The susceptibility of disulfide bonds to modification in keratin fibers undergoing tensile stress.

Cysteine residues perform a dual role in mammalian hairs. The majority help stabilize the overall assembly of keratins and their associated proteins, but a proportion of inter-molecular disulfide bonds are assumed to be associated with hair mechanical flexibility. Hair cortical microstructure is hierarchical, with a complex macro-molecular organization resulting in arrays of intermediate filaments at a scale of micrometres. Intermolecular disulfide bonds occur within filaments and between them and the surrounding matrix. Wool fibers provide a good model for studying various contributions of differently situated disulfide bonds to fiber mechanics. Within this context, it is not known if all intermolecular disulfide bonds contribute equally, and, if not, then do the disproportionally involved cysteine residues occur at common locations on proteins? In this study, fibers from Romney sheep were subjected to stretching or to their breaking point under wet or dry conditions to detect, through labeling, disulfide bonds that were broken more often than randomly. We found that some cysteines were labeled more often than randomly and that these vary with fiber water content (water disrupts protein-protein hydrogen bonds). Many of the identified cysteine residues were located close to the terminal ends of keratins (head or tail domains) and keratin-associated proteins. Some cysteines in the head and tail domains of type II keratin K85 were labeled in all experimental conditions. When inter-protein hydrogen bonds were disrupted under wet conditions, disulfide labeling occurred in the head domains of type II keratins, likely affecting keratin-keratin-associated protein interactions, and tail domains of the type I keratins, likely affecting keratin-keratin interactions. In contrast, in dry fibers (containing more protein-protein hydrogen bonding), disulfide labeling was also observed in the central domains of affected keratins. This central "rod" region is associated with keratin-keratin interactions between anti-parallel heterodimers in the tetramer of the intermediate filament.

Wool fiber curvature is correlated with abundance of K38 and specific keratin-associated proteins.

Curvature in mammalian fibers, such as wool and human hair, is an important feature of the functional trait of coat structure-it affects mechanical resilience and thermo-insulation. However, to examine the relationship between fiber curvature, ultrastructure and protein composition fiber diameter variability has to be minimal. To achieve this we utilised the progeny of straight-wool domestic sheep mutant rams (crimp mutants) and wild-type ewes. Proteomic and structural results of the resulting mutant/wild-type twin pairs confirmed that straight crimp mutant wool had a normal cuticle and the same cortical protein and ultrastructural building blocks as wild-type (crimpy) fibers but differed in the layout of its cortical cells and in the relative proportions of keratin (K) and keratin-associated proteins (KAPs). In the case of the crimp mutants (straight fibers), the orthocortex was distributed in a fragmented, annular ring, with some orthocortical cells near the central medulla, a pattern similar to that of straight hairs from humans and other mammals. Crimp mutant fibers were noted for the reduced abundance of some proteins in the high glycine-tyrosine class normally associated with the orthocortex, specifically the KAP6, KAP7, and KAP8 families, while proteins from the KAP16 and KAP19 were found in increased abundance. In addition to this, the type I keratin, K38, which is also associated with the orthocortex, was also found at lower abundance in the mutant fibers. Conversely, proteins from the ultra-high sulfur class normally associated with the paracortex, specifically the KAP4 and KAP9 families, were found in higher abundance.

A detailed mapping of the readily accessible disulphide bonds in the cortex of wool fibres.

Trichocyte keratin intermediate filament proteins (keratins) and keratin associated proteins (KAPs) differ from their epithelial equivalents by having significantly more cysteine residues. Interactions between these cysteine residues within a mammalian fiber, and the putative regular organization of interactions are likely important for defining fiber mechanical properties, and thus biological functionality of hairs. Here we extend a previous study of cysteine accessibility under different levels of exposure to reducing compounds to detect a greater resolution of statistically non-random interactions between individual residues from keratins and KAPs. We found that most of the cysteines with this non-random accessibility in the KAPs were close to either the N- or C- terminal domains of these proteins. The most accessible non-random cysteines in keratins were present in the head or tail domains, indicating the likely function of cysteine residues in these regions is in readily forming intermolecular bonds with KAPs. Some of the less accessible non-random cysteines in keratins were discovered either close to or within the rod region in positions previously identified in human epithelial keratins as involved in crosslinking between the heterodimers of the tetramer. Our present study therefore provides a deeper understanding of the accessibility of disulfides in both keratins and KAPs and thus proves that there is some specificity to the disulfide bond interactions leading to these inter- and intra-molecular bonds stabilizing the fiber structure. Furthermore, these suggest potential sites of interaction between keratins and KAPs as well as keratin-keratin interactions in the trichocyte intermediate filament.

Application of MALDI-TOF analysis to reveal diversity and dynamics of winemaking yeast species in wild-fermented, organically produced, New Zealand Pinot Noir wine.

Rapid yeast identification is of particular importance in monitoring wine fermentation and assessing strain application in winemaking. We used MALDI-TOF MS analysis supported by 26 S rRNA gene sequence analysis and Saccharomyces-specific PCR testing to differentiate reference and field strains recovered from organic wine production facilities in Waipara, New Zealand, in which Pinot Noir wine was produced by spontaneous fermentations in the vineyard and in the winery. Strains were isolated from each of four key stages of each ferment to evaluate changes in taxonomic diversity. MALDI-TOF MS analysis was confirmed as an excellent yeast identification method, with even closely related Saccharomyces species readily distinguished. A total of 13 indigenous species belonging to eight genera were identified from Pinot Noir ferments, with taxonomic diversity generally reducing as fermentation progressed. However, differences between the taxa recovered were observed between the vineyard and winery ferments, despite the grapes used being from the same batch. Furthermore, some consistent proteomic differences between strains of S. cerevisiae, Hanseniasporum uvarum, Candida californica, Pichia membranifaciens and Starmerella bacillaris correlated with the different fermentation systems used. The high speed, low cost, taxonomic resolution and ability to characterise subtle changes in phenotype that may result from variations in environmental conditions makes MALDI-TOF analysis an attractive tool for further and wider applications in the wine industry. Such applications may include monitoring wine fermentation to actively support the consistency of high-quality wine products, and potentially for the development of such products too.

From Natural Xanthones to Synthetic C-1 Aminated 3,4-Dioxygenated Xanthones as Optimized Antifouling Agents.

Biofouling, which occurs when certain marine species attach and accumulate in artificial submerged structures, represents a serious economic and environmental issue worldwide. The discovery of new non-toxic and eco-friendly antifouling systems to control or prevent biofouling is, therefore, a practical and urgent need. In this work, the antifouling activity of a series of 24 xanthones, with chemical similarities to natural products, was exploited. Nine (1, 2, 4, 6, 8, 16, 19, 21, and 23) of the tested xanthones presented highly significant anti-settlement responses at 50 µM against the settlement of mussel Mytilus galloprovincialis larvae and low toxicity to this macrofouling species. Xanthones 21 and 23 emerged as the most effective larval settlement inhibitors (EC50 = 7.28 and 3.57 µM, respectively). Additionally, xanthone 23 exhibited a therapeutic ratio (LC50/EC50) > 15, as required by the US Navy program attesting its suitability as natural antifouling agents. From the nine tested xanthones, none of the compounds were found to significantly inhibit the growth of the marine biofilm-forming bacterial strains tested. Xanthones 4, 6, 8, 16, 19, 21, and 23 were found to be non-toxic to the marine non-target species Artemia salina (<10% mortality at 50 µM). Insights on the antifouling mode of action of the hit xanthones 21 and 23 suggest that these two compounds affected similar molecular targets and cellular processes in mussel larvae, including that related to mussel adhesion capacity. This work exposes for the first time the relevance of C-1 aminated xanthones with a 3,4-dioxygenated pattern of substitution as new non-toxic products to prevent marine biofouling.

A Multi-Bioassay Integrated Approach to Assess the Antifouling Potential of the Cyanobacterial Metabolites Portoamides.

