Characterization of the Resting Axolemma in the Giant Axon of the Squid

Previous electron microscope studies have shown that the Schwann cell layer is traversed by long and tortuous slit-like channels ~60Å wide, which provide the major route of access to the axolemma surface. In the present work the restriction offered by the resting axolemma to the passage of six small non-electrolyte molecules has been determined. The radii of the probing molecules were estimated from constructed molecular models. The ability of the axolemma to discriminate between the solvent (water) and each probing molecule was expressed in terms of the reflection coefficient σ. σ was then used to calculate an effective pore size for the resting axolemma. The value of 4.25 Å found for the pore radius is in excellent agreement with the 1.5 to 8.5 Å limiting values previously calculated from our measurements of water fluxes. The presence of pores with 4.25 Å radius in the resting axolemma is compatible with restricted diffusion of Na. The present paper leads to the conclusion that the axolemma is the only continuous barrier across which the ionic gradient responsible for the normal functioning of the nerve can be maintained. The combined findings of electron microscopy, water permeability, and molecular restricted filtration indicate that in all probability the axolemma is the "excitable membrane" of the physiologists.     

Determination of Equivalent Pore Radius for Human Red Cells by Osmotic Pressure Measurement

A new method has been developed to measure the equivalent pore radius in cellular membranes, and has been applied to human red cells. When red cells are suddenly introduced into a non-isosmolar concentration of non-lipid-soluble non-electrolyte molecules, water will enter or leave the cell. The rate of cell swelling or shrinking is determined and extrapolated to zero time to give the initial rate of volume change. By suitable adjustment of the concentration of the external solution the initial rate may be brought to zero. The transient equilibrium concentration, determined by interpolation from experimental data, gives a measure of Staverman's reflection coefficient, σ. The zero time method has enabled us to determine σ for nine permeant molecules. σ is directly related to the equivalent pore radius; the experimental data lead to a value of 4.2 Å for the equivalent pore radius in man, in good agreement with the previous figure of 3.5 Å given by Paganelli and Solomon. The zero time method offers a number of advantages over previous methods for determination of this parameter. It requires no measurement of the rate of water entrance into the cell, and is essentially independent of the kinetics of cell swelling. It may be applied to a variety of living cells so that many additional membranes may now be characterized in terms of their equivalent pore radius.     

Kinetics of Diffusion and Convection in 3.2-Å Pores: Exact Solution by Computer Simulation

The kinetics of transport in pores the size postulated for cell membranes has been investigated by direct computer simulation (molecular dynamics). The simulated pore is 11 Å long and 3.2 Å in radius, and the water molecules are modeled by hard, smooth spheres, 1 Å in radius. The balls are given an initial set of positions and velocities (with an average temperature of 313° K) and the computer then calculates their exact paths through the pore. Two different conditions were used at the ends of the pore. In one, the ends are closed and the balls are completely isolated. In the other, the ball density in each end region is fixed so that a pressure difference can be established and a net convective flow produced. The following values were directly measured in the simulated experiments: net and diffusive (oneway) flux; pressure, temperature, and diffusion coefficients in the pore; area available for diffusion; probability distribution of ball positions in the pore; and the interaction between diffusion and convection. The density, viscosity, and diffusion coefficients in the bulk fluid were determined from the theory of hard sphere dense gases. From these values, the “equivalent” pore radius (determined by the same procedure that is used for cell membranes) was computed and compared with the physical pore radius of the simulated pore.     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.     

High membrane permeability for melatonin

The pineal gland, an endocrine organ in the brain, synthesizes and secretes the circulating night hormone melatonin throughout the night. The literature states that this hormone is secreted by simple diffusion across the pinealocyte plasma membrane, but a direct quantitative measurement of membrane permeability has not been made. Experiments were designed to compare the cell membrane permeability to three indoleamines: melatonin and its precursors N-acetylserotonin (NAS) and serotonin (5-HT). The three experimental approaches were (1) to measure the concentration of effluxing indoleamines amperometrically in the bath while cells were being dialyzed internally by a patch pipette, (2) to measure the rise of intracellular indoleamine fluorescence as the compound was perfused in the bath, and (3) to measure the rate of quenching of intracellular fura-2 dye fluorescence as indoleamines were perfused in the bath. These measures showed that permeabilities of melatonin and NAS are high (both are uncharged molecules), whereas that for 5-HT (mostly charged) is much lower. Comparisons were made with predictions of solubility-diffusion theory and compounds of known permeability, and a diffusion model was made to simulate all of the measurements. In short, extracellular melatonin equilibrates with the cytoplasm in 3.5 s, has a membrane permeability of ∼1.7 µm/s, and could not be retained in secretory vesicles. Thus, it and NAS will be “secreted” from pineal cells by membrane diffusion. Circumstances are suggested when 5-HT and possibly catecholamines may also appear in the extracellular space passively by membrane diffusion.     

