Diffusivity of Cu2+ in calcium alginate gel beads

The diffusivity of Cu(2+) in calcium alginate beads calculated by the shrinking core model (SCM) was reevaluated in this work. The results obtained in this work were significantly different than those by the original authors. There were excellent agreements between the results obtained by the SCM in this work and those by the more rigorous linear absorption model (LAM) by the original authors. (c) 1994 John Wiley & Sons, Inc.     

Diffusivity of Cu(2+) in calcium alginate gel beads

A linear absorption model (LAM) is used to describe the process of metal binding to spherically shaped biopolymers particles. The LAM was solved using a numerical algorithm which calculates diffusivities of metal ion in biopolymer gels. It assumes attainment of rapid metal-biopolymer binding equilibrium accompanied by rate limiting diffusion of the metal ions through the gel. The model was tested using batch experiments in which copper (Cu(2+)) binding with calcium alginate beads was investigated. Biopolymer density in the beads was varied between 2% and 5%. The diffusion coefficient of Cu(2+) calculated from the LAM ranged from 1.19 x 10(-9) to 1.48 x 10(-9) m(2) s(-1) (average 1.31 +/- 0.21 x 10(-9) m(2) s(-1)), independent of biopolymer density. The LAM has theoretical advantages over the shrinking core model (shell progressive model). The latter calculated an unreasonable exponential increase in the diffusion coefficient as density of alginate polymer in the bead increased. (c) 1993 John Wiley & Sons, Inc.     

Anisotropic rotational diffusion in model-free analysis for a ternary DHFR complex

Model-free analysis has been extensively used to extract information on motions in proteins over a wide range of timescales from NMR relaxation data. We present a detailed analysis of the effects of rotational anisotropy on the model-free analysis of a ternary complex for dihydrofolate reductase (DHFR). Our findings show that the small degree of anisotropy exhibited by DHFR (D_||/D=1.18) introduces erroneous motional models, mostly exchange terms, to over 50% of the NH spins analyzed when isotropic tumbling is assumed. Moreover, there is a systematic change in S², as large as 0.08 for some residues. The significant effects of anisotropic rotational diffusion on model-free motional parameters are in marked contrast to previous studies and are accentuated by lowering of the effective correlation time using isotropic tumbling methods. This is caused by the preponderance of NH vectors aligned perpendicular to the principal diffusion tensor axis and is readily detected because of the high quality of the relaxation data. A novel procedure, COPED (COmparison of Predicted and Experimental Diffusion tensors) is presented for distinguishing genuine motions from the effects of anisotropy by comparing experimental relaxation data and data predicted from hydrodynamic analyses. The procedure shows excellent agreement with the slow motions detected from the axially symmetric model-free analysis and represents an independent procedure for determining rotational diffusion and slow motions that can confirm or refute established procedures that rely on relaxation data. Our findings show that neglect of even small degrees of rotational diffusion anisotropy can introduce significant errors in model-free analysis when the data is of high quality. These errors can hinder our understanding of the role of internal motions in protein function.     

Truncated structural variants of lipoarabinomannan in Mycobacterium leprae and an ethambutol-resistant strain of Mycobacterium tuberculosis

Current knowledge on the structure of lipoarabinomannan (LAM) has resulted primarily from detailed studies on a few selected laboratory strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmatis. Our previous work was the first to report on the salient structural features of M. tuberculosis clinical isolates and demonstrated significant structural variations. A prime effort is to correlate a particular structural characteristic with observed differences in eliciting an immunobiological response, especially in the context of CD1-restricted presentation of LAM to T cells. T cell clones derived from the cutaneous lesions of leprosy patients have been shown to recognize specifically LAM from Mycobacterium leprae and not from M. tuberculosis Erdman or H37Rv. Herein we provide further fine structural data on LAM from M. leprae (LepLAM) and a tuberculosis clinical isolate, CSU20 (CSU20LAM), which was unexpectedly recognized by the supposedly LepLAM-specific CD1-restricted T cell clones. In comparison with the de facto laboratory LAM standard from M. tuberculosis H37Rv (RvLAM), LepLAM derived from in vivo grown M. leprae is apparently simpler in its arabinan architecture with a high degree of exposed, non-mannose-capped termini. On the other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly heterogeneous population of LAM ranging from rather small and non-mannose-capped to full-length and fully capped variants. LepLAM and CSU20LAM contain a higher level of succinylation than RvLAM, which, in the context of truncated or less elaborated arabinan, may contribute to selective recognition by T cells. LAM from all species could be resolved into discrete forms by isoelectric focusing based apparently on their arabinan heterogeneity. In the light of our current and more recent findings, we reason that all immunobiological data should be cautiously interpreted and that the actual LAM variants that may be present in vivo during infection and pathogenesis need to be taken into consideration.     

