Biomedical subjects

V J Morris

Publications and source records attributed to V J Morris.

At least 19 recordsLinked to original sources

PubMed2004

Atomic force microscopy of emulsion droplets: probing droplet-droplet interactions.

A method has been developed for attaching oil (tetradecane) droplets to the end of an atomic force microscopy (AFM) cantilever and for immobilizing droplets on a glass substrate. This approach has permitted the monitoring of droplet-droplet interactions in aqueous solution as a function of interdroplet separation. Coating the droplet surfaces with added proteins or surfactants has allowed the production of model emulsions. We demonstrate that AFM measurements of droplet deformability are sensitive to interfacial rheology by modifying the interfacial film on a pair of droplets in situ. For droplets coated with the anionic surfactant sodium dodecyl sulfate, screening of the double layer has been found to facilitate coalescence. Direct imaging of the droplets has revealed the presence of regularly spaced concentric rings on the droplet surfaces. Careful experimental studies suggest that these structures may be imaging artifacts and are not perturbations of the droplet surface determined by the composition of the interface.

Alkanes↗

PubMed2004

Watching molecular processes with the atomic force microscope: dynamics of polymer adsorption and desorption at the single molecule level.

The formation of networks is an important step in the synthesis of many biological assemblies. For example, during the synthesis of plant cell walls the factors which dictate the arrangement of the polymeric constituents that make up the cell wall are not yet understood. Factors such as site-directed binding provide a possible theoretical background for beginning to understand the assembly of complex biological structures, but modelling of this process is difficult, time consuming and lacks experimental methods for verification. Through the use of atomic force microscopy (AFM) it has been demonstrated that changes in the binding of a single heterogeneous cell wall polysaccharide to a charged substrate can be followed in real time. Furthermore, subsequent image analysis allows the probability of binding of the molecule to be mapped to produce a real data set which is comparable with those obtained in simulation studies. In addition, these AFM studies have provided new mechanistic clues to the adsorption/desorption process of this polysaccharide.

Adsorption↗

PubMed2002

Allergens of the cupin superfamily.

The cupin family comprises a family of proteins possessing a common beta-barrel structure that is thought to have originated in a prokaryotic ancestor. This structural motif is found as a single domain in fungal spherulins, fern sporulins and the germins/oxalate oxidase proteins of plants, while the globular storage proteins of plants, called legumins (11 S) and euvicilins (7 S), are two-domain cupins. The 11 S globulins are hexameric heteroligomeric proteins of M (r) approximately 360000, with each subunit comprising an acidic 30000-40000- M (r) polypeptide that is disulphide-linked to a 20000- M (r) basic polypeptide. A number of cupins have been identified as major plant food allergens, including the 7 S globulins of soybean (beta-conglycinin), peanut (conarachin; Ara h 1), walnut (Jug r 2) and lentil, and the 11 S globulins of peanut (arachin; Ara h 3), soybean (glycinin) and possibly also coconut and walnut. Other members of the cupin superfamily have not been identified as allergens, with the exception of one germin (germination-specific protein) from pepper. Cupins are generally very stable proteins. A summary of our current knowledge of allergenic seed storage globulins will be presented, together with an overview of cupin structure and stability properties, as illustrated by the allergenic soya globulins, glycinin and beta-conglycinin.

Allergens↗

PubMed2001

Both binding sites of the starch-binding domain of Aspergillus niger glucoamylase are essential for inducing a conformational change in amylose.

