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Item Glycaemic potency reduction by coarse grain structure in breads is largely eliminated during normal ingestion(Cambridge University Press on behalf of The Nutrition Society, 2022-05-28) Srv A; Mishra S; Hardacre A; Matia-Merino L; Goh K; Warren FJ; Monro JAThe hypothesis that coarse grain particles in breads reduce glycaemic response only if the particles remain intact during ingestion was tested. Three breads were formulated: (1) White bread (WB - reference), (2) 75 % of kibbled purple wheat in 25 % white bread matrix (PB) and (3) a 1:1 mixture of 37·5 % kibbled soya beans and 37·5 % of kibble purple wheat in 25 % white bread matrix (SPB). Each bread was ingested in three forms: unchewed (U), as customarily consumed (C) and homogenised (H). Twelve participants ingested 40 g available carbohydrate portions of each bread in each form, with post-prandial blood glucose measured over 120 min. Glycaemic responses to WB were the same regardless of its form when ingested. Unchewed PB had significantly less glycaemic effect than WB, whereas the C and H forms were similar to WB. Based on a glycaemic index (GI) of 70 for WB, the GI values for the C, U and H breads, respectively, were WB: 70·0, 70 and 70, PB: 75, 42 and 61, SPB: 57, 48 and 55 (%) (Least significant difference = 17·43, P < 0·05, bold numbers significantly different from WB). The similar glycaemic response to the H and C forms of the breads, and their difference from the U form, showed that the glycaemia-moderating effect of grain structure on starch digestion was lost during customary ingestion of bread. We conclude that the kibbled-grain structure may not effectively retard starch digestion in breads as normally consumed because it is largely eliminated by ingestive processes including chewing.Item The disordered- and ordered-state structures of κ-carrageenan : an X-ray scattering, molecular dynamics, and density-functional theory study : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physics at Massey University, Palmerston North, New Zealand(Massey University, 2022) Westberry, Benjaminκ-carrageenan is a biopolymer extracted from marine algae. It exists in aqueous solution, at high temperatures and/or low salt concentrations as a ‘disordered-state’, and at low temperatures and in the presence of certain salts as an ‘ordered-state’. The transition between disordered- and ordered-states involves molecular structural changes, which are essential to its interesting viscoelastic properties that are routinely exploited in a plethora of applications. Despite this, the molecular conformations of the disordered- and ordered-states, as well as the details of the transitional pathway connecting them, remain a source of contention. While decades of research have amassed a vast trove of information on the disorder-order transition, an atomistic understanding of the structure in solution has remained elusive. This study takes advantages of recent advances in computational capabilities in order to simulate κ-carrageenan solutions on length scales of ∼10 nm over μs time scales, and thus develop atomistic models of the disordered- and ordered-states. Both models are used to calculate wide-angle X-ray scattering profiles, and these are subsequently validated by comparison to data obtained at a synchrotron facility. The models will be further explored using density functional theory to calculate their expected optical rotation behaviour, which finds that the formation of double-helices from single chains is able to explain the increase in optical rotation measured experimentally when transitioning from the disordered to ordered-state. Structural analysis of both experimentally-verified models find the disordered-state to have a significant amount of residual helical secondary-structure, whereas the ordered-state is mostly double-helical. Crucially, simulations show that the ordered-state arises spontaneously from the so-called disordered-state at a rate dependant on salt concentration, without prior uni-molecular changes. The findings of this research are the most detailed model of the disorder-order transition to-date, and demonstrate that the existing paradigm of a ‘coil-to-helix’ transition is in need of revision.Item Characterisation of de-structured starch and its interactions in whey protein isolate gels : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2022) Ang, Cai LingStarch serves as an important additive to enhance the physico-chemical properties of many food products. With the increased pursuit of natural products, there is an increasing demand for “clean-label” starches. In this study, waxy potato starch was physically-modified at elevated temperatures of 120–150 °C for 30 min at 300 rpm, in a pressurised reactor. The treatment converted native starch granules into their macromolecular chains (denoted as de-structured waxy potato starch, DWPS). This doctoral thesis presents the: (i) method of modifying starch (i.e., the de-structuring process), (ii) the mechanism of starch de-structuring, (iii) the rheological changes in DWPS samples and the shear-thickening mechanism, and (iv) the interactions of these DWPS ingredients with whey protein isolate (WPI) in a protein-based gel system, at different pH and ionic strength. The molar mass (Mᵥᵥ), particle size, rheological properties, degree of branching (DB) and side-chain length distribution of DWPS samples were characterised to elucidate the starch de-structuring mechanism. DWPS treated at 120 °C DWPS showed similar Mᵥᵥ (~3.6 × 10⁸ Da) as its native form (~3.7 × 10⁸ Da) indicating that the treatment at 120 °C resulted in the disassembly of starch granules into their macromolecular chains. Reduction in viscosity, Mᵥᵥ and particle size was observed with an increase in temperature from 120 to 150 °C, suggesting a cleavage in amylopectin chains. The DB and side-chain distribution data suggest that the reduction in Mᵥᵥ is likely due to the cleavage at α-1,4 linkages near the middle of the main amylopectin backbone. Particle size analysis by laser diffraction measurements revealed the presence of large fragment particles (> 1 µm) in DWPS samples, indicating that the starch de-structuring process into its macromolecules was incomplete even at 150 °C for 30 min. The DWPS (5% w/w) samples were found to exhibit a wide range of rheological properties—Newtonian, shear-thinning, shear-thickening and anti-thixotropy behaviours—depending on their treatment temperature (120–150 °C). In particular, 120 °C DWPS exhibited interesting shear-thickening, anti-thixotropy and shear-induced gelation. These rheological properties are different from the shear-thinning and thixotropy behaviours observed in most conventionally gelatinised waxy potato starches treated at 95 °C. The complex shear-induced structures of 120 °C DWPS were attributed to a two-step process: (i) upon shear at the critical shear rate (~10–20 s⁻¹), the shear stress caused a size reduction in the starch fragments and (ii) the increased number of small fragments together with the amylopectin chains in very close proximity could lead to the formation of a complex network probably consisting of amylopectin chains and a large number of fragments (2–20 μm). Shear thickening properties were attributed largely to these soft fragment particles colliding and sliding past each other during shear. The data from this study has also shown that the hydrogen bonding, electrostatic, hydrophobic interactions, or the combination of these interactions did not cause the shear-thickening behaviour. The influence of 4% w/w DWPS on 13% w/w WPI gels was studied by characterising the phase stability of the liquid mixtures, and mechanical properties, microstructure, and water-immersion stability of fine-stranded polymeric and coarse-stranded particulate protein gels at pH 7 and pH 5, respectively. At neutral pH, synergistic gel hardness of WPI was obtained with the incorporation of 140 °C DWPS. The increased gel strength was attributed to the enhanced density of a very fine-stranded gel network. The ability of the gel to retain its shape when immersed in water for 40 h was most noticeable for the composite gels containing either gelatinised starch or DWPS samples (swollen gels but with intact shape). In contrast, pure WPI gel and composite gel containing maltodextrin turned into very weak fluid-like and disintegrated gels, respectively. At pH 5, WPI formed particulate gels. The addition of gelatinised starch or DWPS weakened the particulate protein gels, likely due to phase separation and interrupted protein network with starch polymers acting as inactive fillers. The effects of NaCl and CaCl₂ (i.e., type of salts and ionic strength) on the mechanical and microstructural properties of composite gels containing 13% w/w WPI and 4% w/w 140 °C DWPS were also evaluated. Thermodynamic incompatibility between WPI and 140 °C DWPS was observed upon the addition of NaCl (~75 mM) or CaCl₂ (10–75 mM). The combined effects of such thermodynamic incompatibility with the changes in protein connectivity induced by varied ionic strength led to the formation of distinctive gel structures (inhomogeneous self-supporting gels with a liquid centre and weak gels with paste-like consistency) that were different from thermodynamic compatible homogeneous self-supporting gels (pure WPI and WPI + maltodextrin gels). At ≥ 250 mM NaCl, instead of a paste-like texture, a recovered soft self-supporting gel structure was observed when using 140 °C DWPS. The ability to generate a range of textures in WPI gelation-based foods by using 140 °C DWPS under different ionic conditions, is a feasible strategy for structuring high-protein foods for dysphagia—aimed to be either thickened fluids or soft solids. Additionally, this acquired knowledge is also relevant when formulating food gels for 3-D printing. The desirable rheological properties of DWPS samples and their ability to alter WPI gel structure signify the potential of DWPS as a clean-label ingredient to structure foods of specific needs (e.g., whipping cream for enhanced structure upon shear and high-protein foods for dysphagia sufferers).Item Unravelling the molecular contributions to collagen higher order structure : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand(Massey University, 2019) Visser, Danielle ReneeAbnormal levels of cross-linking in fibrillar collagen strands have been shown to cause a number of