Donald E. Rivett

A Model for the Surface of Keratin Fibers

A model of the epicuticle membrane of keratin fibers shows that it is a heavily crosslinked protein containing approximately 25% by weight of fatty acid, predomi nantly 18-methyleicosanoic acid, acylated to the protein as a thioester. The conclusion that acylated fatty acids reside on the surface of and completely surround individual cuticle cells is supported by an analysis of the amount of fatty acid removed by alcoholic alkali against treatment time and the observed decreasing amount of bound fatty acid found as fiber diameters increase. Allworden sacs form only under acidic conditions, which also cleave bound fatty acids. Prior removal of bound fatty acids facilitates the rapid formation of Allworden sacs.

The role of 18-methyleicosanoic acid in the structure and formation of mammalian hair fibres

Although branched chain fatty acids perform many functions in biological systems, the importance of the anteiso 18 methyleicosanoic acid (MEA) has only recently been recognized. In this first review on MEA its role and distribution is explored. MEA has been found in minor amounts in the fatty acid components of a wide range of biological materials, but the current interest results from it being the major covalently bound fatty acid in mammalian hair fibres, a finding which is unusual because protein-bound fatty acids are typically straight-chain, even-numbered acids (C14-C18). MEA is released by surface restricted reagents indicating that it is located exclusively in or on the surface of the cuticle cells, a conclusion that has been verified by analysis of isolated cuticle cells, X-ray photoelectron spectroscopy (XPS) and secondary-ion mass spectroscopy (SIMS) studies support these results in that they show the surface of the cuticle to be predominantly hydrocarbon. When either neutral hydroxylamine or acidic chlorine solutions are applied to hair and wool fibres fatty acids are liberated, indicating the presence of thioester bonds. Calculations, based on fatty acid and amino acid analysis, indicate that approximately one residue in 10 of the cuticular membrane protein is a fatty acid thioester of cysteine. Removal of this covalently linked fatty acid renders the fibre hydrophilic, thus offering a chemical explanation for many technological and cosmetic treatments of mammalian fibres. Examination of the fibre surface and that of isolated cuticle cells by transmission electron microscopy (TEM) confirms the presence of a thin non-staining continuous layer surrounding the cuticle cells. Alkaline treatments which remove the bound fatty acids were found to disrupt this layer. TEM examination of developing hair fibres has indicated that the fatty acid layer on the upper surface and scale edges of the cuticle cell differs from that of the underside of the cell. Similar structural studies of hair from patients with maple syrup urine disease (MSUD) support the findings that thioester-bound MEA is limited to the upper surface of fibre cuticle cells. The current model proposed for the boundary layer consists of crosslinked protein with surface thioester-linked fatty acids, forming a continuous hydrophobic layer on the upper surface and scale edges of the cells.

Studies on the photooxidation of tryptophan

Abstract 1. 1. From the study of the photooxidation of tryptophan in three different aqueous systems, it has been concluded that the primary reaction is one which leads to the formation of formylkynurenine. Secondary reactions result in the formation of a variety of simpler amino acids together with a complex mixture of aromatic and polymeric materials. 2. 2. A reaction scheme has been proposed to explain the formation of the products detected.

The effect of sequence variations and structure on the cytolytic activity of melittin peptides

The importance of various amino acid residues in melittin for cytolytic function against mammalian cells was assessed by use of a monoclonal antibody to the C-terminal region, synthesis of peptide analogues and chemical modification of specific residues. A monoclonal anti-melittin antibody directed to the basic C-terminal region inhibited cytolytic activity. Consistent with this, deletion of one of the two Lys Arg sequences at the C terminal end of the peptide reduced cytolysis 8-fold, although significant activity was still present. A similar reduction in activity was also found with a synthetic analogue which had the reverse sequence to melittin. In contrast, when the last 6 residues of the C-terminal region were transferred to the N-terminus, a peptide with markedly reduced activity was obtained. Chemical modification of lysine and arginine residues of melittin indicated that lysine was only minimally important for functional activity compared with arginine which was essential. In particular, our results demonstrate that substitution of serine for lysine 7 has no significant effect on the activity of the peptide and suggest that this residue is important only in maintaining the amphipathic helix of the peptide.

