SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.

Increases in renal ε-(γ-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy:Pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, e-(γ-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney e-(γ-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p <0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p <0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular e-(γ-glutamyl) lysine (+ 361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular e-(γ-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca2+ and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.

Representing spray zone with cross flow as a well-mixed compartment in a high shear granulator

The spray zone is an important region to control nucleation of granules in a high shear granulator. In this study, a spray zone with cross flow is quantified as a well-mixed compartment in a high shear granulator. Granulation kinetics is quantitatively derived at both particle-scale and spray zone-scale. Two spatial decay rates, DGSDR (droplet-granule spatial decay rate) ζDG and DPSDR (droplet-primary particle spatial decay rate) ζDP, which are functions of volume fraction and diameter of particulate species within the powder bed, are defined to simplify the deduction. It is concluded that in cross flow, explicit analytical results show that the droplet concentration is subject to exponential decay with depth which produces a numerically infinite depth of spray zone in a real penetration process. In a well-mixed spray zone, the depth of the spray zone is 4/(ζDG + ζDP) and π2/3(ζDG + ζDP) in cuboid and cylinder shape, respectively. The first-order droplet-based collision rates of, nucleation rate B0 and rewetting rate RW0 are uncorrelated with the flow pattern and shape of the spray zone. The second-order droplet-based collision rate, nucleated granule-granule collision rate RGG, is correlated with the mixing pattern. Finally, a real formulation case of a high shear granulation process is used to estimate the size of the spray zone. The results show that the spray zone is a thin layer at the powder bed surface. We present, for the first time, the spray zone as a well-mixed compartment. The granulation kinetics of a well-mixed spray zone could be integrated into a Population Balance Model (PBM), particularly to aid development of a distributed model for product quality prediction.

Shell-side transfer in shell-and-tube heat exchangers

Local mass transfer coefficients were determined by using the electrochemical technique. A simple model of a heat exchanger with segmental nickel tube joined to p.v.c. rods replaced the exchanger tubes. Measurements were made for both no-Ieakage, semi-leakage and total leakage configurations. Baffle-spacings of 47.6 mm, 66.6 mm, 97 mm and 149.2 mm wer studied. Also studied were the overall exchanger pressure drops for each configuration. The comparison of the heat transfer data with this work showed good agreement at high flow rates for the no-leakage case, but the agreement became poor for lower flow rates and leakage configurations. This disagreement was explained by non-analogous driving forces existing in the two systems. The no-leakage data showed length-wise variation of transfer coefficients along the exchanger length. The end compartments showing transfer coefficients inferior by up to 26% compared to tbe internal compartments, depending on Reynolds number. With the introduction of leakage streams this variation however became smaller than the experimental accuracy. A model is outlined to show the characteristic behaviour of individual electrode segments within the compartment. This was able to discriminate between cross and window zones for the no- leakage case, but no such distinction could be made for the leakage case. A flow area was found which, when incorporated in the Reynolds number, enabled the correlation of baffle-cut and baffle-spacing parameters for the no-leakage case . This area is the free flow area determined at the baffle edge. Addition of the leakage area to this flow area resulted in correlation of all commercial leakage geometrical parameters. The procedures used to correlate the pressure drop data from a total of eighteen different configurations on a single curve are also outlined.

The effect of pH on PAMAM dendrimer-siRNA complexation:endosomal considerations as determined by molecular dynamics simulation

