Monitoring of clinical signs in goats with transmissible spongiform encephalopathies

Background  

As there is limited information about the clinical signs of BSE and scrapie in goats, studies were conducted to describe the clinical progression of scrapie and BSE in goats and to evaluate a short clinical protocol for its use in detecting scrapie-affected goats in two herds with previously confirmed scrapie cases. Clinical assessments were carried out in five goats intracerebrally infected with the BSE agent as well as five reported scrapie suspects and 346 goats subject to cull from the two herds, 24 of which were retained for further monitoring. The brain and selected lymphoid tissue were examined by postmortem tests for disease confirmation.     

Comparison of abnormal prion protein glycoform patterns from transmissible spongiform encephalopathy agent-infected deer,elk, sheep,and cattle

Analysis of abnormal prion protein glycoform patterns from chronic wasting disease (CWD)-affected deer and elk, scrapie-affected sheep and cattle, and cattle with bovine spongiform encephalopathy failed to identify patterns capable of reliably distinguishing these transmissible spongiform encephalopathy diseases. However, PrP-res patterns sometimes differed among individual animals, suggesting infection by different or multiple CWD strains in some species.     

T cells infiltrate the brain in murine and human transmissible spongiform encephalopathies

CD4 and CD8 T lymphocytes infiltrate the parenchyma of mouse brains several weeks after intracerebral, intraperitoneal, or oral inoculation with the Chandler strain of mouse scrapie, a pattern not seen with inoculation of prion protein knockout (PrP(-/-)) mice. Associated with this cellular infiltration are expression of MHC class I and II molecules and elevation in levels of the T-cell chemokines, especially macrophage inflammatory protein 1beta, IFN-gamma-inducible protein 10, and RANTES. T cells were also found in the central nervous system (CNS) in five of six patients with Creutzfeldt-Jakob disease. T cells harvested from brains and spleens of scrapie-infected mice were analyzed using a newly identified mouse PrP (mPrP) peptide bearing the canonical binding motifs to major histocompatibility complex (MHC) class I H-2(b) or H-2(d) molecules, appropriate MHC class I tetramers made to include these peptides, and CD4 and CD8 T cells stimulated with 15-mer overlapping peptides covering the whole mPrP. Minimal to modest K(b) tetramer binding of mPrP amino acids (aa) 2 to 9, aa 152 to 160, and aa 232 to 241 was observed, but such tetramer-binding lymphocytes as well as CD4 and CD8 lymphocytes incubated with the full repertoire of mPrP peptides failed to synthesize intracellular gamma interferon (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) cytokines and were unable to lyse PrP(-/-) embryo fibroblasts or macrophages coated with (51)Cr-labeled mPrP peptide. These results suggest that the expression of PrP(sc) in the CNS is associated with release of chemokines and, as shown previously, cytokines that attract and retain PrP-activated T cells and, quite likely, bystander activated T cells that have migrated from the periphery into the CNS. However, these CD4 and CD8 T cells are defective in such an effector function(s) as IFN-gamma and TNF-alpha expression or release or lytic activity.     

Neuronal cell death in transmissible spongiform encephalopathies (prion diseases) revisited: from apoptosis to autophagy

Neuronal autophagy, like apoptosis, is one of the mechanisms of the programmed cell death (PCD). In this review, we summarize the presence of autophagic vacuoles in experimentally induced scrapie, Creutzfeldt–Jakob disease and Gerstmann–Sträussler–Scheinker (GSS) syndrome. Initially, a part of the neuronal cytoplasm was sequestrated by concentric arrays of double membranes; the enclosed cytoplasm appeared relatively normal except that its density was often increased. Next, electron density of the central area dramatically increased. The membranes then proliferated within the cytoplasm in a labyrinth-like manner and the area sequestrated by these membranes enlarged into a more complex structure consisting of vacuoles, electron-dense areas and areas of normally-looking cytoplasm connected by convoluted membranes. Of note, autophagic vacuoles form not only in neuronal perikarya but also in neurites and synapses. Finally, a large area of the cytoplasm was transformed into a collection of autophagic vacuoles of different sizes. On a basis of ultrastructural studies, we suggest that autophagy plays a major role in transmissible spongiform encephalopathies (TSEs) and may even participate in a formation of spongiform change.     

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.     

