_p?n? + ?vn is the particle flux, the dimensionless equation for the colloid density N can be expressed as∂N∂τ=DpDs∇2N−ΓpDs∇(N∇lnC)−∇⋅(VN),[1]where C is the dimensionless solute concentration, τ is the dimensionless time (tD_s/L², where L is a characteristic length scale), and V is the dimensionless fluid velocity, which is defined as V = v_fL/D_s. The first term on the right-hand side of Eq. 1 represents the diffusion of colloids, whereas the latter terms represent the colloid advection driven by a solute gradient.In general, Γ_p is commonly regarded as a size-independent value in the thin Debye layer approximation (κa → ∞). When the thickness of the Debye layer becomes comparable to the size of the particle, the diffusiophoretic mobility becomes size dependent, Γ_p(κa). Following the pioneering work of Prieve and coworkers (9, 10), the size-dependent diffusiophoretic mobility of the particles is expressed asΓp=ϵ2η(kBTZe)2u01−u1/(u0 κa),[2]where ϵ is the permittivity of the medium, η is the viscosity of the medium, k_B is the Boltzmann constant, T is the temperature, Z is the valence of the solute, and e is the elementary charge. Also, u₀ and u₁ are functions of the zeta potential. The lowest order contribution for very large particles or vanishing thickness of the Debye layer (κa → ∞) isu0=2βZeζpkBT+8 ln cosh(Zeζp4kBT).[3]Here, ζ_p is the zeta potential of the particles and β = (D₊ ? D_?)/(D₊ + D_?), where D₊ and D_? are the diffusivities of cations and anions, respectively. The first and second terms on the right-hand side of Eq. 3 represent electrophoresis and chemiphoresis, respectively. For u₁, lengthy expressions can be found in (9) (see Supporting Information for further discussion).A remarkable fact is that even when κa ≈ 100, unlike electrophoretic mobility (20), the predicted diffusiophoretic mobility deviates noticeably from Γ_p(κa → ∞) (10). When κa ≈ 10, which is a general condition for common diffusiophoresis experiments (7, 12, 16, 17), the diffusiophoretic mobility is predicted to be almost an order of magnitude lower, depending on ζ_p (10). Our experimental conditions are in a similar range, κa ≈ 2 ? 40 [κ^?1 = 13.6 nm when c = (c_i + c_o)/2 ≈ 1 mM], indicating that size effects can be significant.The diffusiophoresis of particles with different sizes in a dead-end channel is shown in Fig. 2. Owing to the broad range of κa, we observe strong size-dependent particle focusing, where the larger particles tend to reside deeper in the channel, and they generally tend to concentrate more (Fig. 2A and Movie S3). For instance, the smallest particles (2a = 0.057?μm) show a focusing ratio of about 2, and the terminal peak position x_p is about 50% of the channel length L at t = 300 s, whereas, the largest particles (2a = 1.01?μm) show a focusing ratio as large as 100, and the particles travel nearly to the end of the channel without noticeable dispersion (Fig. 2 D and E).Open in a separate windowFig. 2.Size-dependent particle focusing driven by a solute gradient (NaCl: c_i = 2 mM, c_o = 0.02 mM). (A) Fluorescent images and (B) intensity distributions of colloids with different diameters ranging from 0.06 to 1.01 μm at t = 300 s. The intensity I is normalized by the maximum intensity I_max. (C) Theoretical prediction for colloid density profiles with different diameters at t = 300 s. Plot of (D) peak position (x_p) and (E) focus magnitude (I_max/I_min, I_min is the minimum intensity near the inlet) for various particles at t = 300 s obtained from B. Black curves represent theoretical predictions. (Scale bar: A, 50 μm.)For a given zeta potential (−70 mV), Γ_p is estimated to vary by almost a factor of 5 based on Eq. 2 for our range of particle sizes (Fig. S3). Considering that D_p also changes significantly with size based on the Stokes–Einstein relation, a combination of diffusiophoresis and Brownian diffusion leads to a strong size effect, which is confirmed from a good agreement between the experiment and the theory based on Eqs. 1 and 2 (Fig. 2 B and C). The discrepancy near the entrance of the channel for small particles shown in Fig. 2C mainly comes from the penetration of the fluorescent signal coming from the main channel because of the strong optical density required for small particles.Open in a separate windowFig. S3.Dependence of particle diffusiophoretic mobility Γ_p on the particle radius a and the Debye layer thickness κ^?1. Solid line: ζ_p = ?70??mV, Dotted line: ζ_p = ?80??mV, Dashed line: ζ_p = ?60??mV.Other possible size-dependent phenomena include particle–particle interactions (5), wall interactions (21), hydrodynamic dispersion (22), gravimetric effects (21), electrokinetic lift (23), etc., but we argue that these effects are negligible compared with the finite κa effect (detailed discussion in Supporting Information). Note that the presence of the fluid advection does not significantly influence the particle transport, but only contributes to the lateral inhomogeneity in the early stage, which is confirmed by comparing the theoretical results with and without the solute gradient-induced fluid advection (Figs. S4 and ​andS7;S7; Supporting Information).Open in a separate windowFig. S4.Calculated colloid transport in the presence of a solute gradient. (A) Solute distribution versus time. Particle distribution versus time for (B) small particles (diameter 0.06?μm) and (C) large particles (diameter 1.01??μm). Solid lines indicate calculations with fluid advection induced by solute gradient, whereas dashed lines indicate calculations without fluid advection.Open in a separate windowFig. S7.Numerical simulation results of the side view (x–z plane) of the particle migration along a dead-end channel driven by a solute gradient in the presence of flow advection; z = 0 refers to the centerline of the channel, such that the images represent upper half of the channel. Particle size: (A) 0.06 and (B) 1.01??