The cyclic peptides portoamides produced by the cyanobacterium Phormidium sp. LEGE 05292 were previously isolated and their ability to condition microcommunities by allelopathic effect was described. These interesting bioactive properties are, however, still underexplored as their biotechnological applications may be vast. This study aims to investigate the antifouling potential of portoamides, given that a challenge in the search for new environmentally friendly antifouling products is to find non-toxic natural alternatives with the ability to prevent colonization of different biofouling species, from bacteria to macroinvertebrates. A multi-bioassay approach was applied to assess portoamides antifouling properties, marine ecotoxicity and molecular mode of action. Results showed high effectiveness in the prevention of mussel larvae settlement (EC50 = 3.16 µM), and also bioactivity towards growth and biofilm disruption of marine biofouling bacterial strains, while not showing toxicity towards both target and non-target species. Antifouling molecular targets in mussel larvae include energy metabolism modifications (failure in proton-transporting ATPases activity), structural alterations of the gills and protein and gene regulatory mechanisms. Overall, portoamides reveal a broad-spectrum bioactivity towards diverse biofouling species, including a non-toxic and reversible effect towards mussel larvae, showing potential to be incorporated as an active ingredient in antifouling coatings.

Expression and purification of high sulfur and high glycine-tyrosine keratin-associated proteins (KAPs) for biochemical and biophysical characterization.

Keratin-associated proteins (KAPs) were identified 70 years ago in wool follicles. KAPs are encoded by several multi-gene families and are classified into three different groups: ultra-high sulfur (UHS), high sulfur (HS) and high glycine-tyrosine (HGT). KAPs are the major constituent of the matrix between the hair keratin intermediate filaments (IFs), and stabilise hair structure by extensive disulfide bonding. However, detailed molecular structural information is lacking for KAPs and for KAP interactions with IFs. As a preliminary step towards their biophysical and structural characterization, we have expressed and purified a HS KAP (KAP11.1) and a HGT KAP (KAP6.1). The expression and purification of KAPs is challenging because they are cysteine-rich proteins with unusual amino acid compositions, they tend to be insoluble in isolation and are prone to forming aggregates in solution. Here we describe the high yield production of pure, soluble KAPs in a chaotrope- and detergent-free buffer. This method has the potential to be used for the overproduction of other KAPs and similar cysteine-rich proteins with high isoelectric points.

Diversity of Trichocyte Keratins and Keratin Associated Proteins.

Wool and hair fibres are primarily composed of proteins of which the keratins and keratin associated proteins (KAPs) are the major component. Considerable diversity is known to exist within these two groups of proteins. In the case of the keratins two major families are known, of which there are 11 members in the acidic Type I family and 7 members in the neutral-basic Type II family. The KAPs are even more diverse than the keratins, with 35 families being known to exist when the KAPs found in monotremes, marsupials and other mammalian species are taken into consideration. Human hair and wool are known to have 88 and 73 KAPs respectively, though this number rises for wool when polymorphism within KAP families is included.

Fibre Ultrastructure.

Mammalian hair fibres can be structurally divided into three main components: a cuticle, cortex and sometimes a medulla. The cuticle consists of a thin layer of overlapping cells on the surface of the fibre, constituting around 10% of the total fibre weight. The cortex makes up the remaining 86-90% and is made up of axially aligned spindle-shaped cells of which three major types have been recognised in wool: ortho, meso and para. Cortical cells are packed full of macrofibril bundles, which are a composite of aligned intermediate filaments embedded in an amorphous matrix. The spacing and three-dimensional arrangement of the intermediate filaments vary with cell type. The medulla consists of a continuous or discontinuous column of horizontal spaces in the centre of the cortex that becomes more prevalent as the fibre diameter increases.

The Follicle Cycle in Brief.

This chapter presents a very succinct overview of the cyclic biology of the hair follicle as it transitions from the quiescent telogen stage to the anagen stage in which hairs are actively produced before regressing through the catagen stage to telogen.