Intracellular degradation of genes, most notably within the endo-lysosomal compartment is considered a significant barrier to (non-viral) gene delivery in vivo. Previous reports based on in vitro studies claim that carriers possessing a mixture of primary, secondary and tertiary amines are able to buffer the acidic environment within the endosome, allowing for timely release of their contents, leading to higher transfection rates. In this report, we adopt an atomistic molecular dynamics (MD) simulation approach, comparing the complexation of 21-bp siRNA with low-generation polyamidoamine (PAMAM) dendrimers (G0 and G1) at both neutral and acidic pHs, the latter of which mimics the degradative environment within maturing 'late-endosomes'. Our simulations reveal that the time taken for the dendrimer-gene complex (dendriplex) to reach equilibrium is appreciably longer at low pH and this is accompanied by more compact packaging of the dendriplex, as compared to simulations performed at neutral pH. We also note larger absolute values of calculated binding free energies of the dendriplex at low pH, indicating a higher dendrimer-nucleic acid affinity in comparison with neutral pH. These novel simulations provide a more detailed understanding of low molecular-weight polymer-siRNA behavior, mimicking the endosomal environment and provide input of direct relevance to the "proton sponge theory", thereby advancing the rational design of non-viral gene delivery systems.

Characterization of heparin-binding site of tissue transglutaminase:its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCa, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

Local shell-side coefficients in shell and tube exchanges the use of a mass transfer technique

A diffusion-controlled electrochemical mass transfer technique has been employed in making local measurements of shell-side coefficients in segmentally baffled shell and tube heat exchangers. Corresponding heat transfer data are predicted through the Chilton and Colburn heat and mass transfer analogy. Mass transfer coefficients were measured for baffle spacing lengths of individual tubes in an internal baffle compartment. Shell-side pressure measurements were also made. Baffle compartment average coefficients derived from individual tube coefficients are shown to be in good agreement with reported experimental bundle average heat transfer data for a heat exchanger model of similar geometry. Mass transfer coefficients of individual tubes compare favourably with those obtained previously by another mass transfer technique. Experimental data are reported for a variety of segmental baffle configurations over the shell-side Reynolds number range 100 to 42 000. Baffles with zero clearances were studied at three baffle cuts and two baffle spacings. Baffle geometry is shown to have a large effect on the distribution of tube coefficients within the baffle compartment. Fluid "jetting" is identified with some baffle configurations. No simple characteristic velocity is found to correlate zonal or baffle compartment average mass transfer data for the effect of both baffle cut and baffle spacing. Experiments with baffle clearances typical of commercial heat exchangers are also reported. The effect of leakage streams associated with these baffles is identified. Investigations were extended to double segmental baffles for which no data had previously been published. The similarity in the shell-side characteristics of this baffle arrangement and two parallel single segmental baffle arrangements is demonstrated. A general relationship between the shell-side mass transfer performance and pressure drop was indicated by the data for all the baffle configurations examined.

Local heat transfer coefficient in a baffled shell and tube heat exchanger

Local shell side coefficient measurements in the end conpartments of a model shell and tube heat exchanger have been made using an electrochemical technique. Limited data are also reported far the second compartment. The end compartment average coefficients have been found to be smaller than reported data for a corresponding internal conpartment. The second compartment data. have been shown to lie between those for the end compartments and the reported internal compartment data. Experimental data are reported fcr two port types and two baffle orientations. with data for the case of an inlet compartment impingement baffle also being given . Port type is shown to have a small effect on compartment coefficients, these being largely unaffected. Likewise, the outlet compartment average coefficients are slightly snaller than those for the inlet compartment, with the distribution of individual tube coefficients being similar. Baffle orientation has been shown to have no effect on average coefficients, but the distribution of the data is substantially affected. The use of an impingement baffle in the inlet compartment lessens the efect of baffle orientation on distribution . Recommendations are made for future work.

The development of functional in vitro toxicity tests

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaC0-2 cell model, there was no significant change in the transport of [3H] L-proline by the cell after eo-incubation with either dapsone or cyclophosphamide (50µM) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (100µM). With the test involving the NG115-401 L-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after eo-incubation with either phenytoin or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (50µM) (0.44 ± 0.04 mM), sulphamethoxazole (50µM) (0.43 ± 0.08mM) and carbamazepine (50µM) (0.47 ± 0.034 mM), when eoincubated with the rat microsomal system, compared to the control (0.52 ± 0.07mM). There was no significant depletion in glutathione when the erythrocytes were eoincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50µM) (0.953 ± 0110mM) when co-incubated the rat microsomal system, compared to the control (1.124 ± 0.032mM). Differences were considered statistically significant for p<0.05, using the Student's two tailed 't' test with Bonferroni's correction. There was no significant depletion of glutathione with phenytoin, carbamazepine and sulphamethoxazole when co-incubated with the rat microsomalsystem, compared to the control.