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. I. Theory.

It is shown that fluorescence anisotropy from lipidlike probes in the hexagonal HII phase gives information of (a) orientational order parameters, (b) the wobbling diffusion constant, and (c) the hopping diffusion constant of the probe, DH, equals DL/R2, the lateral diffusion constant over the square of the radius of the hexagonal tubes. Here we consider only lipidlike probes having the absorption transition movement and/or the emission transition moment along the long axis of the molecule. Three models are introduced for analysis of time-resolved data: the "WOBHOP," the "reduced WOBHOP," and the "P2P4HOP" model. The fluorescence anisotropy in response to a very short excitation pulse in each of the three models is a constant plus a number of exponentials. The WOBHOP and reduced WOBHOP models have 3 and 2 exponentials, respectively, and both contain four fitting parameters: r0 (the fundamental anisotropy), (P2) (the second rank orientational order parameter), DW (the wobbling diffusion constant), and DH (the hopping diffusion constant). The P2P4HOP model has eight exponentials and five fitting parameters: the four parameters listed above and (P4) (the fourth rank orientational order parameter). Analysis of fluorescence anisotropy data in the hexagonal HII phase using one of these models allows for obtaining the hopping diffusion constant, and, if the lateral diffusion constant is known, the radius of the hexagonal tubes. Substitution of DH = 0 in each of the three models yields an expression for the fluorescence anisotropy that is used in the literature for lamellar (L alpha or L beta) phases. The fluorescence anisotropy in coexisting L alpha/HII phases is discussed.     

Simulation of a Hard-Sphere Fluid in Bicontinuous Random Media

Abstract

The influence of solid-phase connectivity on size-exclusion partitioning and on diffusion of a dilute hard-sphere fluid in overlapping and nonoverlapping spheres models of porous media is investigated using molecular dynamics and Monte Carlo simulation techniques. Four models are examined, two of which are subject to constrained bicontinuity of the pore and solid phases and two in which the solid spheres in the assemblies are randomly distributed in space. It is shown that at moderate to high porosities, connected (bicontinuous) structures lead to a significant increase in the partition and diffusion coefficients when the particles of the pore fluid are of finite size. The consequences of solid phase connectivity are also clearly illustrated in the long-time decay of the velocity autocorrelation function (VACF) of the diffusing particles, particularly in the vicinity of the percolation threshold. Under these conditions the power law exponents on the long-time tail of the VACF are generally found to be higher in connected models than in random systems and the importance of this result is demonstrated using one of the scaling rules of percolation theory. The simulation results are also compared with the predictions of current theories of partitioning and diffusion in random sphere assemblies and, with reference to experimental data available from the literature, it is shown that bicontinuous models are better representations of real porous media.     

Translational diffusion coefficient of cycloamylose in aqueous sodium hydroxide

Seven cyclic (1 --> 4)-alpha-D-glucan (cycloamylose) samples ranging in weight-average molecular weight from 5 x 10(3) to 1.8 x 10(4) and gamma-cyclodextrin have been studied by sedimentation equilibrium in dimethylsulfoxide (at 25 degrees C) and by dynamic light scattering in 0.5 N aqueous sodium hydroxide (at 25 degrees C), a good solvent for linear amylose. The measured translational diffusion coefficients D in the aqueous NaOH agree fairly closely with previous Monte Carlo results for cyclic (1 --> 4)-alpha-D-glucan chains with excluded volume, when correction is made for the effects of bead diameter and fluctuating hydrodynamic interaction (HI) on the Kirkwood theory on which the computation of D was based. These D data are also explained almost quantitatively by Yamakawa and Fujii's expression for the associated KP ring (based on the Kratky-Porod wormlike chain) with the molecular parameters for linear amylose if the fluctuating HI and excluded-volume effects are taken into account. It is concluded that the translational diffusion behavior of cycloamylose in the aqueous NaOH is consistent with the conformational characteristics derived from the conformational energy of maltose and dilute-solution data for linear amylose.     