Lateral Drug Diffusion in Human Nails

The main objective of the current work is to demonstrate the process of passive lateral diffusion in the human nail plate and its effect on the passive transungual permeation of antifungal drug ciclopirox olamine (CPO). A water soluble dye, methyl red sodium salt (MR) was used to visualize the process of lateral diffusion using a novel suspended nail experiment. The decline in concentration of CPO correlates with that of concentration of MR from the proximal to the distal end of the nail in suspended nail study. Three toenails each were trimmed to 5 mm × 5 mm (25 mm²), 7 mm × 7 mm (49 mm²), and 9 mm × 9 mm (81 mm²) to study the extent and effect of lateral diffusion of the CPO on its in vitro transungual permeation. The permeation flux of CPO decreased as the surface area of the toenail increased. There was a positive correlation between the concentrations of CPO and MR in the area of application and in the peripheral area of the toenails of the three surface areas, confirming the findings in the suspended nail experiment. Profound lateral diffusion of CPO was demonstrated and shown to reduce the in vitro passive transungual drug permeation and prolong the lag-time in human toenails. The study data implies that during passive in vitro transungual permeation experiments, the peripheral nail around the area of drug application has to be kept to a minimum, in order to get reliable data which mimics the in vivo situation.KEY WORDS: ciclopirox olamine, lateral diffusion, passive, topical, transungual     

Small-angle X-ray scattering of pig thyroglobulin in solution (Short Communication)

Small-angle X-ray scattering measurements on native pig thyroglobulin in phosphate buffer, pH6.9, yield a radius of gyration of 6.4nm (64Å), a particle volume of approx. 1.5×10³nm³ (1.5×10⁶ų), an axial ratio of 2.2:1 (assuming an ellipsoidal shape), and a solvation of 0.63g of solvent/g of protein.     

Sublingual Diffusion of Epinephrine Microcrystals from Rapidly Disintegrating Tablets for the Potential First-Aid Treatment of Anaphylaxis: In Vitro and Ex Vivo Study

For the first-aid treatment of anaphylaxis, epinephrine (Epi) 0.3 mg intramuscular (IM) injection in the thigh is the drug of choice. Epi auto-injectors are widely recommended for anaphylaxis treatment in community settings but not necessarily carried or used as prescribed when anaphylaxis occurs. We therefore developed rapidly disintegrating sublingual tablets (RDSTs) as an alternative noninvasive dosage form. Our objective in this study was to evaluate the effect of reducing Epi particle size on its in vitro and ex vivo diffusion, with the goal of enhancing Epi sublingual absorption from Epi RDSTs. Epi particle size was reduced by top-bottom technique using a microfluidizer for one pass at 30,000 Psi. The micronized Epi crystals (Epi-MC) were characterized using Zetasizer, Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Epi RDSTs were formulated and manufactured using our previously developed method. In vitro and ex vivo diffusion of Epi 10, 20, and 40 mg RDSTs and Epi-MC 10 and 20 mg RDSTs (n = 4) were evaluated using Franz cells. Epi 10 mg solution was used as a control. Mean (±standard deviation (SD)) Epi particle size was successfully reduced from 131.8 ± 10.5 to 2.5 ± 0.4 μm. Cumulative Epi diffused and influx from 40 mg Epi RDSTs and 20 mg Epi-MC RDSTs were not significantly different from each other in vitro and ex vivo (p > 0.05). Also, Epi permeability from 20 mg Epi-MC RDSTs was significantly higher than from the rest (p < 0.05). Epi-MC RDSTs improved Epi diffusion twofold and might have the potential to reduce the Epi dose needed in RDSTs by 50%.KEY WORDS: adrenaline, anaphylaxis, diffusion, epinephrine, sublingual     

Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.     

Dynamics of the Serine Chemoreceptor in the Escherichia coli Inner Membrane: A High-Speed Single-Molecule Tracking Study

We investigated the mobility of the polar localized serine chemoreceptor, Tsr, labeled by the fluorescent protein Venus in the inner membrane of live Escherichia coli cells at observation rates up to 1000 Hz. A fraction (7%) of all Tsr molecules shows free diffusion over the entire cell surface with an average diffusion coefficient of 0.40 ± 0.01 μm² s⁻¹. The remaining molecules were found to be ultimately confined in compartments of size 290 ± 15 nm and showed restricted diffusion at an inner barrier found at 170 ± 10 nm. At the shortest length-scales (<170 nm), all Tsr molecules diffuse equally. Disruption of the cytoskeleton and rounding of the cells resulted in an increase in the mobile fraction of Tsr molecules and a fragmenting of the previously polar cluster of Tsr consistent with a curvature-based mechanism of Tsr cluster maintenance.     