The interaction of the two binding sites of the starch-binding domain (SBD) of Aspergillus niger glucoamylase 1 (GA-I) with substrate has been investigated by using atomic force microscopy (AFM) and UV difference spectroscopy in combination with site-specific mutants of both SBD and GA-I. The SBD possesses two binding sites with distinct affinities towards the soluble linear substrate maltoheptaose; dissociation constants (K(d)) of 17 and 0.95 microM were obtained for W563 K (binding site 2 mutant) and W590 K (binding site 1 mutant), respectively, compared to an apparent K(d) of 23 microM for the wild-type SBD. Further, the two sites are almost but not totally independent of each other for binding, since abolishing one site does not prevent the amylose chain binding to the other site. Using AFM, we show that the amylose chains undergo a conformational change to form loops upon binding to the SBD, using either the recombinant wild-type SBD or a catalytically inactive mutant of GA-I. This characteristic conformation of amylose is lost when one of the SBD binding sites is eliminated by site-directed mutagenesis, as seen with the mutants W563 K or W590 K. Therefore, although each binding site is capable of simple binding to a ligand, both sites must be functional in order to induce a gross conformational change of the amylose molecules. Taken together these data suggest that for the complex with soluble amylose, SBD binds to a single amylose chain, site 1 being responsible for the initial recognition of the chain and site 2 being involved in tighter binding, leading to the circularisation of the amylose chain observed by AFM. Binding of the SBD to the amylose chain results in a novel two-turn helical amylose complex structure. The binding of parallel amylosic chains to the SBD may provide a basis for understanding the role of the SBD in facilitating enzymatic degradation of crystalline starches by glucoamylase 1.

Amino Acid Substitution↗

PubMed2001

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin.

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.

Amino Acid Sequence↗

PubMed2001

Formation of thermally induced aggregates of the soya globulin beta-conglycinin.

The effect of ionic strength (I) on the formation of thermally induced aggregates by the 7S globular storage protein of soya, beta-conglycinin, has been studied using atomic force microscopy. Aggregates were only apparent when I> or =0.1, and had a fibrous appearance, with a height (diameter) of 8-11 nm. At high ionic strength (I=1.0) the aggregates appeared to associate into clumps. When aggregate formation was studied at I=0.2, it was clear that aggregation only began at temperatures above the main thermal transition for the protein at 75 degrees C, as determined by differential scanning calorimetry. This coincided with a small change in secondary structure, as indicated by circular dichroism spectroscopy, suggesting that a degree of unfolding was necessary for aggregation to proceed. Despite prolonged heating the size of the aggregates did not increase indefinitely, suggesting that certain beta-conglycinin isoforms were able to act as chain terminators. At higher protein concentrations (1% w/v) the linear aggregates appeared to form large macroaggregates, which may be the precursors of protein gel formation. The ability of beta-conglycinin to form such distinctive aggregates is discussed in relation to the presence of acidic inserts in certain of the beta-conglycinin subunits, which may play an important role in limiting aggregate length.

Hot Temperature↗

PubMed2001

Investigating the nature of branching in pectin by atomic force microscopy and carbohydrate analysis.

Atomic force microscopy (AFM) has been used to investigate the nature of the long branches attached to pectin which were described in a previous report [Round, A. N.; MacDougall, A. J.; Ring, S. G.; Morris, V. J. Carbohydr. Res. 1997, 303, 251-253]. Analysis of the AFM images and comparison with neutral sugar and linkage analyses of the two pectin fractions suggest that the distribution and total amount of branches observed do not correspond with the pattern of neutral sugar distribution. It is thus postulated that the long chains consist of polygalacturonic acid, attached via an as yet undetermined linkage to the pectin backbone, with the neutral sugars present as short, undetected branches. This explanation would have important implications for the nature of 'in situ' pectin networks within plant cell walls and models of gelation in commercial extracted pectin, and the existence of significant branching will markedly influence the viscosity of extracted pectins.

Carbohydrates↗

PubMed2001

Structure and conformation of a novel genetically engineered polysaccharide P2.

A new exocellular polysaccharide (P2) has been produced by the manipulation of a glycosyl transferase gene (aceP) involved in the biosynthesis of the polysaccharide acetan by the bacterium Acetobacter xylinum strain CKE5. The P2 polysaccharide has been studied by methylation analysis, reductive cleavage, and 1H and 13C NMR spectroscopy. The data are consistent with the structure predicted when the aceP gene is deactivated: [Molecular structure: see text]. The effect of cooling on proton NMR line width indicates a coil-helix transition in P2 at about 70 degrees C.

Azotobacter↗

PubMed2001

Microstructure of aggregated and nonaggregated kappa-carrageenan helices visualized by atomic force microscopy.

