Should Deceased Donors be Tested for vCJD?

The 1997 Bovine Spongiform Encephalopathy enquiry and the 2001 Hepatitis C litigation judgement set the UK scene for evoking the precautionary principle and the legal precedent that liability for defective transfusion products should not be dependent on medical negligence, but on the mere fact of defectiveness. Animal models indicate that vCJD in humans, with infection via the oral route, is likely to be associated with infectivity within the lymphoreticular system (LRS). This is likely to appear prior to the involvement of the central nervous system and thus infectivity is likely to be present in the LRS before the onset of clinical disease. A number of relevant epidemiological studies using LRS tissue have shown a low, but measurable, existence of the carrier state in vCJD. Two possible cases of transmission of the abnormal prion of vCJD by blood transfusion suggested that tissues might also transmit and that testing of LRS tissue from deceased potential tissue donors should be considered as a first measure towards the prevention of vCJD transmission by tissues designated for use in transplantation. A variety of different tissues could be used as representative of the LRS, but each is associated with problems of feasibility and practicality. Assays for vCJD have not been validated in the context of donor screening rather than epidemiological studies nor on deceased donors. However, given the number of vCJD cases in the UK, significant attention should be paid to the logistical, ethical, social and other issues associated with undertaking vCJD testing of tissue donors, with a view to introducing testing of deceased tissue donors for vCJD disease or latency.     

Antibodies and protein misfolding: From structural research tools to therapeutic strategies

Protein misfolding disorders, including the neurodegenerative conditions Alzheimer's disease (AD) and Parkinson's disease (PD) represent one of the major medical challenges or our time. The underlying molecular mechanisms that govern protein misfolding and its links with disease are very complex processes, involving the formation of transiently populated but highly toxic molecular species within the crowded environment of the cell and tissue. Nevertheless, much progress has been made in understanding these events in recent years through innovative experiments and therapeutic strategies, and in this review we present an overview of the key roles of antibodies and antibody fragments in these endeavors. We discuss in particular how these species are being used in combination with a variety of powerful biochemical and biophysical methodologies, including a range of spectroscopic and microscopic techniques applied not just in vitro but also in situ and in vivo, both to gain a better understanding of the mechanistic nature of protein misfolding and aggregation and also to design novel therapeutic strategies to combat the family of diseases with which they are associated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

William Borrer's lichens in the Supplement to the English Botany 1829–1866

Fifty-seven lichens were documented in Hooker's Supplement to the English Botany , published in five volumes from 1829 to 1866, when British lichenology was, until 1851, largely in a state of stagnation. These included 26 new lichens described by William Borrer, one of which, Verrucaria Hookeri (now Dacampia hookeri ), has proved to be a lichenicolous lichen. The merit of his work is discussed. One new species, namely Strigula Babingtonii , was described by the Reverend Miles Joseph Berkeley, but this is now regarded as a non-lichenized ascomycete fungus. An attempt was made to trace the whereabouts of all the type specimens, and only one could not be located. The current taxonomic status of the new species was investigated by the critical study of original references. No names require correction, but the authorities for Bacidia incompta (Borrer) Anzi and Verrucaria fuscella (Turner) Winch & Thornhill need emendation. The copyright dates of the plates are given, which should be accepted as those of publication.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 154 , 381–392.     

Two-rung model of a left-handed beta-helix for prions explains species barrier and strain variation in transmissible spongiform encephalopathies

In this study, a new beta-helical model is proposed that explains the species barrier and strain variation in transmissible spongiform encephalopathies. The left-handed beta-helix serves as a structural model that can explain the seeded growth characteristics of beta-sheet structure in PrP(Sc) fibrils. Molecular dynamics simulations demonstrate that the left-handed beta-helix is structurally more stable than the right-handed beta-helix, with a higher beta-sheet content during the simulation and a better distributed network of inter-strand backbone-backbone hydrogen bonds between parallel beta-strands of different rungs. Multiple sequence alignments and homology modelling of prion sequences with different rungs of left-handed beta-helices illustrate that the PrP region with the highest beta-helical propensity (residues 105-143) can fold in just two rungs of a left-handed beta-helix. Even if no other flanking sequence participates in the beta-helix, the two rungs of a beta-helix can give the growing fibril enough elevation to accommodate the rest of the PrP protein in a tight packing at the periphery of a trimeric beta-helix. The folding of beta-helices is driven by backbone-backbone hydrogen bonding and stacking of side-chains in adjacent rungs. The sequence and structure of the last rung at the fibril end with unprotected beta-sheet edges selects the sequence of a complementary rung and dictates the folding of the new rung with optimal backbone hydrogen bonding and side-chain stacking. An important side-chain stack that facilitates the beta-helical folding is between methionine residues 109 and 129, which explains their importance in the species barrier of prions. Because the PrP sequence is not evolutionarily optimised to fold in a beta-helix, and because the beta-helical fold shows very little sequence preference, alternative alignments are possible that result in a different rung able to select for an alternative complementary rung. A different top rung results in a new strain with different growth characteristics. Hence, in the present model, sequence variation and alternative alignments clarify the basis of the species barrier and strain specificity in PrP-based diseases.     