μm.All of the demonstrated results so far were driven by a single solute gradient with a contrast of 100 (NaCl: c_i = 2 mM, c_o = 0.02 mM), which may be limited for practical applications, especially with in vivo drug delivery where a strong solute gradient may lead to an osmotic shock. Thus, to seek broader insights of diffusiophoresis so as to gain further control over the movement of colloidal particles, we focus on the role of individual mechanisms that contribute to the overall transport. Recall that diffusiophoresis has two contributions, one from chemical potential differences (chemiphoresis), and the other from differences between the diffusivities of anions and cations, which creates a local electric field (electrophoresis). Considering that the diffusivity of K⁺ is nearly identical to that of Cl⁻ (D_K⁺/D_Cl^? = 0.97, cf. D_Na⁺/D_Cl^? = 0.66; ref. 24), we suggest that there can be an alternative strategy to control colloid transport besides a single solute gradient: by displacing a solute with another solution containing different species. Analogous to a liquid junction potential (25, 26), the interdiffusion of multicomponent solutes having equimolar concentration also generates a spontaneous electric field that can allow electrophoresis. For instance, the interdiffusion of NaCl and KCl solutions can generate an electric field of  ～ 40 V/m, which is comparable to the single solute (NaCl) gradient case (Supporting Information).Using this strategy, we can create a local electric field that gives rise to the electrophoresis of the particles in the channelwise direction so that the colloid transport into the channel is enhanced by locating NaCl inside the dead-end channel and displacing it with KCl, which flows through the main channel. As illustrated in Fig. 3 A and B (Movie S4), we show that this strategy allows particle focusing and fast colloidal transport that is comparable to the single solute gradient case (black curves). This “displacement” strategy is desired when a constant osmolarity of the solution is required, such as with in vivo transport systems.Open in a separate windowFig. 3.Interdiffusion of equimolar multicomponent solutes (c_i = c_o = 2 mM) for inducing diffusiophoresis in a dead-end channel under constant osmolarity. (A) Fluorescent intensity distribution at 300 s and (B) trace of front position, x_f = x(i = 0.2I_i), for different solute configurations (blue: inner = KCl, outer = NaCl; red: inner = NaCl, outer = KCl). Results of a single solute with a gradient (NaCl: c_i = 2 mM, c_o = 0.02 mM) are presented in black for comparison.Likewise, if we place NaCl and KCl in the opposite configuration, the electric field is now generated in a reverse direction, which is expected to slow down the colloid transport. Indeed, such an initial distribution of electrolyte completely prevents the colloidal particles from going into the channel until the electric field has vanished (blue curve in Fig. 3B; Movie S4). This effect lasts for over a minute, which is the same order of magnitude as the solute diffusion time, L²/D_s ～ 100 s. This approach may have a significant implication in programmable drug delivery applications where such an electric field can serve as a trigger for drug release (27).We have demonstrated an effective way of delivering colloidal particles into dead-end channels by imposing solute gradients, either by a single solute gradient, or by multispecies interdiffusion. We further demonstrated that size-dependent diffusiophoresis can be obtained by controlling κa, a fact that is commonly ignored. A key observation regarding the size-dependent diffusiophoresis is that there is a tendency for the larger particles to focus more and transport farther into the channel, which suggests many technological implications such as particle sorting that are otherwise difficult to achieve in dead-end geometries. As demonstrated in Fig. 4 A–D (Movie S5), particle sorting from a mixture of two (or more) particles having different sizes can be simply achieved. This feature is enabled by the fact that particle–particle interactions are very weak for phoretically driven particles such that the particles are easily separated without interfering with neighboring particles (5).Open in a separate windowFig. 4.Control of colloidal particles in dead-end channels via a solute gradient for various applications. (A–D) Size-dependent particle sorting from mixture of particles in a dead-end channel driven by a solute gradient (NaCl: c_i = 2 mM, c_o = 0.02 mM). The mixture consists of polystyrene particles having diameters of 0.21 and 1.01 μm dyed with different fluorophores. (A–C) Fluorescent images of (A) diameter = 0.21 μm particles and (B) 1.01 μm particles at t = 300 s, and (C) intensity distributions of A and B along the channel. D is an overlaid image of A and B. (E–G) Control of lipid vesicles for drug delivery applications. Fluorescent images of (E) SUVs (mean diameter  ≈  56 nm) and (F) LUVs (mean diameter  ≈  861 nm), and (G) intensity distributions of the vesicles along the channel at t = 300 s. (Scale bars: 50 μm.)In addition to particle sorting, size-dependent diffusiophoresis can be important especially in pharmaceuticals because it makes it possible to manipulate the final concentration and location of particles within deep pores with controlled dispersion based on their sizes. As an example, delivery of lipid vesicles (lipid composition is provided in Materials and Methods) in deep pores can be manipulated via imposing a solute gradient across the pore, where the peak penetration depth and the concentration ratio of the vesicles are controllable based on the size of the vesicles (Fig. 4 E–G and Movie S6). This demonstration implies the possible application of size-dependent diffusiophoresis in site-specific delivery systems where localized targeting of particles with minimal dispersion to the periphery is desired (14).     