Effect of amphotericin B on wild-type and mutated prion proteins in cultured cells: putative mechanism of action in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts remain on the nature of the responsible agent of these diseases, it is clear that a protein called PrP(Sc) [the scrapie isoform of prion protein (PrP)] plays a central role. PrP(Sc) represents a conformational variant of PrP(C) (the cellular isoform of PrP), the normal host protein. Polyene antibiotics, such as amphotericin B, have been shown to delay the accumulation of PrP(Sc) and to increase the incubation time of the disease after experimental transmission in laboratory animals. Unlike agents such as Congo red, the inhibitory effect of amphotericin B on PrP(Sc) generation has not been observed in infected cultures. Using transfected cells expressing wild-type or mutated mouse PrPs, we show here that amphotericin B is able to interfere with the generation of abnormal PrP isoforms in culture. Its action seems related to a modification of PrP trafficking through the association of this glycosylphosphatidylinositol-anchored protein with detergent-resistant microdomains. These results represent a first step toward the comprehension of the mechanism of action of amphotericin B in transmissible spongiform encephalopathies.     

Extracellular circulating nucleic acids in human plasma in health and disease

The concentration of extracellular DNA and RNA in blood plasma of healthy donors, trauma patients, patients with breast and lung cancer, nonmalignant breast tumors and nonmalignant lung diseases were estimated. Significant amounts of extracellular RNA were found in plasma of trauma patients. The concentration of DNA and RNA in plasma of trauma patients correlates with the extent of posttraumatic organ failure. Extracellular RNA was not found in the plasma of breast cancer patients and patients with nonmalignant breast tumors, whereas a very high concentration of extracellular RNA was found in patients with malignant and nonmalignant diseases of lung.     

Relationship between clinical signs and postmortem test status in cattle experimentally infected with the bovine spongiform encephalopathy agent

Background  

Various clinical protocols have been developed to aid in the clinical diagnosis of classical bovine spongiform encephalopathy (BSE), which is confirmed by postmortem examinations based on vacuolation and accumulation of disease-associated prion protein (PrP^d) in the brain. The present study investigated the occurrence and progression of sixty selected clinical signs and behaviour combinations in 513 experimentally exposed cattle subsequently categorised postmortem as confirmed or unconfirmed BSE cases. Appropriate undosed or saline inoculated controls were examined similarly and the data analysed to explore the possible occurrence of BSE-specific clinical expression in animals unconfirmed by postmortem examinations.     

Detection of circulating immune complexes by the enzyme-linked immunosorbent assay in sera from cattle infected with Fasciola hepatica

Polyethylene glycol-precipitated immune complexes (PIC) from the sera of 5 calves with Fasciola hepatica worm burdens ranging between 27 and 70 flukes were examined for parasite antigen content at 2, 4, 6, 8, 10, and 16 wk postinfection (PI) by the enzyme-linked immunosorbent assay (ELISA). Three assays were devised using an affinity-processed rabbit antibody to worm excretory/secretory (FhES) antigens. The PIC plate assay detected parasite antigen by adherence of anti-FhES antibody to PIC incubated overnight on ELISA plates, and tests were visualized using anti-rabbit peroxidase-linked antibody. The serum complex and PIC capture assay utilized the anti-FhES immunoglobulin as an antigen capture antibody linked to the solid phase. The attached complexes were then detected by the adhering bovine antibody, either soluble complexes in serum or as PIC. All assays showed circulating immune complex (CIC) values elevated at 6-8 wk PI, which generally coincided with increased host circulating antibody to FhES antigens. The greatest detection rate for all of the immune complex (IC) detection assays occurred with the PIC capture assay. It detected antigen in almost 90% of sera tested at 6 and 8 wk PI. Both the serum complex and PIC capture assay detected greatest amounts of CIC in those animals with the largest worm burdens, whereas the PIC plate assay showed no such trend. This study shows that F. hepatica antigen detection in CIC can be used to aid immunologic diagnosis of fascioliasis.     

Synthesis of nucleic acids and proteins in the appendix of chickens infected with Eimeria tenella]

A comparative study was carried out of the synthesis of DNA, RNA and protein in blind appendages of the intestine of healthy and infected with E. tenella chickens. It was established that the inclusion of P32 into DNA and C14 - orotic acid into RNA increases in 2-10 days after the infection. An intensive inclusion of C14-lysine into protein of blind appendages was observed in 2-6 days after the infection of chickens. Under the effect of the parasite an activation of metabolism of DNA, RNA and protein takes place in blind appendages of the host's intestine.     