Seed dispersal curves: Behavior of the tail of the distribution

Summary Seed dispersal is important both to plant fitness and to plant population structure. We suggest that the tail of the seed dispersal curve is at least as important biologically as the modal portion of the curve, and we present a relatively simple, four-parameter model, based on diffusion principles, for the tail of the seed distribution. This model includes two types of qualitative behavior: algebraic tails (which tend to be longer and have greater reach) and exponential tails (which are shorter and have less reach). We have selected 68 data sets from the literature, each giving a seed shadow that could be categorized statistically as (1) exponential, (2) algebraic, (3) neither, or (4) both models fit adequately. Algebraic shapes for seed-shadow tails were common in this sample, and tail behavior was not generally specific to a particular dispersal mode. This result may suggest that algebraic tails are generally favored by selection and can be achieved by several means, but limitations of existing data sets and of statistical methodology preclude final judgement. Smaller complete samples of seed distances would provide a better basis for the analysis of tails than do the present form of data sets (consisting of counts of seeds in discrete distance categories).     

Measuring the diffusion of linguistic change

We examine situations in which linguistic changes have probably been propagated via normal contact as opposed to via conquest, recent settlement and large-scale migration. We proceed then from two simplifying assumptions: first, that all linguistic variation is the result of either diffusion or independent innovation, and, second, that we may operationalize social contact as geographical distance. It is clear that both of these assumptions are imperfect, but they allow us to examine diffusion via the distribution of linguistic variation as a function of geographical distance. Several studies in quantitative linguistics have examined this relation, starting with Séguy (Séguy 1971 Rev. Linguist. Romane 35, 335-357), and virtually all report a sublinear growth in aggregate linguistic variation as a function of geographical distance. The literature from dialectology and historical linguistics has mostly traced the diffusion of individual features, however, so that it is sensible to ask what sort of dynamic in the diffusion of individual features is compatible with Séguy's curve. We examine some simulations of diffusion in an effort to shed light on this question.     

Bounds for generalized Dreitlein-Smoes models

The analysis of a previous paper obtaining bounds on the total population number of species (chemical or biological) described by the recently proposed Dreitlein-Smoes model of oscillatory kinetic systems, including diffusion, is extended to generalized models of the Dreitlein-Smoes type, describing a system ofS components withS>2. The results for such generalized models are analogous to those of the previous case. It is found that the effects of diffusion serve to restrict the region in the concentration space available to limitcycle type oscillations.     

Role of Prolactin Receptors in Lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations in the tumor suppressor genes encoding Tuberous Sclerosis Complex (TSC) 1 and TSC2. The protein product of the TSC2 gene is a well-known suppressor of the mTOR pathway. Emerging evidence suggests that the pituitary hormone prolactin (Prl) has both endocrine and paracrine modes of action. Here, we have investigated components of the Prl system in models for LAM. In a TSC2 (+/-) mouse sarcoma cell line, down-regulation of TSC2 using siRNA resulted in increased levels of the Prl receptor. In human LAM cells, the Prl receptor is detectable by immunohistochemistry, and the expression of Prl in these cells stimulates STAT3 and Erk phosphorylation, as well as proliferation. A high affinity Prl receptor antagonist consisting of Prl with four amino acid substitutions reduced phosphorylation of STAT3 and Erk. Antagonist treatment further reduced the proliferative and invasive properties of LAM cells. In histological sections from LAM patients, Prl receptor immuno reactivity was observed. We conclude that the Prl receptor is expressed in LAM, and that loss of TSC2 increases Prl receptor levels. It is proposed that Prl exerts growth-stimulatory effects on LAM cells, and that antagonizing the Prl receptor can block such effects.     

Improving parameter estimation for cell surface FRAP data

Fluorescence Recovery After Photobleaching (FRAP) using the confocal laser scanning microscope has become a standard method used to determine the diffusion coefficient and mobile fraction of cell surface proteins. A common experimental approach is to bleach a stripe on the cell surface and fit the ensuing FRAP curve to a 1D diffusion model. This model is derived from the time course of recovery to an infinitely long stripe bleached on an infinite flat plane. This choice of model dictates the use of a long bleach stripe. We demonstrate that, in the case of a long bleach stripe, the finite extent of the cell leads to significant errors in parameter estimation. We further show that these errors are reduced when a relatively small stripe is bleached. Unfortunately, diffusion to such a region is fundamentally two dimensional and therefore applying the 1D model of diffusion leads to significant errors. We derive an equation suitable for fitting to FRAP data acquired from small bleach regions and analyze its accuracy using simulated data. We propose that the use of a small bleach region along with a two dimensional diffusion model is the ideal protocol for cell surface FRAP.     