In situ measurement of solute transport in the bone lacunar-canalicular system

Solute transport through the bone lacunar-canalicular system is believed to be essential for osteocyte survival and function but has proved difficult to measure. We report an approach that permits direct measurement of real-time solute movement in intact bones. By using fluorescence recovery after photobleaching, the movement of a vitally injected fluorescent dye (sodium fluorescein) among individual osteocytic lacunae was visualized in situ beneath the periosteal surface of mouse cortical bone at depths up to 50 microm with laser scanning confocal microscopy. Transport was analyzed by using a two-compartment mathematical model of solute diffusion that accounted for the characteristic anatomical features of the lacunar-canalicular system. The diffusion coefficient of fluorescein (376 Da) was determined to be 3.3 +/- 0.6 x 10(-6) cm2/sec, which is 62% of its diffusion coefficient in water and is similar to diffusion coefficients measured for comparably sized molecules in cartilage. The diffusion of fluorescein in bone is also consistent with the presence of an osteocyte pericellular matrix whose structure resembles that proposed for the endothelial glycocalyx [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239-255]. To our knowledge, this is the first instance where the dynamics of molecular movement has been measured directly in the bone lacunar-canalicular system. This in situ imaging approach should also facilitate the analysis of convection-based transport mechanisms in bones of living animals.     

