Bonner sphere measurements of 241Am-B and 241Am-F neutron energy spectra unfolded using high-resolution a priori data.

High-resolution neutron energy spectra, covering the entire energy range of interest, for two standard radionuclide neutron sources ((241)Am-B and (241)Am-F) have been derived from Bonner sphere measurements by using high-resolution a priori data in the unfolding process. In each case, two a priori spectra were used, one from a two-stage calculation and also one from a combination of the calculated spectrum with a high-resolution measured spectrum. The unfolded spectra are compared with those published elsewhere and show significant differences from the ISO- and IAEA-recommended spectra for (241)Am-B and (241)Am-F, respectively. Values for the fluence-average energy and fluence-to-dose-equivalent conversion coefficients are presented for the new spectra, and the implications of the new spectra for the emission rates of the sources when measured by the manganese bath technique are also determined.

Bioprospecting keratinous materials.

The concept of bioprospecting for bioactive peptides from keratin-containing materials such as wool, hair, skin and feathers presents an exciting opportunity for discovery of novel functional food ingredients and nutraceuticals, while value-adding to cheap and plentiful natural sources. The published literature reports multiple examples of proline-rich peptides with productive bio-activity in models of human disease including tumour formation, hypertension control and Alzheimer's disease. Bioactive peptides have been identified from food and other protein sources however the bioactivity of keratin-related proteins and peptides is largely unknown. Considering the high representation of proline-rich peptides among proven bioactive peptides, the proline-rich character of keratinous proteins supports current research. A selection of mammalian (cow epidermis, sheep wool) and avian (chicken feather) keratinous materials were subjected to enzymatic hydrolysis using established processing methods. A bio-assay of determining inhibition of early stage amyloid aggregation involved using a model fibril-forming protein - reduced and carboxymethylated bovine K-casein (RCMk-CN) and quantitation of fibril development with the amyloid-specific fluorophore, Thioflavin T (ThT). The assay was fully validated for analytical repeatability and used together with appropriate positive controls. Peptide library products derived from chicken feather (n=9), sheep wool (n=9) and bovine epidermis (n=9) were screened in the fibril inhibition assay based on K-casein. 3 of 27 products exhibited interesting levels of bio-activity with regard to fibril inhibition. HPLC profiles provide an indication of the complexity of the assemblage of peptides in the three active products. We conclude the bioprospecting research using keratinous materials shows promise for discovery of useful bioactive peptides.

Recent developments in radionuclide neutron source emission rate measurements at the National Physical Laboratory.

This paper outlines the key features of the new manganese bath facility of the National Physical Laboratory and describes the measurements performed to characterise and validate the facility. Other developments have focused on the use of MCNP to calculate the fraction of neutrons captured by nuclei other than manganese and the fraction escaping from the bath. This has highlighted the large difference in the oxygen(n,alpha) capture fraction when different cross-section evaluations are applied. This can change the value obtained for the neutron emission rate of a source by up to 0.8%.

Characterization and utilization of a Bonner sphere set based on gold activation foils.

Bonner sphere (BS) sets which use activation foils as the central thermal neutron sensor have advantages over active BS systems in certain environments, for example, pulsed fields, or fields with high photon components. In such environments, they may be the only type of neutron spectrometer which can be used. This paper describes work, using both measurements and calculations, to validate the response functions for a BS set based on gold activation foils. As an illustration of the use of such a system, a measurement is described of the contaminant neutron spectrum in the treatment room of a 21 MV hospital linear accelerator providing photon beams for radiotherapy.

The content of 250Cf and 248Cm in 252Cf neutron sources and the effect on the neutron emission rate.

One of the most common radionuclide neutron sources used for the calibration of detectors is (252)Cf. However, these sources also contain (250)Cf, which is present in the material from which the sources are made, and (248)Cm, which is formed as the daughter of (252)Cf via alpha-decay. Both decay by spontaneous fission with longer half-lives than (252)Cf. Consequently, as the source becomes older, the emission rate does not follow the decay curve of (252)Cf. Fits have been made to emission rate measurements of (252)Cf sources at NPL spanning over 30 y to deduce their (250)Cf and (248)Cm content. The emission rate of a source can be significantly underestimated if the presence of (250)Cf and (248)Cm is not taken into account, and this has been investigated for a typical (252)Cf source. The importance of this problem to other calibration laboratories and users of (252)Cf sources is emphasised.

Modelling of neutron survey instrument performance and experimental validation of those calculated response data.