Improving diagnosis and oral vaccination strategies against bovine tuberculosis

In this work peptide antigens [ESAT-6,p45 in water (1ml, 1mg/ml)] have been adsorbed onto 10mg inorganic substrates (hydroxyapatite (MHA P201;P120, CHA), polystyrene, calcium carbonate and glass microspheres) and in vitro release characteristics were determined. The aim of formulation was to enhance the interaction of peptides with antigen presenting cells and to achieve rapid peptide release from the carrier compartment system in a mildly acidic environment. Hydroxyapatite microparticle P201 has a greater surface area and thus has the largest peptide adsorption compared to the P120. CHA gave a further higher adsorption due to larger surface area than that available on microparticles. These particles were incorporated into the BOVIGAMTM assay to determine if they improve the sensitivity. After overnight incubation the blood plasma was removed and the amount of IFN-g in each plasma sample was estimated. CHA and MHA P201 gave a significantly higher immune response at low peptide concentration compared to the free peptide, thus indicating that these systems can be used to evaluate Tuberculosis (TB) amongst cattle using the BOVIGAMTM assay. Badgers are a source of TB and pass infection to cattle. At the moment vaccination against TB in badgers is via the parenteral route and requires a trained veterinary surgeon as well as catching the badgers. This process is expensive and time consuming; consequently an oral delivery system for delivery of BCG vaccines is easier and cheaper. The initial stage involved addition of various surfactants and suspending agents to disperse BCG and the second stage involved testing for BCG viability. Various copolymers of Eudragit were used as enteric coating systems to protect BCG against the acidic environment of the stomach (SGF, 0.1M HCl pH 1.2 at 37oC) while dissolving completely in the alkaline environment of the small intestine (SIF, IM PBS solution pH 7.4 at 37oC). Eudragit L100 dispersed in 2ml PBS solution and 0.9ml Tween 80 (0.1%w/v) gave the best results remaining intact in SGF loosing only approximately 10-15% of the initial weight and dissolving completely within 3 hours. BCG was incorporated within the matrix formulation adjusted to pH 7 at the initial formulation stage containing PBS solution and Tween 80. It gave viability of x106 cfu/ml at initial formulation stage, freezing and freeze-drying stages. After this stage the matrix was compressed at 4 tons for 3 mins and placed in SGF for 2 hours and then in SIF until dissolved. The BCG viability dropped to x106 cfu/ml. There is potential to develop it further for oral delivery of BCG vaccine.

High-performance liquid chromatography and pharmacokinetics of some antibiotics

The principles of High Performance Liquid Chromatography (HPLC) and pharmacokinetics were applied to the use of several clinically-important drugs at the East Birmingham Hospital. Amongst these was gentamicin, which was investigated over a two-year period by a multi-disciplinary team. It was found that there was considerable intra- and inter-patient variation that had not previously been reported and the causes and consequences of such variation were considered. A detailed evaluation of available pharmacokinetic techniques was undertaken and 1- and 2-compartment models were optimised with regard to sampling procedures, analytical error and model-error. The implications for control of therapy are discussed and an improved sampling regime is proposed for routine usage. Similar techniques were applied to trimethoprim, assayed by HPLC, in patients with normal renal function and investigations were also commenced into the penetration of drug into peritoneal dialysate. Novel assay techniques were also developed for a range of drugs including 4-aminopyridine, chloramphenicol, metronidazole and a series of penicillins and cephalosporins. Stability studies on cysteamine, reaction-rate studies on creatinine-picrate and structure-activity relationships in HPLC of aminopyridines are also reported.