Features of Large Hinge-Bending Conformational Transitions. Prediction of Closed Structure from Open State

We performed a detailed analysis of conformational transition pathways for a set of 10 proteins, which undergo large hinge-bending-type motions with 4–12 Å RMSD (root mean-square distance) between open and closed crystal structures. Anisotropic network model-Monte Carlo (ANM-MC) algorithm generates a targeted pathway between two conformations, where the collective modes from the ANM are used for deformation at each iteration and the conformational energy of the deformed structure is minimized via an MC algorithm. The target structure was approached successfully with an RMSD of 0.9–4.1 Å when a relatively low cutoff radius of 10 Å was used in ANM. Even though one predominant mode (first or second) directed the open-to-closed conformational transition, changes in the dominant mode character were observed for most cases along the transition. By imposing radius of gyration constraint during mode selection, it was possible to predict the closed structure for eight out of 10 proteins (with initial 4.1–7.1 Å and final 1.7–2.9 Å RMSD to target). Deforming along a single mode leads to most successful predictions. Based on the previously reported free energy surface of adenylate kinase, deformations along the first mode produced an energetically favorable path, which was interestingly facilitated by a change in mode shape (resembling second and third modes) at key points. Pathway intermediates are provided in our database of conformational transitions (http://safir.prc.boun.edu.tr/anmmc/method/1).     

Self Crowding of Globular Proteins Studied by Small-Angle X-Ray Scattering

Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D₂O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10^-4 cm³mol/g² for Mb in phosphate buffer, 1.6 ×10^-4 cm³mol/g² for BPTI in phosphate buffer and 9.2 ×10^-4 cm³mol/g² for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.     

KINETOPLAST DEOXYRIBONUCLEIC ACID OF THE HEMOFLAGELLATE TRYPANOSOMA LEWISI

Cesium chloride centrifugation of DNA extracted from cells of blood strain Trypanosoma lewisi revealed a main band, ρ = 1.707, a light satellite, ρ = 1.699, and a heavy satellite, ρ = 1.721. Culture strain T. lewisi DNA comprised only a main band, ρ = 1.711, and a light satellite, ρ = 1.699. DNA isolated from DNase-treated kinetoplast fractions of both the blood and culture strains consisted of only the light satellite DNA. Electron microscope examination of rotary shadowed preparations of lysates revealed that DNA from kinetoplast fractions was mainly in the form of single 0.4 µ circular molecules and large masses of 0.4 µ interlocked circles with which longer, often noncircular molecules were associated. The 0.4 µ circular molecules were mainly in the covalently closed form: they showed a high degree of resistance to thermal denaturation which was lost following sonication; and they banded at a greater density than linear DNA in cesium chloride-ethidium bromide gradients. Interpretation of the large masses of DNA as comprising interlocked covalently closed 0.4 µ circles was supported by the findings that they banded with single circular molecules in cesium chloride-ethidium bromide gradients, and following breakage of some circles by mild sonication, they disappeared and were replaced by molecules made up of low numbers of apparently interlocked 0.4 µ circles. When culture strain cells were grown in the presence of either ethidium bromide or acriflavin, there was a loss of stainable kinetoplast DNA in cytological preparations. There was a parallel loss of light satellite and of circular molecules from DNA extracted from these cells.     

Identification of a Tsetse Fly Salivary Protein with Dual Inhibitory Action on Human Platelet Aggregation

Background

Tsetse flies (Glossina sp.), the African trypanosome vectors, rely on anti-hemostatic compounds for efficient blood feeding. Despite their medical importance, very few salivary proteins have been characterized and functionally annotated.