Gelation of kappa-carrageenan is believed to involve two steps: helix formation on cooling and a further specific cation (salt) induced side-by-side aggregation of helices. Samples that should contain aggregated and also nonaggregated "helices" of kappa-carrageenan were prepared in aqueous solutions, spread onto freshly cleaved mica surfaces, and visualized under butanol using atomic force microscopy. In the presence of an excessive amount of a gel-promoting salt, KCl, kappa-carrageenan appeared to form rigid rodlike structures considered as large aggregates of double helices. Even when the side-by-side interhelical aggregation was suppressed by diluting random coiled solutions prior to cooling, by adding an aggregation-impeding salt, NaI, or by transforming kappa-carrageenan into the tetramethylammonium (TMA) salt, branched rodlike structures were still evident, suggesting that the side-by-side aggregation of helices is not a prerequisite for kappa-carrageenan to form a network structure, at least locally. Even in the absence of factors that promote side-by-side aggregation, kappa-carrageenan helices appeared to be capable of associating and forming gel networks.

Carbohydrate Conformation↗

PubMed2000

Observations on the crystallization of oligogalacturonates.

Oligogalacturonates were produced by the limited enzymic hydrolysis of polygalacturonic acid and purified by ion-exchange chromatography. The fractions obtained were of limited polydispersity, determined by analytical ion-exchange chromatography. Oligomers with an average degree of polymerization of 10-15 were readily crystallized from aqueous salt solutions at neutral pH as single crystals. Crystal morphology of the salts examined, Na+, K+ and Ca2+ were characteristic of the salt. The wide-angle X-ray diffraction patterns obtained for the sodium salt were consistent with published fibre diffraction data of this salt form.

Chromatography, Ion Exchange↗

PubMed1999

Generation of a novel polysaccharide by inactivation of the aceP gene from the acetan biosynthetic pathway in Acetobacter xylinum.

The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with beta-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.

Amino Acid Sequence↗

PubMed1998

Evidence for intermolecular binding between deacetylated acetan and the glucomannan konjac mannan.

Binary mixtures of deacetylated acetan and konjac mannan form thermoreversible gels under conditions for which the individual components do not gel. Such synergistic behaviour is normally attributed to intermolecular binding between the two polysaccharides. X-ray diffraction data obtained from oriented fibres prepared from deacetylated acetan-konjac mannan gels provides direct evidence for intermolecular binding between the two polysaccharides. The novel heterotypic junction zones appear to be six-fold helices with a pitch of 5.6 +/- 0.1 nm.

Acetobacter↗

PubMed1998

Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy.

The molecular weight of the extracellular polysaccharide (CR1/4) produced by Acetobacter xylinum strain CR1/4 has been shown to be dependent upon growth conditions. Under normal growth conditions a high molecular weight polysaccharide ( > 1 x 10(6) Da) is produced. Maintaining the pH at 5 results in an order of magnitude increase in the total yield of polysaccharide, but also an order of magnitude decrease in molecular weight. Analysis of the CR1/4 polysaccharides by the techniques of atomic force microscopy and static light scattering suggests that they are double helices. In solution the molecules behave as stiff coils with a Kuhn statistical segment length of 325 nm.

Carbohydrate Conformation↗

PubMed1997

Cloning of the aceF gene encoding the phosphomannose isomerase and GDP-mannose pyrophosphorylase activities involved in acetan biosynthesis in Acetobacter xylinum.

The aceF gene from Acetobacter xylinum was identified and cloned from a genomic DNA library. The complete DNA sequence was determined and computer analysis of the translated gene sequence revealed homology with the deduced amino acid sequence of xanB from Xanthomonas campestris. Therefore aceF is likely to encode a bifunctional enzyme with mannose-6-phosphate isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. PMI and GMP activities were detected in strains of Escherichia coli expressing the cloned aceF gene.

Amino Acid Sequence↗

PubMed1997

Effect of deacetylation on the synergistic interaction of acetan with locust bean gum or konjac mannan.