Mathematical Models for the Effects of pH, Temperature, and Sodium Chloride on the Growth of Bacillus stearothermophilus in Salty Carrots

Estimating the shelf life and safety of any food product is an important part of food product development. Predictive food microbiology reduces the time and expense associated with conventional challenge and shelf life testing. The purpose of this study was to characterize and model germination, outgrowth, and lag (GOL) time and the exponential growth rate (EGR) of Bacillus stearothermophilus in salty carrot medium (SCM) as a function of pH, temperature, and NaCl concentration. B. stearothermophilus is a spore-forming thermophilic organism associated with flat sour spoilage of canned foods. A split-split plot design was used to measure the effects and interactions of pH (5.5 to 7.0), temperature (45 to 60(deg)C), and NaCl (0 to 1%) on the growth kinetics of B. stearothermophilus in SCM. A total of 96 experiments were analyzed, with individual curve parameters determined by using the Gompertz equation. Quadratic polynomial models for GOL time and EGR of B. stearothermophilus in terms of temperature, pH, and NaCl were generated by response surface analysis. The r(sup2) values for the GOL time and EGR models were 0.917 and 0.916, respectively. These models provide an estimate of bacterial growth in response to combinations of the variables studied within the specified ranges. The models were used to predict GOL times and EGRs for additional experimental conditions. The accuracy of these predictions validated the model's predictive ability in SCM.     

Topology and thermodynamics of gaseous ligands diffusion paths in human neuroglobin

The physiological role of recently discovered human neuroglobin (Ngb) is still unknown. Sound hypothesis says that it protects brain during hypoxia. In this paper the advanced potential of mean force by implicit ligand sampling (PMF/ILS) method is used to study the free energy landscape of Ngb for O(2), NO and CO ligands. The multiple diffusion paths are discovered and four ligand binding cavities are determined. The data show that certain regions are easily accessible by O(2) and NO but are protected from CO. Free energy landscapes provide realistic data for stochastic models of ligand diffusion in proteins.     

Laser-assisted microdissection of the kidney: Fundamentals and applications

Laser-assisted microdissection (LAM) permits the procurement of relatively pure cell populations from histological sections. When applied to the kidney, LAM combined with molecular biological techniques has expanded our understanding of renal biology and pathology. Both frozen and fixed renal tissues can be microdissected. However, sample type and tissue processing can influence the quality of molecular data generated. Data analysis may also be complicated by relative variations in gene expression levels. Importantly, preliminary studies have shown that molecular data obtained following LAM on the kidney can offer new diagnostic and prognostic information. Thus, LAM and molecular markers may eventually become incorporated into the routine kidney biopsy examination.     

Enantiomeric free radicals and enzymatic control of stereochemistry in a radical mechanism: the case of lysine 2,3-aminomutases

The product of yjeK in Escherichia coli is a homologue of lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4, and both enzymes catalyze the isomerization of (S)- but not (R)-alpha-lysine by radical mechanisms. The turnover number for LAM from E. coli is 5.0 min(-1), 0.1% of the value for clostridial LAM. The reaction of E. coli LAM with (S)-alpha-[3,3,4,4,5,5,6,6-(2)H8]lysine proceeds with a kinetic isotope effect (kH/kD) of 1.4, suggesting that hydrogen transfer is not rate-limiting. The product of the E. coli enzyme is (R)-beta-lysine, the enantiomer of the clostridial product. Beta-lysine-related radicals are observed in the reactions of both enzymes by electron paramagnetic resonance (EPR). The radical in the reaction of clostridial LAM has the (S)-configuration, whereas that in the reaction of E. coli LAM has the (R)-configuration. Moreover, the conformations of the beta-lysine-related radicals at the active sites of E. coli and clostridial LAM are different. The nuclear hyperfine splitting between the C3 hydrogen and the unpaired electron at C2 shows the dihedral angle to be 6 degrees, unlike the value of 77 degrees reported for the analogous radical bound to the clostridial enzyme. Reaction of (S)-4-thialysine produces a substrate-related radical in the steady state of E. coli LAM, as in the action of the clostridial enzyme. While (S)-beta-lysine is not a substrate for E. coli LAM, it undergoes hydrogen abstraction to form an (S)-beta-lysine-related radical with the same stereochemistry of hydrogen transfer from C2 of (S)-beta-lysine to the 5'-deoxyadenosyl radical as in the action of the clostridial enzyme. The resulting beta-lysyl radical has a conformation different from that at the active site of clostridial LAM. All evidence indicates that the opposite stereochemistry displayed by E. coli LAM is determined by the conformation of the lysine side chain in the active site. Stereochemical models for the actions of LAM from C. subterminale and E. coli are presented.     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