Methodology/Principal Findings

Here we report on the functional characterisation of a 5′nucleotidase-related (5′Nuc) saliva protein of the tsetse fly Glossina morsitans morsitans. This protein is encoded by a 1668 bp cDNA corresponding at the genomic level with a single-copy 4 kb gene that is exclusively transcribed in the tsetse salivary gland tissue. The encoded 5′Nuc protein is a soluble 65 kDa glycosylated compound of tsetse saliva with a dual anti-hemostatic action that relies on its combined apyrase activity and fibrinogen receptor (GPIIb/IIIa) antagonistic properties. Experimental evidence is based on the biochemical and functional characterization of recombinant protein and on the successful silencing of the 5′nuc translation in the salivary gland by RNA interference (RNAi). Refolding of a 5′Nuc/SUMO-fusion protein yielded an active apyrase enzyme with K_m and V_max values of 43±4 µM and 684±49 nmol Pi/min×mg for ATPase and 49±11 µM and 177±37 nmol Pi/min×mg for the ADPase activity. In addition, recombinant 5′Nuc was found to bind to GPIIb/IIIa with an apparent K_D of 92±25 nM. Consistent with these features, 5′Nuc potently inhibited ADP-induced thrombocyte aggregation and even caused disaggregation of ADP-triggered human platelets. The importance of 5′Nuc for the tsetse fly hematophagy was further illustrated by specific RNAi that reduced the anti-thrombotic activities in saliva by approximately 50% resulting in a disturbed blood feeding process.

Conclusions/Significance

These data show that this 5′nucleotidase-related apyrase exhibits GPIIb/IIIa antagonistic properties and represents a key thromboregulatory compound of tsetse fly saliva.     

Porosity of the Yeast Cell Wall and Membrane

The limiting sizes of molecules that can permeate the intact cell wall and protoplast membrane of Saccharomyces cerevisiae were determined from the inflection points in a triphasic pattern of passive equilibrium uptake values obtained with a series of inert probing molecules varying in molecular size. In the phase identified with the yeast protoplast, the uptake-exclusion threshold corresponded to a monodisperse ethylene glycol of molecular weight = 110 and Einstein-Stokes hydrodynamic radius (r(ES)) = 0.42 nm. In the cell wall phase, the threshold corresponded to a polydisperse polyethylene glycol of number-average molecular weight ( M(n)) = 620 and average radius (r(ES)) = 0.81 nm. The third phase corresponded to complete exclusion of larger molecules. The assessment of cell wall porosity was confirmed by use of a second method involving analytical gel chromatographic analyses of the molecular weight distribution for a single polydisperse polyglycol before and after uptake by the cells, which indicated a quasi-monodisperse threshold for the cell wall of M(n) = 760 and r(ES) = 0.89 nm. The results were reconciled with two situations in which much larger protein molecules previously have been reported able to penetrate the yeast cell wall.     

Temporal Evolution of Parotid Volume and Parotid Apparent Diffusion Coefficient in Nasopharyngeal Carcinoma Patients Treated by Intensity-Modulated Radiotherapy Investigated by Magnetic Resonance Imaging: A Pilot Study

Purpose

To concurrently quantify the radiation-induced changes and temporal evolutions of parotid volume and parotid apparent diffusion coefficient (ADC) in nasopharyngeal carcinoma (NPC) patients treated by intensity-modulated radiotherapy by using magnetic resonance imaging (MRI).

Materials and Methods

A total of 11 NPC patients (9 men and 2 women; 48.7 ± 11.7 years, 22 parotid glands) were enrolled. Radiation dose, parotid sparing volume, severity of xerostomia, and radiation-to-MR interval (RMI) was recorded. MRI studies were acquired four times, including one before and three after radiotherapy. The parotid volume and the parotid ADC were measured. Statistical analysis was performed using SPSS and MedCalc. Bonferroni correction was applied for multiple comparisons. A P value less than 0.05 was considered as statistically significant.

Results

The parotid volume was 26.2 ± 8.0 cm³ before radiotherapy. The parotid ADC was 0.8 ± 0.15 × 10⁻³ mm²/sec before radiotherapy. The parotid glands received a radiation dose of 28.7 ± 4.1 Gy and a PSV of 44.1 ± 12.6%. The parotid volume was significantly smaller at MR stage 1 and stage 2 as compared to pre-RT stage (P < .005). The volume reduction ratio was 31.2 ± 13.0%, 26.1 ± 13.5%, and 17.1 ± 16.6% at stage 1, 2, and 3, respectively. The parotid ADC was significantly higher at all post-RT stages as compared to pre-RT stage reciprocally (P < .005 at stage 1 and 2, P < .05 at stage 3). The ADC increase ratio was 35.7 ± 17.4%, 27.0 ± 12.8%, and 20.2 ± 16.6% at stage 1, 2, and 3, respectively. The parotid ADC was negatively correlated to the parotid volume (R = -0.509; P < .001). The parotid ADC was positively associated with the radiation dose significantly (R² = 0.212; P = .0001) and was negatively associated with RMI significantly (R² = 0.203; P = .00096) significantly. Multiple regression analysis further showed that the post-RT parotid ADC was related to the radiation dose and RMI significantly (R² = 0.3580; P < .0001). At MR stage 3, the parotid volume was negatively associated with the dry mouth grade significantly (R² = 0.473; P < .0001), while the parotid ADC was positively associated with the dry mouth grade significantly (R² = 0.288; P = .015).