Three moderator-type neutron survey instruments have been modelled for energy and angle dependence of the response, in greater detail than before. These response data have been verified by comparison with published experimental measurements and measurements made specifically for this project. Influences on the instrument response have also been investigated. These have included its mode-of-use and perturbations caused by variations in the instrument manufacture. The implications of these new response data have been assessed by an extensive programme of folding the responses with workplace energy distributions.

Angle dependence of response characteristics of neutron survey instruments.

Neutron area survey instruments are designed to have an approximately isotropic response. In practice, the response cannot be perfectly isotropic for instruments that do not have spherical symmetry, and for all instruments it is modified by the inclusion of batteries, electronics, handles, etc. This affects the ability of the survey instrument to measure accurately an isotropic dose equivalent quantity. Measurements of the angle dependence of response for four of the most commonly used designs of survey instrument (Harwell 0949, Mark 7 NRM, NM2 and Studsvik 2202D) have been performed in a low-scatter room using radionuclide and monoenergetic neutron sources. The Monte Carlo code MCNP has been used to model the responses and to investigate their sensitivity to the polyethylene density, counting gas pressure and other manufacturing tolerances. Preliminary modelling results are presented here.

Practical implications of neutron survey instrument performance.

Improvements have been made to the Monte Carlo modelling used to calculate the response of the neutron survey instruments most commonly used in the UK, for neutron energies up to 20 MeV. The improved modelling of the devices includes the electronics and battery pack, allowing better calculations of both the energy and angle dependence of response. These data are used to calculate the response of the instruments in rotationally and fully isotropic, as well as unidirectional fields. Experimental measurements with radionuclide sources and monoenergetic neutron fields have been, and continue to be made, to test the calculated response characteristics. The enhancements to the calculations have involved simulation of the sensitivity of the response to variations in instrument manufacture, and will include the influence of the user and floor during measurements. The practical implications of the energy and angle dependence of response, variations in manufacture, and the influence of the user are assessed by folding the response characteristics with workplace energy and direction distributions.

A novel protocol to identify mutations in patients with wiskott-Aldrich syndrome.

Mutations of WASP (Wiskott-Aldrich syndrome protein) underlie the severe immunodeficiency/platelet disorder Wiskott-Aldrich syndrome (WAS) and its milder variant X-linked thrombocytopenia (XLT). The affected gene, a 12-exon structure on the X-chromosome, is expressed exclusively in blood cells. The encoded product WASP is a 502-amino-acid scaffolding protein that functions in stimulus-induced nucleation of actin filaments to form dynamic cell surface projections. To date, more than 150 mutations have been identified in 300 WAS/XLT kindred worldwide, generally through methodologies that include sophisticated exon screening steps such as single-strand conformation analysis. We report here a simpler protocol, which was designed for use in clinical settings to identify the mutations of newly diagnosed patients. The approach relies on directly sequencing amplified exons according to a staggered schedule based on statistical evaluation of previous cases. In a 2 1/2-year trial, samples from 28 consecutive patients were analyzed; these included 3 "blindly labeled" previously studied cases. The mutations that were identified include a broad spectrum (8 missense, 3 nonsense, 5 splice site mutations, 11 small insertion/deletions, 1 large deletion) and were broadly distributed (in 10 of the 12 exons). All mutations were verified and no discrepancies were encountered. Per patient, a mean of six DNA sequencing reactions and 6-7 h of staff effort sufficed for mutation identification and verification, indicating that the protocol is cost-effective. This cumulative experience demonstrates the suitability, reliability, and versatility of the new protocol.

Effect of the proteasome inhibitor ALLnL on cisplatin sensitivity in human ovarian tumor cells.

Small molecules suppressing proteasome function inhibit the post-translational ubiquitination of selected proteins. Ubiquitin H2A is an example of an abundant chromatin-associated protein that is known to be ubiquitinated, which is important for several proteins involved in the repair of DNA damage. We therefore studied the effect of the proteasome inhibitor, N-acetyl leucyl-leucyl norlucinal (ALLnL), on cisplatin sensitivity in three human ovarian tumor cell lines. The proteasome inhibitor ALLnL was administered for 4 h before cells were subsequently exposed to cisplatin for 1 h. Our results showed that ALLnL, at its respective IC20 concentration, increased cellular sensitivity to cisplatin in an additive manner in human ovarian cancer A2780, A2780/CP70, and OVCAR3 cells. We also demonstrated that ALLnL caused a 50% increase in total cellular accumulation of cisplatin, and reduced the rate of cisplatin efflux by about 50%. In addition, DNA damage levels were increased after ALLnL treatment. By contrast, DNA repair was inhibited 2 to 3-fold in ALLnL-pretreated cells, as compared to the controls. Furthermore, our study showed that ALLnL deubiquitinated nucleosomal histone H2A in these cells in a concentration-dependent fashion, as assessed by Western blot analysis. These data suggest that sublethal levels of exposure to agents that inhibit proteasome function may alter the subcellular pharmacology of platinum in human ovarian carcinoma cells.