human and animal diseases. Cross-linking is a vital step in fibrillogenesis and contributes greatly to the structural integrity of collagenous tissues. Conversely, defects in cross-link formation can significantly alter fibrillar organisation and lead to pathogenesis. Because collagen cross-links form on collagen-specific hydroxylated lysine residues, an understanding of the link between hydroxylysine and cross-link concentrations is needed to determine whether the level of hydroxylysine, the stereochemistry of these hydroxylysine residues, or other post-translational modifications such as glycosylation affect the level of cross-linking in tissue. While some research has been done to elucidate the connection between the two in different tissue types from the same animal, little has been undertaken to relate hydroxylation and glycosylation of lysine and hydroxylysine to the concentration and types of cross-links in different species. Furthermore, no research has been done to compare the relative distribution of diastereomers of hydroxylysine even within the same species. In order to make a valid comparison, collagen needs to be purified from skin to a high degree and separated into different collagen types and sub- structures as much as possible. To achieve this, the extraction and purification of collagen from the skins of four different mammalian species displaying different skin tensile strengths has been optimised. Different extraction methods were used to prevent the loss of specific features of the collagens that were characterised that may otherwise be lost. Amino acid analysis revealed that while the ratios of the two hydroxylysine diastereomers differed between different animals and extraction methods, the differences were not significant. Mass spectral analysis of cross-links showed that goat skin differed from the other three animals in its cross-link profile. Amino acid analysis combined with mass spectral analysis revealed that on average 70% of proline residues were hydroxylated, a figure much higher than previously thought. Mass spectral analysis also revealed that there are some differences between the glycosylation pattern of different animals, and the ratios of the different types of collagen which are extracted from each animal. While these findings need to be confirmed, they challenge some long held beliefs about the collagen molecule and provide a firm foundation for future work.Item Use of small angle x-ray scattering in investigations of leather and the cornea : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Manawatū, New Zealand(Massey University, 2018) Kelly, S. J. R.Collagen is the most abundant protein in the body and the major structural component of skin and the cornea, where it provides strength and is an important physical and chemical barrier against the environment. The biological function of collagen lies predominantly in its mechanical properties where its structural arrangement greatly influences the tissue characteristics. Understanding collagen structure, its properties and how these are affected by processing, is essential for the manufacture of skin products with superior function and when considering collagen in abnormal corneal tissue. Leather is derived from skins of various animals, providing aesthetically pleasing products that are strong and hard wearing because of their collagen structure. Collagen is comprised of fibrils which have been studied here in leather produced from skins of ovine (sheep), bovine (cattle) and cervine (deer) origins. Small angle X-ray scattering (SAXS) was used to evaluate the collagen fibril structure and alignment in leather, processed normally and by stretch-tanning, along with tear and bend testing. The average collagen fibril direction at standard sampling points in all species was perpendicular to the backbone, with the average fibril orientation relative to the backbone being 44° in cervine, 66° in bovine and 79° in ovine. The orientation index (OI) suggests the relative alignment of the fibrils, where 1 is perfectly aligned and 0 is randomly aligned. The OI was lowest in cervine (0.24), suggesting a more mesh-like arrangement, increasing in bovine (0.38) and highest in ovine (0.44) where fibrils lay more parallel to one another. There was considerable and unpredictable variability in collagen arrangements in each species but a significant difference in tear strength with ovine leather (21 N/mm) being weakest, and cervine leather (53 N/mm) stronger than bovine leather (43 N/mm), making ovine leather not suitable for high value applications like footwear. Previous correlations between leather strength and fibril alignment suggest greater alignment led to greater strength. When fibrils were aligned artificially by stretch-tanning, the OI in ovine leather increased from 0.48 to 0.79 as did the strength from 27 to 43 N/mm, making it comparable to bovine leather strength. Measurements of the bend modulus of stretch-tanned ovine leather, which was stiffer than the non-stretch tanned leather (15 vs. 34 kPa), when conditioned under increasing relative humidity environments, during which water was incorporated into leather’s collagen structure, resulted in a 66% reduction in