Pharmacokinetics and metabolism of MZPES, a novel lipophilic antifolate

m-Azidopyrimethamine ethanesulphonate salt (MZPES) is a new potent dihydrofolate reductase inhibitor designed to be both lipophilic and rapidly biodegradable. The drug is active against some methotrexate-refractory cell lines and against a broad spectrum of malignant cells in murine models. The pharmacokinetics of the drug were evaluated in the mouse, rat and man. A specific analytical method was developed to allow determination of MZP (the free base of MZPES) and its putative metabolite m-amino-pyrimethamine (MAP) in plasma, urine, faeces and tissues. Analytical methodology involved solvent extraction followed by reversed-phase ion-pair high pressure liquid chromatography. Mice were dosed at 10 and 20 mg/kg IP and 10 mg/kg PO. Absorption was rapid from both sites with a mean plasma elimination half-life of 4 hours. Oral bio-availability, relative to intraperitoneal injection, exceeded 95% in the mouse. MZP attained concentrations in mouse tissues 4 to 14 fold greater than those found in plasma and penetrated the blood-brain barrier effectively. Following intraperitoneal administration of MZP to the rat, the recovery of MZP and MAP in urine and faeces was 14% during 72 hours. MZPES was formulated for a phase I clinical evaluation as a 1% w/v aqueous solution and was administered by IV infusion in 5% dextrose over 1 hour. The drug obeyed 2-compartment kinetics with a central compartment volume of 27 litres and a volume of distribution of 118 litres. Plasma distribution and elimination half-lives were 0.27 and 34 hours respectively and plasma clearance was 7.5 L/hr. MZP was removed from plasma more rapidly than the prototypic lipophilic dihydrofolate reductase inhibitor metoprine (half-life 216 hours). The pharmacokinetics of MZPES showed no dose-dependency over the dose-range studied (27 to 460 mg/m2). The dose-limiting toxicity was nausea and vomiting. The short half-life of the drug should allow easy assessment of the optimum dose and schedule of administration.

Biological barriers to the nasal delivery of peptide drugs

The nasal absorption of larger peptide and protein drugs is generally low. The importance of the mucus layer and enzymic degradation in reducing absorption were investigated. Reversed-phase high-performance liquid chromatographic (HPLC) methods were developed to assay a variety of compounds. Pig gastric mucus (PGM) was selected to investigate the importance of the mucus layer. A method of treating and storing PGM was developed and evaluated which was representative of the gel in vivo. The nature of the mucus barrier was evaluated in vitro with three-compartment diffusion cells and a series of compounds with differing physicochemical properties. Mucus retarded the diffusion of all the compounds with molecular weight and charge exerting a marked effect. Binding to mucus was investigated by a centrifugation method. All of the compounds tested were found to bind to mucus with the exception of the negatively charged molecule benzoic acid. The small peptides did not demonstrate greater binding to mucus than any of the other compounds evaluated. The effect of some absorption enhancers upon the rate of diffusion of tryptophan through mucus was determined in vi tro. At the concentrations employed the enhancers EDTA, N-acetylcysteine and taurodeoxycholic acid exerted no effect, whilst taurocholic acid and cholic acid, were found to slightly reduce the rate of diffusion. The intracellular and luminal proteolytic activity of the nose was investigated in the sheep animal model with a nasal mucosal homogenate and a nasal wash preparation respectively and a series of chemically similar peptides. Hydrolysis was also investigated with the proteolytic enzymes carboxypeptidase A, cytosolic leucine aminopeptidase and microsomal leucine aminopeptidase. Sheep nasal mucosa possesses significant peptide hydrolase activity capable of degrading all the substrates tested. Considerable variation in susceptibility was observed. Degradation occurred excl us i ve ly at the pept ide bond between the aromatic amino ac id and glycine, indicating some specificity for aromatic amino acids. Hydrolysis profiles indicated the presence of both aminopeptidase and carboxypeptidase enzymes. The specific activity of the microsomal fraction was found to be greater than the cytosolic fraction. Hydrolysis in the nasal wash indicated the presence of either luminal or loosely-bound proteases, which can degrade peptide substrates. The same specificity for aromatic amino acids was observed and aminopeptidase activity demonstrated. The specific activity of the nasal wash was smaller than that of the homogenate.