A chromosomal rearrangement hotspot can be identified from population genetic variation and is coincident with a hotspot for allelic recombination

Insights into the origins of structural variation and the mutational mechanisms underlying genomic disorders would be greatly improved by a genomewide map of hotspots of nonallelic homologous recombination (NAHR). Moreover, our understanding of sequence variation within the duplicated sequences that are substrates for NAHR lags far behind that of sequence variation within the single-copy portion of the genome. Perhaps the best-characterized NAHR hotspot lies within the 24-kb-long Charcot-Marie-Tooth disease type 1A (CMT1A)-repeats (REPs) that sponsor deletions and duplications that cause peripheral neuropathies. We investigated structural and sequence diversity within the CMT1A-REPs, both within and between species. We discovered a high frequency of retroelement insertions, accelerated sequence evolution after duplication, extensive paralogous gene conversion, and a greater than twofold enrichment of SNPs in humans relative to the genome average. We identified an allelic recombination hotspot underlying the known NAHR hotspot, which suggests that the two processes are intimately related. Finally, we used our data to develop a novel method for inferring the location of an NAHR hotspot from sequence variation within segmental duplications and applied it to identify a putative NAHR hotspot within the LCR22 repeats that sponsor velocardiofacial syndrome deletions. We propose that a large-scale project to map sequence variation within segmental duplications would reveal a wealth of novel chromosomal-rearrangement hotspots.     

Intelligence can be detected but is not found attractive in videos and live interactions

Self-reported mate preferences suggest intelligence is valued across cultures, consistent with the idea that human intelligence evolved as a sexually selected trait. The validity of self-reports has been questioned though, so it remains unclear whether objectively assessed intelligence is indeed attractive. In Study 1, 88 target men had their intelligence measured and based on short video clips were rated on intelligence, funniness, physical attractiveness and mate appeal by 179 women. In Study 2 (N = 763), participants took part in 2 to 5 speed-dating sessions in which their intelligence was measured and they rated each other's intelligence, funniness, and mate appeal. Measured intelligence did not predict increased mate appeal in either study, whereas perceived intelligence and funniness did. More intelligent people were perceived as more intelligent, but not as funnier. Results suggest that intelligence is not important for initial attraction, which raises doubts concerning the sexual selection theory of intelligence.     

Carrier erythrocytes from cattle can be prepared from cells stored in a hypotonic, porous, dialyzed state

Hypotonic dialysis of bovine erythrocytes to 160-180 mosmol/kg produces cells with metastable pores. Cells stored for 3 weeks in a hypotonic dialyzed state can be used to prepare carrier erythrocytes. Pores generated by nonlytic hypotonic conditions are initially greater than 40 A in radius but shrink to less than 20 A with storage beyond 1 week. A metastable pore allows for hemoglobin isolation from hypotonically dialyzed cells.     

Difference in conformational diversity between nucleic acids with a six-membered 'sugar' unit and natural 'furanose' nucleic acids

Natural nucleic acids duplexes formed by Watson-Crick base pairing fold into right-handed helices that are classified in two families of secondary structures, i.e. the A- and B-form. For a long time, these A and B allomorphic nucleic acids have been considered as the 'non plus ultra' of double-stranded nucleic acids geometries with the only exception of Z-DNA, a left-handed helix that can be adopted by some DNA sequences. The five-membered furanose ring in the sugar-phosphate backbone of DNA and RNA is the underlying cause of this restriction in conformational diversity. A collection of new Watson-Crick duplexes have joined the 'original' nucleic acid double helixes at the moment the furanose sugar was replaced by different types of six-membered ring systems. The increase in this structural and conformational diversity originates from the rigid chair conformation of a saturated six-membered ring that determines the orientation of the ring substituents with respect to each other. The original A- and B-form oligonucleotide duplexes have expanded into a whole family of new structures with the potential for selective cross-communication in a parallel or antiparallel orientation, opening up a new world for information storage and for molecular recognition-directed self-organization.     

Relationship between temperature and the ratio of single-stranded to double-stranded nucleic acids in identified mollusk giant neurons

Using the microspectrofluorimetric technique, the temperature dependence of the synthetic activity of the nucleus and of some cytoplasmic areas in the giant identified neurons of Lymnaea stagnalis was investigated after Acridine Orange staining. The elevation of the environmental temperature from 4 degrees to 20 degrees C resulted in the increased synthetic activity of the nucleus and cytoplasm of giant neurons. In these conditions, the highest synthetic activity was registered in the apical area of the neuron cytoplasm, providing presumably, the cell neurosecretory activity. The synthetic activity of the axon perinuclear area, producing compounds for the axonal transport, increases at the elevated temperatures to a lesser degree, because it has a relatively high level as well at low temperatures.