stiffness. Examination of clinically normal sheep corneas were used to determine effects of common preservatives on collagen structures using SAXS. Compared to the control, frozen cornea, there was a significant increase in the fibril diameter and D-spacing of collagen in corneas stored for 5 days in all the preservatives studied (5% glutaraldehyde, 10% formalin, Triton X and phosphate buffered saline). Corneas from cats with corneal opacities (Florida spots) that were studied using histology, transmission electron microscopy (TEM) and SAXS showed that there was less collagen in the stroma of the lesions. Here collagen fibrils had larger and more variable diameters (32 nm vs. the normal 27 nm), and a greater relative alignment (OI) compared to normal corneas (0.43 vs. 0.29, respectively). These changes explain the opacity of the lesions as corneal transparency depends on regular small fibril diameters which are aligned orthogonally. The above studies have demonstrated the usefulness of SAXS in characterizing collagen in natural, pathological, and mechanically and chemically altered collagen-based samples.Item Primary structure study of phosphofructokinase from Streptococcus lactis : a thesis presented [in] fulfilment of the requirement for the Master of Science in biochemistry at Massey University(Massey University, 1990) Qian, XiaoPhosphofructokinase is an important regulatory enzyme in the glycolytic pathway catalysing the phosphorylation of Fructose-6-phosphate to Fructose-1, 6-bisphosphate. Phosphofructokinase from Streptococcus lactis was isolated by chromatographic methods including DEAE Cellulose ion-exchange and Cibacron Blue Sepharose dye-ligand chromatographies. Phosphofructokinase from Streptococcus lactis was digested with trypsin, chymotrypsin, Staphylococcus aureus V8 protease and CNBr and the peptides obtained were sequenced and aligned against the sequences of Escherichia coli and Bacillus stearothermophilus phosphofructokinases. 326 out of 328 amino acid residues of Streptococcus lactis phosphofructokinase were obtained in this study. The comparison of Streptoccus lactis phosphofructokinase with Escherichia coli and Bacillus stearothermophilus phosphofructokinases showed that the sequence similarities among them are above 50%. Most of the secondary structures are conserved in Streptococcus lactis phosphofructokinase. The two 2-helices at the carboxyl terminal of bacterial phosphofructokinases are longer in Streptococcus lactis than in Escherichia coli and Bacillus stearothermophilus. The residues involved in binding of Fructose-6-phosphate/Fructose-1, 6-bisphosphate are the same in the bacterial phosphofructokinase and the residues involved in binding of ATP/ADP and binding of effectors have high degree of similarity.Item Role of N-terminal domains of p400 ATPase in the ATM interaction and DNA damage response : a thesis presented in partial fulfillment of the requirements for a the degree of Master of Science (MSc) in Genetics at Massey University, Manawatū, New Zealand(Massey University, 2016) Weber, Lauren ElizabethEfficient repair of damaged DNA and preservation of genomic integrity is integral in the maintenance of proper cellular function and prevention of unrestricted cell proliferation. One critical threat to the stability of the genome is the double strand break (DSB), arguably one of the most cytotoxic lesions to DNA. Interference with the DSB repair mechanism can lead to dysregulation of cellular systems and the prospective development of malignancies. Two critical proteins in DBS repair are the Ataxia Telangiectasia Mutated (ATM) kinase, a serine/threonine kinase from the Phosphatidylinositol 3-Kinase-related Kinase (PIKK) family, and p400, an ATPase chromatin remodeler. ATM is one of the first responders to DSBs and is responsible for the phosphorylation of a multitude of protein substrates including the histone variant H2AX. Beyond its phosphorylation ability, ATM has been proposed as a potential shuttle for other repair machinery, aiding in the early and efficient recruitment of proteins to the DNA damage foci. One such proposed protein is p400. The exact role of p400 in DSB repair is unknown but previous studies show that there is a decrease in repair efficiency in its absence. A prospective interaction is supported by previous studies in which p400 and p400 N-terminal derivatives co-immunoprecipitate with ATM in vivo in HEK293T cells. This study aimed to confirm the interaction of ATM and p400 N-terminal derivatives in vitro and explore the functional implications of the association in vivo in U2OS cells. It was not possible to isolate full-length p400 derivatives in vitro and thus no conclusive results were obtained. Functional assays revealed the ability of one p400 fragment, F1, to inhibit DNA repair and cell proliferation after DNA double-strand break induction with bleomycin. Ectopic expression of the other two p400 N-terminal fragments, F2 and F3, induced an inhibition of cell proliferation under standard growth conditions. Although no conclusive results were acquired, a trend emerged suggesting that N-terminal fragment F1 is able to interfere with ATM protein-protein interactions resulting in a decrease in the efficiency of the DNA damage response and repair. These results implicate F1 as a potential target for further research in both DNA repair and cancer therapy.Item The structure and performance of collagen biomaterials : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering, Massey University, Palmerston North, New Zealand(Massey University, 2016) Wells, HannahType I collagen materials are used in a wide range of industrial applications. Some examples include leather for shoes and upholstery, acellular dermal matrix (ADM) materials for surgical applications, and bovine pericardium for the fabrication of heart valve replacements. The structure of these materials is based on a matrix of collagen fibrils, largely responsible for the physical properties and strength of the materials. How the collagen fibrils themselves contribute to the overall bulk properties of these materials is not fully understood. The first part of this work investigates a collagen structure defect in leather, known as looseness. Looseness occurs in around 5-10% of bovine leather, and is a result of the collagen fibril layers separating during processing from raw kkin to leather. A greater understanding of why looseness develops in leather and a method of detecting looseness early in processing is needed to save tanners a significant amount on wasted processing time and costs. In addition, an environmentally safe method of disposing of defect and waste leather is sort after since the current method of disposing to landfill is causing environmental concern due to the possibility of chromium leaching from leather into the soil as it biodegrades. Synchrotron based small angle X-ray scattering (SAXS) revealed that loose leather has a more aligned and layered collagen fibril arrangement, meaning there is less fibril overlap, particularly in the grain-corium boundary region. This results in larger gaps in the internal structure of loose leather compared with tight. These gaps could be detected using ultrasonic imaging in partially processed pickle and wet-blue hides as well as leather. Incorporating an ultrasound system into the leather processing line could be a viable method for identifying hides deemed to develop looseness earlier in processing, and these could be diverted down a separate processing line or removed. Disposing of waste leather by first forming biochar prior to land fill proved to be an effective way of reducing chromium from leaching into the environment. XAS revealed that heating leather to temperatures above 600°C in the absence of oxygen formed a char where chromium was bound in the stable form of chromium carbide. The stability of this structure makes chromium less available to form the toxic hexavalent form in the environment and presents a possible alternative option for environmentally safe disposal of leather. The second part to this work looks at the correlation between collagen fibril structure in a range of biomaterials in relation to material strength. Leather, ADM and pericardium are three type I collagen based materials which rely on sufficient strength to carry out their industrial and medical applications. These three materials were studied to try and identify collagen fibril characteristics that relate to high material strength. SAXS on a range of leather samples from various species revealed that collagen fibril diameter had only a small influence over material strength in bovine leather, and no correlation to strength in leather from other species. Therefore it can be said that the influence of fibril orientation on leather strength takes precedence over that of fibril diameter. Fibril diameter, d-spacing and orientation were studied in pericardium using SAXS while simultaneously applying strain. It was revealed collagen materials undergo two distinct stages of deformation when strain is applied and incrementally increased. The first stage, at low strain, involves a re-orientation of fibrils to become more aligned. When strain is increased further, the fibrils themselves take up the strain, causing fibrils to stretch and decrease in diameter. The Poisson ratio of the collagen fibrils was calculated to be 2.1 ± 0.7. This high Poisson's ratio indicates the fibrils decrease in diameter at a faster rate than they elongate with strain, and as a result the volume of the fibrils decreases. This feature of collagen could help explain some of the unique behaviours and strength of collagen based materials and could be useful for optimizing industrial applications of collagen materials. ADM materials, derived from human, porcine and bovine skin was the third collagen material studied. SAXS revealed that each species of ADM material had a slightly different collagen fibril arrangement when viewing the samples perpendicular to the surface. Human ADM was highly isotropic in arrangement, porcine was largely anisotropic, and bovine was somewhere in between the two. Bovine has a more layered fibril arrangement edge on and was the strongest material, followed by human ADM, and porcine was significantly weaker. Bovine was also the most porous material of the three. The discovery of the variations in strength, porosity and fibril arrangement between the three types of ADM materials may help medical