A pulsed field gradient NMR was used to study lateral diffusion in the cholesterol-containing oriented bilayers of saturated (dipalmitoyl- and dimyristoyl-) phosphatidylcholines, upon their limiting hydration. Similar dependences of lateral diffusion coefficients on temperature and cholesterol concentration were observed, which agree with phase diagram showing the presence of the regions of disordered and ordered liquid-crystalline phases and a two-phase region. Under the same conditions, the lateral diffusion coefficient of dipalmitoylphosphatidylcholine is lower, which agrees qualitatively with its larger molecular weight. The comparison of data for dipalmitoylphosphatidylcholine with previous results for dipalmitoylsphingomyelin-cholesterol bilayers under the same conditions, in spite of similarity of phase diagrams, shows large (two–three times) differences in the lateral diffusion coefficient and a different profile of its dependence on cholesterol concentration. The comparison of data for dimyristoylphosphatidylcholine with previous results shows that the values of lateral diffusion coefficient and the shape of its dependence on cholesterol concentration coincide at high concentrations (>15 mol%) but differ at lower concentrations The revealed disagreement may be caused by the fact that the measurements were carried out at different water content in the system. At limiting hydration (more than 35% of water), the lateral diffusion coefficient for lipids decreases when cholesterol concentration rises, while at water content about 25% (as a result of equilibrium hydration from vapors) the lateral diffusion coefficient of phosphatidylcholine may be independent of cholesterol concentration. This is the consequence of the denser packing of molecules in the bilayer at reduced water content, an effect that competes with the ordering effect of cholesterol.     

The automated, accurate and reproducible determination of steady-state permeation parameters from percutaneous permeation data

Procedures for the in vitro determination of percutaneous permeation with Franz diffusion cells are widely accepted. However, the calculation of relevant endpoints, such as the steady-state flux (J) and the permeation coefficient (P(app)), still depends on visual data inspection or an approximation of the steady-state flux as the maximum observed absorption rate. As both these approaches must be considered inappropriate, an automated and reproducible algorithm to analyse permeation data is presented. The method detects both lag-times and non-linear data resulting from substance accumulation in the acceptor compartment of static diffusion cells. It was evaluated by using simulated data, and data from experiments with caffeine and testosterone on bovine udder skin and human reconstituted epidermis (SkinEthic), which represent model barriers with high and low barrier strengths, respectively. It was shown that the algorithm is a suitable method for the identification of steady-state ranges in permeation data. If used on data generated with appropriate experimental approaches, it is a reproducible and time-saving alternative to the visual analysis of diffusion data.     

Molecular mapping of functional domains of the leukocyte receptor for endothelium, LAM-1

The human lymphocyte homing receptor LAM-1, like its murine counterpart MEL-14, functions as a mammalian lectin, and mediates the binding of leukocytes to specialized high endothelial cells in lymphoid organs (HEV). LAM-1 is a member of a new family of cell adhesion molecules, termed selectins or LEC-CAMs, which also includes ELAM-1 and PAD-GEM (GMP-140/CD62). To localize the regions of LAM-1 that are involved in cell adhesion, we developed chimeric selectins, in which various domains of PAD-GEM were substituted into LAM-1, and used these chimeric proteins to define the domain requirements for carbohydrate binding, and to localize the regions recognized by several mAb which inhibit the adhesion of lymphocytes to lymph node HEV. The binding of PPME or fucoidin, soluble complex carbohydrates that specifically define the lectin activity of LAM-1 and MEL-14, required only the lectin domain of LAM-1. The LAM1-1, LAM1-3, and LAM1-6 mAb each strongly inhibit the binding of lymphocytes to HEV in the in vitro frozen section assay, and defined three independent epitopes on LAM-1. Blocking of PPME or fucoidin binding by LAM1-3 indicated that this site is identical, or in close proximity, to the carbohydrate binding site, and analysis of the binding of LAM1-3 to chimeric selectins showed that the epitope detected by LAM1-3 is located within the lectin domain. Although the LAM1-6 epitope is also located in the lectin domain, LAM1-6 did not affect the binding of PPME or fucoidin. The LAM1-1 epitope was located in, or required, the EGF domain, and, importantly, binding of LAM1-1 significantly enhanced the binding of both PPME and fucoidin. These results suggest that adhesion mediated by LAM-1 may involve cooperativity between functionally and spatially distinct sites, and support previous data suggesting a role for the EGF domain of LAM-1 in lymphocyte adhesion to HEV.     

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in Lα phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15° and 45°C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.Abbreviations D _t translational diffusion coefficient - FRAP fluorescence recovery after photobleaching - MSPE a membrane-spanning phosphatidylethanolamine derived from a glycerol-dialkyl-glycerol tetraether lipid isolated from Sulfolobus solfataricus - NBD 4-nitrobenz-2-oxa-1,3-diazolyl - PE phosphatidylethanolamine - POPC 1-palmitoyl-2-oleoylphosphatidylcholine - POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine