Transfer films and their effect on signal errors for helical scan data tape recording systems

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A predictor of membrane class:discriminating α-helical and β-barrel membrane proteins from non-membranous proteins

Accurate protein structure prediction remains an active objective of research in bioinformatics. Membrane proteins comprise approximately 20% of most genomes. They are, however, poorly tractable targets of experimental structure determination. Their analysis using bioinformatics thus makes an important contribution to their on-going study. Using a method based on Bayesian Networks, which provides a flexible and powerful framework for statistical inference, we have addressed the alignment-free discrimination of membrane from non-membrane proteins. The method successfully identifies prokaryotic and eukaryotic α-helical membrane proteins at 94.4% accuracy, β-barrel proteins at 72.4% accuracy, and distinguishes assorted non-membranous proteins with 85.9% accuracy. The method here is an important potential advance in the computational analysis of membrane protein structure. It represents a useful tool for the characterisation of membrane proteins with a wide variety of potential applications.

Alpha helical trans-membrane proteins:enhanced prediction using a Bayesian approach

Membrane proteins, which constitute approximately 20% of most genomes, are poorly tractable targets for experimental structure determination, thus analysis by prediction and modelling makes an important contribution to their on-going study. Membrane proteins form two main classes: alpha helical and beta barrel trans-membrane proteins. By using a method based on Bayesian Networks, which provides a flexible and powerful framework for statistical inference, we addressed alpha-helical topology prediction. This method has accuracies of 77.4% for prokaryotic proteins and 61.4% for eukaryotic proteins. The method described here represents an important advance in the computational determination of membrane protein topology and offers a useful, and complementary, tool for the analysis of membrane proteins for a range of applications.

Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.

Structural characterization of recombinant human CD81 produced in Pichia pastoris

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

Design and characterisation of novel blends of poly(lactic acid)

This thesis is concerned with the effect of polymer structure on miscibility of the three component blends based on poly(lactic acid) (PLA) with using blending techniques. The examination of novel PLA homologues (pre-synthesised poly(a-esters)), including a range of aliphatic and aromatic poly(a-esters) is an important aspect of the work. Because of their structural simplicity and similarity to PLA, they provide an ideal system to study the effect of polyester structures on the miscibility of PLA polymer blends. The miscibility behaviour of the PLA homologues is compared with other aliphatic polyesters (e.g. poly(e-caprolactone) (PCL), poly(hydroxybutyrate hydroxyvalerate) (P(HB-HV)), together with a series of cellulose-based polymers (e.g. cellulose acetate butyrate (CAB)). The work started with the exploration the technique used for preliminary observation of the miscibility of blends referred to as “a rapid screening method” and then the miscibility of binary blends was observed and characterised by percent transmittance together with the Coleman and Painter miscibility approach. However, it was observed that symmetrical structures (e.g. a1(dimethyl), a2(diethyl)) promote the well-packing which restrict their chains from intermingling into poly(L-lactide) (PLLA) chains and leads the blends to be immiscible, whereas, asymmetrical structures (e.g. a4(cyclohexyl)) behave to the contrary. a6(chloromethyl-methyl) should interact well with PLLA because of the polar group of chloride to form interactions, but it does not. It is difficult to disrupt the helical structure of PLLA. PLA were immiscible with PCL, P(HB-HV), or compatibiliser (e.g. G40, LLA-co-PCL), but miscible with CAB which is a hydrogen-bonded polymer. However, these binary blends provided a useful indication for the exploration the novel three component blends. In summary, the miscibility of the three-component blends are miscible even if only two polymers are miscible. This is the benefit for doing the three components blend in this thesis, which is not an attempt to produce a theoretical explanation for the miscibility of three components blend system.

Flexibility of short-strand RNA in aqueous solution as revealed by molecular dynamics simulation:are A′-RNA and A-RNA distinct conformational structures?

We use molecular dynamics simulations to compare the conformational structure and dynamics of a 21-base pair RNA sequence initially constructed according to the canonical A-RNA and A'-RNA forms in the presence of counterions and explicit water. Our study aims to add a dynamical perspective to the solid-state structural information that has been derived from X-ray data for these two characteristic forms of RNA. Analysis of the three main structural descriptors commonly used to differentiate between the two forms of RNA namely major groove width, inclination and the number of base pairs in a helical twist over a 30 ns simulation period reveals a flexible structure in aqueous solution with fluctuations in the values of these structural parameters encompassing the range between the two crystal forms and more. This provides evidence to suggest that the identification of distinct A-RNA and A'-RNA structures, while relevant in the crystalline form, may not be generally relevant in the context of RNA in the aqueous phase. The apparent structural flexibility observed in our simulations is likely to bear ramifications for the interactions of RNA with biological molecules (e.g. proteins) and non-biological molecules (e.g. non-viral gene delivery vectors). © CSIRO 2009.

The tribology of laminated magnetic recording heads

This thesis investigates the mechanisms that lead to pole tip recession (PTR) in laminated magnetic recording heads (also known as "sandwich heads"). These heads provide a platform for the utilisation of advanced soft magnetic thin films in practical recording heads suitable for high frequency helical scan tape recording systems. PTR results from a differential wear of the magnetic pole piece from the tape-bearing surface of the head. It results in a spacing loss of the playback or read signal of 54.6dB per recording wavelength separation of the poles from the tape. PTR depends on the material combination used in the head, on the tape type and the climate - temperature and relative humidity (r.h.). Five head materials were studied: two non-magnetic substrate materials- sintered multi granular CaTi03 and composite CaTi03/ZrTi04/Ti02 and three soft magnetic materials- amorphous CoNbZr, and nanocrystalline FeNbSiN and FeTaN. Single material dummy heads were constructed and their wear rates measured when cycling them in a Hi-8 camcorder against commercially available metal particulate (MP) and metal evaporated (ME) tapes in three different climates: 25°C/20%r.h., 25°C/80%r.h. and 40°C/80%r.h. X-ray photoelectron spectroscopy (XPS) was used to examine changes the head surface chemistry. Atomic force microscopy (AFM) was used to examine changes in head and tape surface topography. PTR versus cycling time of laminated heads of CaTi03/ZrTiO4/Ti02 and FeTaN construction was measured using AFM. The principal wear mechanism observed for all head materials was microabrasion caused by the mating body - the tape surface. The variation in wear rate with climate and tape type was due to a variation in severity in this mechanism, except for tape cycling at 40°C in which gross damage was observed to be occurring to the head surface. Two subsidiary wear mechanisms were found: third body scratching (all materials) and grain pullout (both ceramics and FeNbSiN). No chemical wear was observed, though tribochemical reactions were observed on the metal head surfaces. PTR was found to be caused by two mechanisms - the first differential microabrasion of the metal and substrate materials and which was characterised by a low (~10nm) equilibrium value. The second was by deep ploughing by third body debris particles, thought mainly to be grain pullout particles. This level of PTR caused by this mechanism was often more severe, and of a non-equilibrium nature. It was observed more for ME tape, especially at 40°C/80%r.h. and 25°c/20%r.h. Two other phenomena on the laminated head pole piece were observed and commented upon: staining and ripple texturing.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

Differences in β-amyloid ( β A4) deposition in human patients with Down's syndrome and sporadic Alzheimer's disease

The density of diffuse, primitive, classic and compact β-amyloid ( β A4) deposits was estimated in the hippocampus and adjacent gyri in human patients with Down's syndrome (DS) and sporadic Alzheimer's disease (AD). The objective of the study was to determine whether there were differences in β A4 deposition in DS and sporadic AD and whether these differences could be attributed to overexpression of the amyloid precursor gene (APP) in DS. Total β A4 deposit density was greater in DS than AD in all brain regions studied but the DS/AD density ratios varied between brain regions. In the majority of brain regions, the ratio of primitive to diffuse β A4 deposits was greater in DS but the ratio of classic to diffuse deposits was greater in AD. The data were consistent with the hypothesis that overexpression of the APP gene in DS may lead to increased β A4 deposition. However, local brain factors also appear to be important in β A4 deposition in DS. Overexpression of the APP gene may also be responsible for increased production of paired helical filaments (PHF) and result in enhanced formation of primitive β A4 deposits in DS. In addition, increased formation of classic deposits in AD suggests that factors necessary for the production of a compact amyloid core are enhanced in AD compared with DS. © 1994.

Alzheimer's disease: the relationship between the density of senile plaques, neurofibrillary tangles and A4 protein in human patients

The numerical density of senile plaques (SP) and neurofibrillary tangles (NFT) as revealed by the Glees silver method was compared with SP and NFT revealed by the Gallyas method and with amyloid (A4) deposits in immunostained sections in 6 elderly cases of Alzheimer's disease. The density of NFT was generally greater and A4 lower in tissue from hippocampus compared with the neocortex suggesting that A4 deposition was less important than the degree of paired helical filament (PHF) related damage in the hippocampus. The density of Glees SP was positively correlated Gallyas SP weakly correlated with A4 deposit number. A stepwise multiple regression analysis which included A4 deposit and Gallyas SP density and accounted for 54% of the variation in Glees SP density. Hence, different populations of SP were revealed by the different staining methods. The results suggested that the Glees method may stain a population of SP in a region of cortex where both amyloid deposition and neurofibrillary changes have occurred.

The spatial patterns of β/A4 deposit subtypes in Down's syndrome

The spatial patterns of diffuse, primitive and classic β/A4 deposits were studied in coronal sections of the hippocampus and adjacent gyri in 11 cases of Down's syndrome (DS) varying in age from 38 to 67 years. The objectives of the study were first, to compare the spatial patterns of β/A4 deposits revealed in DS with those reported in cases of Alzheimer's disease (AD) and second, to study how the spatial patterns of β/A4 deposits may develop in the tissue. The spatial patterns revealed in DS exhibited a number of similarities with those reported in AD: (1) the range and frequency of the different types of spatial pattern revealed were similar, (2) β/A4 deposits occurred in clusters and in many cortical tissues, the clusters were distributed in a regular pattern parallel to the pia, (3) the clusters of diffuse and primitive β/A4 deposits occurred in an alternating pattern along the cortex, and (4) the clusters of classic β/A4 deposits were not correlated with the clusters of the diffuse and primitive deposits. Primitive deposits may develop from the diffuse deposits in regions of the cortex where extracellular paired helical filaments were formed. However, clusters of the classic β/A4 deposits, which are formed in older cases, appear to develop independently of the diffuse and primitive deposits. © 1994 Springer-Verlag.

Distribution of A4 protein and neurofibrillary change in the hippocampus in Alzheimer's disease

The principal components of classical senile plaques (SP) in Alzheimer's disease (AD) appear to be A4/beta protein and paired helical filaments (PHF). A4 deposits may evolve into classical SP in brain regions vulnerable to the formation of PHF. We have investigated the diatribution of A4 deposits using an immunostain and the neurofibrillary change using the Gallyas stain in various regions of the hippocampus. This region is particularly affected in AD and also has relatively restricted inputs and outputs. In 6 patients we found a significant preponderance of A4 deposits in the adjacent parahippocampal gyrus (PHG) compared with all regions of the hippocampus. However, plaque-like clusters of PHF (Gallyas plaques) were more abundant in the subiculum while neurofibrillary tangles (NFT) were more abundant in the subiculum and region CA1 compared with the PHG and other hippocampal regions. Hence, A4 deposits appear to be concentrated in the region providing a major input into the hippocampus while the neurofibrillary changes are characteristic of the major output areas (subiculum and CA1). Hence, the data suggest that A4 formation and the neurofibrillary changes may occur in regions of the hippocampus that are connected anatomically.

Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

Dipole moments and Kerr effect of poly(n-vinylcarbazole) and its complexes with trinitrofluorenone

N-vinylcarbazole was polymerised using the free radical catalyst (azo-bisisobutyronitrile) and cationic catalysts (boron-trifluoride etherate and aluminium chloride). The polymers produced were characterised by molecular weight measurements and powder x-ray diffraction. The tacticity of the polymer samples was determined using proton and carbon-13 nuclear magnetic resonance spectroscopy. Measurements of their static dielectric permittivity and electro-optical birefringence (Kerr effect) in solution in 1,4-dioxane were carried out over a range of temperatures. The magnitudes of the dipole moments and Kerr constants were found to vary with changes in the tacticity of poly(N-vinylcarbazole). The results of these measurements support the view that the stereostructure of poly(N-vinylcarbazole) is sensitive to the mechanism of polymerisation. These results, together with proton and carbon-13 N.M.R. data, are discussed in terms of the possible conformations of the polymer chains and the relative orientation of the bulky carbazole side groups. The dielectric and molecular Kerr effect studies have also been carried out on complexes formed between 2,4,7-trinitro-9-fluorenone (TNF) and different stereoregular forms of poly(N-vinylcarbazole) in solution in 1,4-dioxane. The differences in the molar Kerr constants between pure (uncomplexed) and complexed poly(N-vinylcarbazole) samples were attributed to changes in optical anisotropy and dipole moments. A molecular modelling computer program Desktop Molecular Modeller was used to examine the 3/1 helical isotactic and 2/1 helical syndiotactic forms of poly(N-vinylcarbazole). These models were used to calculate the pitch distances of helices and the results were interpreted in terms of van der Waal's radii on TNF. This study indicated that the pitch distance in 3/1 isotactic helices was large enough to accommodate the bulky TNF molecules to form sandwich type charge transfer complexes whereas the pitch distance in syndiotactic poly(N-vinylcarbazole) was smaller and would not allow a similar type of complex formation.