professionals select the most suitable material for specific surgical procedures and could lead to a greater number of successful surgeries taking place.Item The effects of cross linking on collagen type 1 nanostructure and nanostructural response to uniaxial tension : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Manawatu, New Zealand(Massey University, 2016) Kayed, HananCollagen type I, is a fibrillar protein with a complex hierarchical structure, forming the extracellular matrices of an extensive range of organs and tissues. Applications for treated collagen materials vary vastly from commercial uses to the medical field for bioprosthetics and tissue grafts. Glycosaminoglycan (GAG), cross links naturally bridge fibrils, whilst glutaraldehyde is widely used as a synthetic linking agent in medical and other industries. No consensus has been reached regarding what contribution, if any, such cross links have on collagen structure and mechanical responses to applied stresses. This research investigated the role of GAG and glutaraldehyde cross links on the nanostructure and nanostructural response of type I collagen fibrils under uniaxial strain. Bovine pericardium was decellularised, producing native samples, or further treated with glutaraldehyde or chondroitinase ABC to produce glutaraldehyde cross linked or GAG-depleted collagen samples respectively. Synchrotron small angle X-ray scattering (SAXS), and atomic force and polarised light microscopy provided quantitative and qualitative information on collagen nanostructure. Uniaxial tensile experiments in conjunction with SAXS were performed to monitor structural changes with applied strain. Glutaraldehyde cross links constrained fibrils into more networked isotropic structures and demonstrated a mechanical function, recruiting 45% of fibrils into stretching which experienced strains of up to 6.4%. Comparison of native with chondroitinase ABC-treated samples showed GAGs do not constrain fibrils into alignment and have potential fibril lubricating effects; 12% of fibrils in native tissue experienced strains up to 4.1%, and 36% of fibrils experienced strains up to 4.6% in the GAG-depleted tissue. A higher degree of fibril sliding occurs in native tissue. Interestingly, whilst adult pericardia are more cross linked and fibrils of neonatal pericardia are more aligned, both tissues share similar propensities to form more isotropic structures with glutaraldehyde treatment. These findings build a comprehensive picture as to the function cross linking has in collagen structure and mechanical response at the nano-level, where such knowledge may prove useful for the preparation of collagen materials for specific applications.Item Structural studies on hydrophobins from Neurospora : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy(Massey University, 2004) Winefield, Robert DouglasBackground: Hydrophobins are a group of low molecular weight, cysteine-rich, fungal cell-wall proteins with unique biophysical properties. Principal among these is the ability of hydrophobin monomers to self-assemble into insoluble, chemically resistant amphipathic films at the interface between hydrophobic and hydrophilic surfaces. This enables fungi to coat their hyphae and fruiting bodies with a hydrophobic layer that prevents these structures from becoming waterlogged. Proposed industrial and medical applications have sought to exploit these protein's polymeric hydrophobins to reverse the wettability of a surface upon binding. The hydrophobin protein EAS (product of the gene eas) coats macroconidia produced by the model ascomycete Neurospora crassa, making this species an ideal subject for structural studies on hydrophobins. Results: (1) Genes homologous to eas were detected in each of the Neurospora species examined. EAS proteins isolated from each of the conidiating species proved to be identical to that known in N. crassa. The aconidiate homothallic Neurospora species also possess copies of eas, essentially identical to that from N. crassa, but transcription studies implied that the gene is inactive in these species. (2) I attempted to express EAS in its native form and I succeeded in generating recombinant Pichia pastoris and Escherichia coli as isolates. However, I did not detect the expression of EAS in any of these isolates. This was despite the fact that the Pichia isolates were actively transcribing the recombinant gene. (3) EAS was chemically digested according to Wu and Watson (1997). Mass spectrometric analysis of these digests revealed that the four intramolecular disulfide bridges in EAS exist between Cys9-Cys60, Cys18-Cys54, Cys19-Cys45, and Cys61-Cys80. This arrangement is identical to that recently determined for the class II hydrophobin HFB2. (4) Atomic force microscopic analysis of rodlet films deposited on hydrophilic mica and hydrophobic graphite revealed the presence of a central cleft in the hydrophobic and hydrophilic sides of individual rodlets. This cleft is believed to be the boundary between the long protofilaments that are bundled together to form polymeric rodlets. Also seen were shorter oval structures, consistent with short protofilaments detected during real-time analysis of amyloid polymerisation.