Conclusion

Our pilot study successfully demonstrates the concurrent changes and temporal evolution of parotid volume and parotid ADC quantitatively in NPC patients treated by IMRT. Our results suggest that the reduction of parotid volume and increase of parotid ADC are dominated by the effect of acinar loss rather than edema at early to intermediate phases and the following recovery of parotid volume and ADC toward the baseline values might reflect the acinar regeneration of parotid glands.     

The Water and Nonelectrolyte Permeability Induced in Thin Lipid Membranes by the Polyene Antibiotics Nystatin and Amphotericin B

Nystatin and amphotericin B increase the permeability of thin (<100 A) lipid membranes to ions, water, and nonelectrolytes. Water and nonelectrolyte permeability increase linearly with membrane conductance (i.e., ion permeability). In the unmodified membrane, the osmotic permeability coefficient, P_f, is equal to the tagged water permeability coefficient, (P_d)_w; in the nystatin- or amphotericin B-treated membrane, P_f/(P_d)_w ≈ 3. The unmodified membrane is virtually impermeable to small hydrophilic solutes, such as urea, ethylene glycol, and glycerol; the nystatin- or amphotericin B-treated membrane displays a graded permeability to these solutes on the basis of size. This graded permeability is manifest both in the tracer permeabilities, P_d, and in the reflection coefficients, σ (Table I). The "cutoff" in permeability occurs with molecules about the size of glucose (Stokes-Einstein radius 4 A). We conclude that nystatin and amphotericin B create aqueous pores in thin lipid membranes; the effective radius of these pores is approximately 4 A. There is a marked similarity between the permeability of a nystatin- or amphotericin B-treated membrane to water and small hydrophilic solutes and the permeability of the human red cell membrane to these same molecules.     

Fluorescently Labeled Methyl-Beta-Cyclodextrin Enters Intestinal Epithelial Caco-2 Cells by Fluid-Phase Endocytosis

Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB) on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10⁻⁸ cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.     

Arterial Spin Labeling Imaging for the Parotid Glands of Patients with Sj?gren’s Syndrome

Sjögren’s syndrome (SS) is characterized by hypofunction of the salivary and lacrimal glands. The salivary function is largely dependent upon the blood supply in the glands. However, the diseased states of the gland perfusion are not well understood. The arterial spin labeling (ASL) technique allows noninvasive quantitative assessment of tissue perfusion without the need for contrast agent. Here, we prospectively compared the perfusion properties of the parotid glands between patients with SS and those with healthy glands using ASL MR imaging. We analyzed salivary blood flow (SBF) kinetics of 22 healthy parotid glands from 11 volunteers and 28 parotid glands from 14 SS patients using 3T pseudo-continuous ASL imaging. SBF was determined in resting state (base SBF) and at 3 sequential segments after gustatory stimulation. SBF kinetic profiles were characterized by base SBF level, increment ratio at the SBF peak, and the differences in segments where the peak appeared (SBF types). Base SBFs of the SS glands were significantly higher than those of healthy glands (59.2 ± 22.8 vs. 46.3 ± 9.0 mL/min/100 g, p = 0.01). SBF kinetic profiles of the SS glands also exhibited significantly later SBF peaks (p < 0.001) and higher SBF increment ratios (74 ± 49% vs. 47 ± 39%, p = 0.04) than the healthy glands. The best SBF criterion (= 51.2 mL/min/100 mg) differentiated between control subjects and SS patients with 71% sensitivity and 82% specificity. Taken together, these results showed that the SS parotid glands were mostly hyperemic and the SS gland responses to gustatory stimulation were stronger and more prolonged than those of the healthy glands. The ASL may be a promising technique for assessing the diseased salivary gland vascularization of SS patients.     

Pore dimensions and the role of occupancy in unitary conductance of Shaker K channels

K channels mediate the selective passage of K⁺ across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K⁺ transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker’s reported ∼20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ∼0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K⁺ is set to ∼4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ∼8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K⁺], beyond that of P475D, suggesting an ∼200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K⁺ transport rates to ∼1 kT. Thus, although Shaker’s pore sustains ion translocation as the BK channel’s does, higher energetic costs of ion stabilization or higher friction with the ion’s rigid hydration cage in its narrower aqueous cavity may entail higher resistance.