In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding.

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.

Intermediate filament structure: hard alpha-keratin.

Structurally there are four classes of intermediate filaments (IF) with distinct but closely related axial organisations. One of these, hard alpha-keratin IF, has been studied to clarify several apparently exceptional features which include the number of molecules in the IF cross-section and the mode by which the axial organisation of its constituent molecules is stabilised. Using the dark-field mode of the STEM at the Brookhaven National Laboratory (USA) mass measurements were obtained from unstained IF isolated from hair keratin. The data thus obtained show that the number of chains in cross-section is about 30 (+/-3: standard deviation) and is very similar to the numbers determined in previous STEM experiments for the dominant filament type in other classes of IF (about 32). Furthermore, re-analysis of the low-angle equatorial X-ray diffraction pattern reveals, in contrast to earlier work, solutions that are compatible with the number of chains in cross-section indicated by the STEM data. The absence of the head-to-tail overlap between parallel molecules characteristic of most of IF may be compensated in hard alpha-keratin by a network of intermolecular disulfide bonds. It is concluded that native IF of hard alpha-keratin and desmin/vimentin--and probably many other kinds of IF as well--contain about 32 chains in cross-section, and that the axial structures of these various kinds of IF differ in small but significant ways, while generally observing the same basic modes of aggregation.

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.

Hair keratinization in health and disease.

The cells of the epidermis and its derivative, the hair follicle, undergo processes of terminal differentiation that involves the synthesis and assembly of classes of protein and enzymes to form the stratum corneum of the epidermis, and the hair fiber and its cuticle. Using genetic linkage and DNA sequencing methods, we now know that mutations in several genes encoding epidermal keratins or a transglutaminase enzyme cause ichthyosis-related diseases. Similar methods have now suggested that mutations in hair keratin genes underlie some cases of monilethrix, and a deficiency in a cuticle lipid metabolizing enzyme causes maple syrup urine disease. It is to be expected that further application of these methods will elucidate the molecular bases of other genetic hair diseases.

Investigation of structural proteins in human hair defects using anagen follicles.

An alternative to using hair specimens for the study of inherited hair shaft defects has been to explore protein compositions in the context of hair formation. Hair follicles were obtained from patients with a hair disorder and the presumptive hair shaft (PHS) separated by microdissection for protein solubilization and electrophoretic experiments aimed at providing a new basis to help explain the mechanism of hair shaft defects. Two-dimensional electrophoresis (fluorographs) of labelled extracts was used to examine the major hair structural proteins and other polypeptide(s) found associated with PHS extracts in normal and aberrant specimens. Changes in either the intermediate filaments (IFs) or matrix polypeptides were not normally found in defective PHS specimens. The polypeptides showing greatest variation were associated with a PHS-specific component which was recently found in normal specimens. The variation in this polypeptide was manifested as multiple spots of different molecular weights. An investigation of the role of PHS-associated polypeptides as a likely part of the hair cross-linking mechanisms, involved examination of a PHS extract from a Menkes' patient. The observations suggest that formation of hair fibre shaft defects may be related to the status of PHS-associated polypeptides which could in turn be influenced by the presence of copper in the hair follicle.

Locations of synthesis of hair structural proteins in human anagen follicles.

Live tissue containing cells of the presumptive hair shaft (PHS), was obtained by microdissection of human anagen hair follicles. Whole PHS, as well as PHS further dissected into three levels of hair development, were subsequently used in protein solubilization procedures. Single- and two-dimensional polyacrylamide gel electrophoresis (PAGE) of radiolabelled extracts, with fluorography, enabled comparison of hair and PHS keratin proteins. Fluorographs demonstrated the major classes of protein comprising the intermediate filaments (IF) and matrix of hair keratins. In addition, extracts from various levels of PHS tissue have provided direct evidence for the locations of synthesis of these protein moieties. Thus, IF and matrix proteins are synthesized sequentially in PHS. A previously unobserved polypeptide component, not present in hair extracts, has been identified and found to vary in relative proportions when compared with the major IF and matrix protein classes as PHS cells differentiate to form hair. In the absence of reliable methods for extraction of human hair, PHS provides an alternative source of material, despite the extra burden of follicle collection and specimen preparation. The procedures described provide a new basis for studying human hair defects and are potentially useful for comparing human hair proteins in a variety of situations.