PRNP gene variation in Pakistani cattle and buffaloes

Bovine spongiform encephalopathy (BSE) is a neurodegenerative prion protein misfolding disorder of cattle. BSE is of two types, classical BSE and atypical BSE which in turn is of two types, H-type BSE and L-type BSE. Both H-type BSE and L-type BSE are primarily sporadic prion disorders. However, one case of H-type BSE has recently been associated with E211K polymorphism in the prion protein gene (PRNP). Two polymorphisms in the bovine PRNP are also associated with susceptibility to classical BSE: a 23 bp insertion/deletion (indel) in the PRNP promoter region and a 12 bp indel in the first intron. No information regarding BSE susceptibility in Pakistani cattle is available. The present study aimed at achieving this information. A total of 236 cattle from 7 breeds and 281 buffaloes from 5 breeds were screened for E211K polymorphism and 23 bp and 12 bp indels employing triplex PCR. The E211K polymorphism was not detected in any of the animals studied. The 23 bp insertion allele was underrepresented in studied cattle breeds while the 12 bp insertion allele was overrepresented. Both 23 bp and 12 bp insertion alleles were overrepresented in studied buffalo breeds. Almost 90% of alleles were insertion alleles across all studied buffalo breeds. The average frequency of 23 bp and 12 bp insertion alleles across all studied cattle breeds was found to be 0.1822 and 0.9407, respectively. There were significant differences between Pakistani and worldwide cattle in terms of allele, genotype and haplotype frequencies of 23 bp and 12 bp indels. The higher observed frequency of 12 bp insertion allele suggests that Pakistani cattle are relatively more resistant to classical BSE than European cattle. However, the key risk factor for classical BSE is the dietary exposure of cattle to contaminated feedstuffs.     

Posttranslational modification impact on the mechanism by which amyloid‐β induces synaptic dysfunction

Oligomeric amyloid‐β (Aβ) 1‐42 disrupts synaptic function at an early stage of Alzheimer's disease (AD). Multiple posttranslational modifications of Aβ have been identified, among which N‐terminally truncated forms are the most abundant. It is not clear, however, whether modified species can induce synaptic dysfunction on their own and how altered biochemical properties can contribute to the synaptotoxic mechanisms. Here, we show that a prominent isoform, pyroglutamated Aβ3(pE)‐42, induces synaptic dysfunction to a similar extent like Aβ1‐42 but by clearly different mechanisms. In contrast to Aβ1‐42, Aβ3(pE)‐42 does not directly associate with synaptic membranes or the prion protein but is instead taken up by astrocytes and potently induces glial release of the proinflammatory cytokine TNFα. Moreover, Aβ3(pE)‐42‐induced synaptic dysfunction is not related to NMDAR signalling and Aβ3(pE)‐42‐induced impairment of synaptic plasticity cannot be rescued by D1‐agonists. Collectively, the data point to a scenario where neuroinflammatory processes together with direct synaptotoxic effects are caused by posttranslational modification of soluble oligomeric Aβ and contribute synergistically to the onset of synaptic dysfunction in AD.     

Copper binding sites in the C-terminal domain of mouse prion protein: A hybrid (QM/MM) molecular dynamics study

We present a hybrid QM/MM Car-Parrinello molecular dynamics study of the copper-loaded C-terminal domain of the mouse prion protein. By means of a statistical analysis of copper coordination in known protein structures, we localized the protein regions with the highest propensity for copper ion binding. The identified candidate structures were subsequently refined via QM/MM simulations. Their EPR characteristics were computed to make contact with the experimental data and to probe the sensitivity to structural and chemical changes. Overall best agreement with the experimental EPR data (Van Doorslaer et al., J Phys Chem B 2001; 105: 1631-1639) and the information currently available in the literature is observed for a binding site involving H187. Moreover, a reinterpretation of the experimental proton hyperfine couplings was possible in the light of the present computational findings.     

The “Genetic Program”: Behind the Genesis of an Influential Metaphor

The metaphor of the “genetic program,” indicating the genome as a set of instructions required to build a phenotype, has been very influential in biology despite various criticisms over the years. This metaphor, first published in 1961, is thought to have been invented independently in two different articles, one by Ernst Mayr and the other by François Jacob and Jacques Monod. Here, after a detailed analysis of what both parties meant by “genetic program,” I show, using unpublished archives, the strong resemblance between the ideas of Mayr and Monod and suggest that their idea of genetic program probably shares a common origin. I explore the possibility that the two men met before 1961 and also exchanged their ideas through common friends and colleagues in the field of molecular biology. Based on unpublished correspondence of Jacob and Monod, I highlight the important events that influenced the preparation of their influential paper, which introduced the concept of the genetic program. Finally, I suggest that the genetic program metaphor may have preceded both papers and that it was probably used informally before 1961.     

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings. The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.