Effects of Processing on the Bound and Free Fatty Acid Levels in Wool

Bound and free fatty acids in degreased wool fibers were affected to varying degrees by processing treatments. Scouring and dyeing both removed significant amounts of bound and free fatty acids from wool. Free fatty acids were reduced by dissolution into the treatment liquor, whereas bound fatty acids were hydrolyzed under the hot aqueous conditions. Chlorination at pH levels below 3 released over 50% of the bound fatty acids. Chlorine treatments cleave only thioesters but not oxygen esters or amides under these conditions, indicating that a significant proportion of the bound fatty acid is linked to wool by a thio ester bond.

Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin

BACKGROUND The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein. OBJECTIVES We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin. STUDY DESIGN The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human kappa myeloma and lymphoma cells, and on human free kappa Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2). RESULTS Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone. CONCLUSION The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.

A TRANSMISSION ELECTRON MICROSCOPE STUDY OF COVALENTLY BOUND FATTY ACIDS IN THE CELL MEMBRANES OF WOOL FIBERS

A transmission electron microscope study has shown that an unstained layer, thought to contain covalently bound fatty acids, completely surrounds the cuticle cells of wool fibers. Alcoholic alkali and chlorine treatments, which both release covalently bound fatty acids, result in the disappearance of the unstained layer. This layer is thought to be an integral part of the cuticle cell membrane. Similar unstained layers between cortical cells are different from the unstained cuticle membrane, because they remain unmodified by alcoholic alkali treatments.

In vitro mucosal digestion of synthetic gliadin-derived peptides in celiac disease

Two celiac-active synthetic peptides derived from the A-gliadin structure corresponding to residues 8–19 (LQPQNPSQQQPQ) and to 11–19 were digestedin vitro with small intestinal mucosa from children with celiac disease in remission and from normal children. The products of digestion were separated into two fractions on the basis of Mr<400 and Mr>400 by gel permeation chromatography and subjected to amino acid analysis. After digestion of the dodecapeptide with celiac mucosa, 71±14% (molar) of the total digestion products remained in the Mr>400 fraction. Glutamine, proline, serine, and asparagine were the major amino acids present. Glutamine, proline, and leucine were the major amino acids in the Mr<400 fraction. The Mr>400 fraction from the celiac mucosal digestion of the nonapeptide was of similar composition to the corresponding fraction from the dodecapeptide and represented 78±15% of the total products. Digestion of the two peptides with normal mucosa gave lower amounts of products in the Mr>400 fraction, but they were of similar composition to the corresponding fractions from the celiac mucosal digestion. Peptides such as NPSQQQP and QNPSQQQ may be present in the Mr>400 fractions since glutamine and proline are present in the approximate ratio of 2∶1, respectively. The results indicate a defect in the mucosal digestion of peptides which are active in an animal model of celiac disease.

A study of the electrochemistry of tryptophan, peptides containing tryptophan, and related compounds (indoles) at mercury electrodes

Abstract The electrochemistry of nineteen indole compounds of the general formula has been studied at mercury electrodes by differential pulse polarography, dc polarography, cyclic voltanunetry, cathodic stripping voltammetry, electrocapillary curves and controlled potential electrolysis in aqueous buffered media. Provided R1 = H, and the double bond is present between R2 and R3, a reversible oxidation process is observed at mercury electrodes which involves the formation of a mercury(II)-indole complex. Analysis of the wave shape, pH dependence, and concentration dependence of the oxidation process leads to the conclusion that the overall reaction is described by the equation 2InH + Hg ⇌ HgIn2 + 2H+ + 2 e− (process 1) where InH = indole with R1 = H. For some compounds a surface controlled second oxidation process is observed at more positive potentials than process one when high concentrations of indole are used. The presence of reactant and product adsorption is revealed by electrocapillary curves, cyclic voltammetric experiments and by the concentration dependence. Strong product adsorption can be used as the basis of a highly sensitive, but non-specific method for the determination of indole by differential pulse cathodic stripping voltammetry.

The presence of glycoproteins in the cell membrane complex of a variety of keratin fibres

Cell membrane complex preparations have been extracted using formic acid from human hair and nail, and from the hair of sheep, alpaca, rabbit, rat, cat, and dog. On analysis they were found to have similar amino acid compositions and they all contained carbohydrate. The sugars were typical of those found in membrane glycoproteins and all preparations reacted with peroxidase-conjugated lectins.