Interstitial flow as a guide for lymphangiogenesis

The lymphatic system is important in tissue fluid balance regulation, immune cell trafficking, edema, and cancer metastasis, yet very little is known about the sequence of events that initiate and coordinate lymphangiogenesis. Here, we characterize the process of lymphatic regeneration by uniquely correlating interstitial fluid flow and lymphatic endothelial cell migration with lymphatic function. A new model of skin regeneration using a collagen implant in a mouse tail has been developed, and it shows that (1) interstitial fluid channels form before lymphatic endothelial cell organization and (2) lymphatic cell migration, vascular endothelial growth factor-C expression, and lymphatic capillary network organization are initiated primarily in the direction of lymph flow. These data suggest that interstitial fluid channeling precedes and may even direct lymphangiogenesis (in contrast to blood angiogenesis, in which fluid flow proceeds only after the vessel develops); thus, a novel and robust model is introduced for correlating molecular events with functionality in lymphangiogenesis.     

Defective transport as a mechanism of acquired resistance to methotrexate in patients with acute lymphocytic leukemia.

Although the mechanisms of resistance to methotrexate (MTX) are known in experimental tumors made resistant to this drug, little information is available regarding acquired resistance to MTX in patients. A competitive displacement assay using the fluorescent lysine analogue of MTX, N-(4-amino-4-deoxy-N10-methylpteroyl)-N epsilon-(4'-fluorescein-thiocarbamyl)-L-lysine (PT430), was developed as a sensitive method of detection of transport resistance to MTX in cell lines, as well as in blast cells from patients with leukemia. Rapid uptake of PT430 at high concentrations (20 mumol/L) in leukemic blasts resulted in achievement of steady-state levels within 2 hours. Subsequent incubation with the folate antagonists, MTX and trimetrexate (TMTX), which differ in the mode of carrier transport, produced characteristic patterns of PT430 displacement. Flow cytometric analysis of the mean fluorescence intensity in the human CCRF-CEM T-cell lymphoblastic leukemia cell line and its MTX-resistant subline clearly identified the presence of transport deficiency in the resistant subline. Analysis of blasts from 17 patients with leukemia, nine with no prior chemotherapy and eight previously treated with chemotherapy, found evidence of MTX transport resistance in two of the four patients who were treated with MTX and considered to be clinically resistant to the drug. The finding that blast cells of some patients with leukemia considered clinically resistant to MTX is due to decreased MTX transport has important implications for clinical use of this drug and for new drug development.     

Conformation gating as a mechanism for enzyme specificity

Acetylcholinesterase, with an active site located at the bottom of a narrow and deep gorge, provides a striking example of enzymes with buried active sites. Recent molecular dynamics simulations showed that reorientation of five aromatic rings leads to rapid opening and closing of the gate to the active site. In the present study the molecular dynamics trajectory is used to quantitatively analyze the effect of the gate on the substrate binding rate constant. For a 2.4-Å probe modeling acetylcholine, the gate is open only 2.4% of the time, but the quantitative analysis reveals that the substrate binding rate is slowed by merely a factor of 2. We rationalize this result by noting that the substrate, by virtue of Brownian motion, will make repeated attempts to enter the gate each time it is near the gate. If the gate is rapidly switching between the open and closed states, one of these attempts will coincide with an open state, and then the substrate succeeds in entering the gate. However, there is a limit on the extent to which rapid gating dynamics can compensate for the small equilibrium probability of the open state. Thus the gate is effective in reducing the binding rate for a ligand 0.4 Å bulkier by three orders of magnitude. This relationship suggests a mechanism for achieving enzyme specificity without sacrificing efficiency.     