Behaviour of concrete frame structures under localised fire scenarios

This thesis encompasses an investigation of the behaviour of concrete frame structure under localised fire scenarios by implementing a constitutive model using finite-element computer program. The investigation phase included properties of material at elevated temperature, description of computer program, thermal and structural analyses. Transient thermal properties of material have been employed in this study to achieve reasonable results. The finite-element computer package of ANSYS is utilized in the present analyses to examine the effect of fire on the concrete frame under five various fire scenarios. In addition, a report of full-scale BRE Cardington concrete building designed to Eurocode2 and BS8110 subjected to realistic compartment fire is also presented. The transient analyses of present model included additional specific heat to the base value of dry concrete at temperature 100°C and 200°C. The combined convective-radiation heat transfer coefficient and transient thermal expansion have also been considered in the analyses. For the analyses with the transient strains included, the constitutive model based on empirical formula in a full thermal strain-stress model proposed by Li and Purkiss (2005) is employed. Comparisons between the models with and without transient strains included are also discussed. Results of present study indicate that the behaviour of complete structure is significantly different from the behaviour of individual isolated members based on current design methods. Although the current tabulated design procedures are conservative when the entire building performance is considered, it should be noted that the beneficial and detrimental effects of thermal expansion in complete structures should be taken into account. Therefore, developing new fire engineering methods from the study of complete structures rather than from individual isolated member behaviour is essential.

Polarized secretion of leukemia inhibitory factor

Background: The direction of cytokine secretion from polarized cells determines the cytokine's cellular targets. Leukemia inhibitory factor LIF) belongs to the interleukin-6 IL-6) family of cytokines and signals through LIFR/gp130. Three factors which may regulate the direction of LIF secretion were studied: the site of stimulation, signal peptides, and expression levels. Stimulation with IL-1 beta is known to promote IL-6 secretion from the stimulated membrane apical or basolateral) in the human intestinal epithelial cell line Caco-2. Since LIF is related to IL-6, LIF secretion was also tested in Caco-2 following IL-1 beta stimulation. Signal peptides may influence the trafficking of LIF. Two isoforms of murine LIF, LIF-M and LIF-D, encode different signal peptides which have been associated with different locations of the mature protein in fibroblasts. To determine the effect of the signal peptides on LIF secretion, secretion levels were compared in Madin-Darby canine kidney MDCK) clones which expressed murine LIF-M or LIF-D or human LIF under the control of an inducible promoter. Low and high levels of LIF expression were also compared since saturation of the apical or basolateral route would reveal specific transporters for LIF. Results: When Caco-2 was grown on permeable supports, LIF was secreted constitutively with around 40% secreted into the apical chamber. Stimulation with IL-1 beta increased LIF production. After treating the apical surface with IL-1 beta, the percentage secreted apically remained similar to the untreated, whereas, when the cells were stimulated at the basolateral surface only 20% was secreted apically. In MDCK cells, an endogenous LIF-like protein was detected entirely in the apical compartment. The two mLIF isoforms showed no difference in their secretion patterns in MDCK. Interestingly, about 70% of murine and human LIF was secreted apically from MDCK over a 400-fold range of expression levels within clones and a 200,000-fold range across clones. Conclusion: The site of stimulation affected the polarity of LIF secretion, while, signal peptides and expression levels did not. Exogenous LIF is transported in MDCK without readily saturated steps.