It has been discovered that deacetylation of the bacterial polysaccharide acetan promotes synergistic interactions with either locust bean gum (LBG) or konjac mannan (KM). Acetan is similar in structure to xanthan, and adopts a similar 5-fold conformation in the solid state. Like xanthan, it shows a thermally reversible order (helix)-disorder (coil) transition in solution. Both polymers have a cellulosic backbone with charged (anionic) sidechains attached at O-3 of alternate glucosyl residues, but the sidechains in acetan are longer (pentasaccharide rather than trisaccharide) and do not contain pyruvic substituents. Acetan has two sites of acetylation, one at O-6 of the inner mannosyl residue of the carbohydrate sidechains (as in xanthan) and the other on the polymer backbone (believed to be at O-6 of the branched glucosyl residues). Solutions of acetan or deacetylated acetan were equilibrated against 10 mM potassium chloride (to stabilise the ordered conformation) and were mixed (at 25 degrees C) with solutions of LBG or KM, also equilibrated against 10 mM potassium chloride. Unlike xanthan, native acetan showed no evidence of synergistic interaction with either LBG or KM. After deacetylation, however, large enhancements were observed in dilute-solution viscosity, and thermoreversible gels were formed at higher concentrations. With KM as co-synergist, gel melting was accompanied by an intense endotherm in differential scanning calorimetry. The magnitude of this endotherm increased with storage time at 25 degrees C, reaching a final value of delta H approximately 15.9 J/g (in comparison with delta H approximately 5.0 J/g for the order-disorder transition of deacetylated acetan alone). It is suggested that interaction occurs by formation of heterotypic junctions between the acetan backbone and unsubstituted regions of the plant polysaccharide, and that the acetate groups on native acetan promote solubility and hence inhibit association.

Acetobacter↗

PubMed1997

Effect of o-acyl substituents on the functional behaviour of Rhizobium meliloti succinoglycan.

The effects of selective removal of acetyl or succinyl substituents on the functionality of succinoglycan polysaccharide have been studied by comparing the behaviour of the polysaccharides isolated from native Rhizobium meliloti strain Rm1021, and genetically modified R. meliloti species. Removal of the succinyl groups was found to dramatically improve pseudoplasticity of the aqueous succinoglycan samples and also increase the cooperativity of the order-disorder transition exhibited by the polysaccharide. Removal of the acetyl substituent led to a decrease in the order-disorder transition temperature, whereas the removal of the succinyl groups led to an increase.

Calorimetry, Differential Scanning↗

PubMed1997

Atomic force microscopy of plant cell walls, plant cell wall polysaccharides and gels.

Methods developed for the routine imaging of polysaccharides by atomic force microscopy (AFM) have been used to image plant polysaccharides from higher plants (pectin) and algae (carrageenan). These methods have been extended to image K-carrageenan association in hydrated films. Finally, AFM has been used to image polysaccharide architecture in moist plant cell walls. Simple experimental and image processing methods have been used to enhance molecular structure in 'rough' cell wall surfaces.

Cell Wall↗

PubMed1996

The cpsABCDE genes involved in polysaccharide production in Streptococcus salivarius ssp. thermophilus strain NCBF 2393.

A 4074-bp EcoRI fragment of Streptococcus salivarius ssp. thermophilus (S. thermophilus) chromosomal DNA containing genes involved in exocellular polysaccharide (EPS) was identified and cloned. The nucleotide sequence of this fragment was determined and found to contain one partial and four complete open reading frames. These were designated cpsA, cpsB, cpsC, cpsD and cpsE and encoded proteins of > 130, 243, 230, 246 and 455 amino acids, respectively, that showed homology with the genes of the cps cluster, involved in polysaccharide biosynthesis, in Streptococcus pneumoniae Type 19F. The cpsA gene is predicted to encode a transcriptional regulator, while cpsC and cpsD are predicted to encode proteins involved in polysaccharide polymerization and export. The cpsE gene is likely to encode the phosphate-prenyl glycosyl-1-phosphate transferase catalyzing the first step in polysaccharide biosynthesis in S. thermophilus. Southern blot analysis revealed that cpsE is found only in polysaccharide producing strains of S. thermophilus.

Amino Acid Sequence↗