Apoptosis as a mechanism for liver disease progression

Hepatocyte injury is ubiquitous in clinical practice, and the mode of cell death associated with this injury is often apoptosis, especially by death receptors. Information from experimental systems demonstrates that hepatocyte apoptosis is sufficient to cause liver hepatic fibrogenesis. The mechanisms linking hepatocyte apoptosis to hepatic fibrosis remain incompletely understood, but likely relate to engulfment of apoptotic bodies by professional phagocytic cells and stellate cells, and release of mediators by cells undergoing apoptosis. Inhibition of apoptosis with caspase inhibitors has demonstrated beneficial effects in murine models of hepatic fibrosis. Recent studies implicating Toll-like receptor 9 in liver injury and fibrosis are also of particular interest. Engulfment of apoptotic bodies is one mechanism by which the TLR9 ligand (CpG DNA motifs) could be delivered to this intracellular receptor. These concepts suggest therapy focused on interrupting the cellular mechanisms linking apoptosis to fibrosis would be useful in human liver diseases.     

Cardio-oesophageal reflex in humans as a mechanism for 'linked angina'

The aim of this study was to investigate the hypothesis thatoesophageal acid stimulation reduces coronary blood flow inhumans as a result of the presence of a cardiooesophageal reflexwhich may provide a mechanism for ‘linked angina’.We studied the effect of oesophageal acid stimulation on coronaryblood flow in 35 syndrome X patients and 24 heart transplantpatients. A line tube was positioned into the patient's distaloesophagus. An intracoronary Doppler catheter was positionedin the proximal left anterior descending coronary artery forcoronary blood flow measurements. Oesophageal instillation of0·1 M hydrochloric acid was performed (60 ml over 5 min)and the measurements were repeated. The coronary blood flowwas significantly reduced by acid oesophageal stimulation inthe syndrome X group [pre-acid 78·9 ± 36·ml . min^–1, post-acid 50·8 ± 32·9ml . min^–1 (P=0·0001)]. However, coronary bloodflow in the heart transplant group, in whom the heart is denervated,was unaffected by acid infusion. We conclude that oesophagealacid stimulation can produce angina and significantly reducecoronary blood flow in humans. The lack of any significant effectin the heart transplant group, in whom the heart is denervated,suggests a neural reflex. (Eur Heart J 1996; 17: 407–413)     

Blood flow and cell-free layer in microvessels

Blood is modeled as a suspension of red blood cells using the dissipative particle dynamics method. The red blood cell membrane is coarse-grained for efficient simulations of multiple cells, yet accurately describes its viscoelastic properties. Blood flow in microtubes ranging from 10 to 40 μm in diameter is simulated in three dimensions for values of hematocrit in the range of 0.15-0.45 and carefully compared with available experimental data. Velocity profiles for different hematocrit values show an increase in bluntness with an increase in hematocrit. Red blood cell center-of-mass distributions demonstrate cell migration away from the wall to the tube center. This results in the formation of a cell-free layer next to the tube wall corresponding to the experimentally observed Fahraeus and Fahraeus-Lindqvist effects. The predicted cell-free layer widths are in agreement with those found in in vitro experiments; the results are also in qualitative agreement with in vivo experiments. However, additional features have to be taken into account for simulating microvascular flow, e.g., the endothelial glycocalyx. The developed model is able to capture blood flow properties and provides a computational framework at the mesoscopic level for obtaining realistic predictions of blood flow in microcirculation under normal and pathological conditions.     

Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate

Dividing cultured cells contain much larger pools of the four dNTPs than resting cells. In both cases the sizes of the individual pools are only moderately different. The same applies to mitochondrial (mt) pools of cultured cells. Song et al. [Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, Mathews CK (2005) Proc Natl Acad Sci USA 102:4990-4995] reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.     

Evidence for a carrier-mediated mechanism for thiamine transport to human jejunal basolateral membrane vesicles

Recent studies from our laboratory have demonstrated the presence of a pH-dependent, amiloride-sensitive, electroneutral carrier-mediated exchange for thiamine absorption in the human small intestinal brush-border membrane vesicles. However, the mechanism of thiamine transport across the human small intestinal basolateral membrane is not understood. The present study was aimed to characterize the mechanism of thiamine transport across the basolateral membranes of the human jejunum. Basolateral membrane vesicles (BLMV) were purified from mucosal scrapings of organ donors, utilizing a Percoll continuous density gradient centrifugation technique. The results showed [³H] thiamine uptake into BLMV to be: (1) markedly stimulated in the presence of an outwardly directed H⁺ gradient (pH 5.5_in/7.5_out); (2) significantly inhibited by amiloride in a dose-dependent manner; (3) sensitive to temperature and medium osmolarity and insensitive to changes in membrane potential; (4) not influenced by the addition of 1 mM Mg²⁺-ATP, inside and outside the vesicles in the presence of Na⁺ and K⁺; (5) inhibited by structural analogs—amprolium, oxythiamin, and unlabeled thiamine (100 M); (6) not affected by organic cations, eg, TEA, N-methyl-nicotinamide (NMN), and choline. (7) saturable as a function of concentration (apparent K _m of 0.76 ± 0.21 M and a V _max of 1.38 ± 0.35 pmol/mg protein/10 sec). These results indicate the presence of a proton gradient-dependent specialized carrier-mediated exchange mechanism for thiamine transport across the human jejunum basolateral membranes.     

Two-phase model for prediction of cell-free layer width in blood flow

This study aimed to develop a numerical model capable of predicting changes in the cell-free layer (CFL) width in narrow tubes with consideration of red blood cell aggregation effects. The model development integrates to empirical relations for relative viscosity (ratio of apparent viscosity to medium viscosity) and core viscosity measured on independent blood samples to create a continuum model that includes these two regions. The constitutive relations were derived from in vitro experiments performed with three different glass-capillary tubes (inner diameter = 30, 50 and 100 μm) over a wide range of pseudoshear rates (5–300 s^− 1). The aggregation tendency of the blood samples was also varied by adding Dextran 500 kDa. Our model predicted that the CFL width was strongly modulated by the relative viscosity function. Aggregation increased the width of CFL, and this effect became more pronounced at low shear rates. The CFL widths predicted in the present study at high shear conditions were in agreement with those reported in previous studies. However, unlike previous multi-particle models, our model did not require a high computing cost, and it was capable of reproducing results for a thicker CFL width at low shear conditions, depending on aggregating tendency of the blood.     

Universal fractal scaling in stream chemistry and its implications for solute transport and water quality trend detection

The chemical dynamics of lakes and streams affect their suitability as aquatic habitats and as water supplies for human needs. Because water quality is typically monitored only weekly or monthly, however, the higher-frequency dynamics of stream chemistry have remained largely invisible. To illuminate a wider spectrum of water quality dynamics, rainfall and streamflow were sampled in two headwater catchments at Plynlimon, Wales, at 7-h intervals for 1–2 y and weekly for over two decades, and were analyzed for 45 solutes spanning the periodic table from H⁺ to U. Here we show that in streamflow, all 45 of these solutes, including nutrients, trace elements, and toxic metals, exhibit fractal 1/f^α scaling on time scales from hours to decades (α = 1.05 ± 0.15, mean ± SD). We show that this fractal scaling can arise through dispersion of random chemical inputs distributed across a catchment. These 1/f time series are non–self-averaging: monthly, yearly, or decadal averages are approximately as variable, one from the next, as individual measurements taken hours or days apart, defying naive statistical expectations. (By contrast, stream discharge itself is nonfractal, and self-averaging on time scales of months and longer.) In the solute time series, statistically significant trends arise much more frequently, on all time scales, than one would expect from conventional t statistics. However, these same trends are poor predictors of future trends—much poorer than one would expect from their calculated uncertainties. Our results illustrate how 1/f time series pose fundamental challenges to trend analysis and change detection in environmental systems.