Temperature influences the interaction of ruminant PrPTSE with soil

Ovine scrapie and cervid chronic wasting disease can be transmitted in the absence of animal-to-animal contact, and environmental reservoirs of infectivity have been implicated in their spread and persistence. Investigating environmental factors that influence the interaction of disease-associated PrP with soils is imperative to understanding what is likely to be the complex role of soil in disease transmission. Here, we describe the effects of soil temperature on the binding/desorption and persistence of both ovine scrapie- and bovine BSE-PrP^TSE. Binding of PrP^TSE to a sandy loam soil at temperatures of 4°C, 8–12°C and 25–30°C demonstrated that an increase in temperature resulted in (1) a decrease in the amount of PrP^TSE recovered after 24 h of interaction with soil, (2) an increase in the amount of N-terminal cleavage of the prion protein over 11 d and (3) a decrease in the persistence of PrP^TSE on soil over an 18 mo period.     

Can plants serve as a vector for prions causing chronic wasting disease?

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrP^TSE) for 24 h in three replicate studies with PrP^TSE in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrP^C) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrP^TSE) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrP^TSE from the roots to the stem to be detectable by the methods employed. Undigested PrP^TSE did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.     

Plasminogen-Based Capture Combined with Amplification Technology for the Detection of PrPTSE in the Pre-Clinical Phase of Infection

Background

Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP^TSE) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP^TSE concentrations in the femtomolar range.

Methodology/Principal Findings

We have developed a three-step assay that firstly captures PrP^TSE from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP^TSE detection by western blot. We achieved a PrP^TSE capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP^TSE in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP^TSE in human plasma spiked with a 10⁻⁸ dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP^TSE in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

Conclusion/Significance

We have developed a sensitive and specific amplification assay allowing the detection of PrP^TSE in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP^TSE in blood of patients displaying positivity in large scale screening tests.     

Cellular prion protein in human plasma–derived extracellular vesicles promotes neurite outgrowth via the NMDA receptor–LRP1 receptor system

Exosomes and other extracellular vesicles (EVs) participate in cell–cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α₂-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α₂-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrP^C) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrP^C-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor–related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R–LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrP^C. Finally, EVs harvested from rat astrocytes carried PrP^C and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrP^C with the NMDA-R–LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.     

Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrP^Sc. Real-time quaking-induced conversion can amplify very small amounts of PrP^Sc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD₅₀) is reached approximately 10¹⁰/g brain (values varies 10^8.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrP^Sc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD₅₀ unit was equivalent to 0.06–0.27 fg of PrP^Sc. The SD₅₀ values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.     

Persistence of F-Specific RNA Coliphages in Surface Waters from a Produce Production Region along the Central Coast of California

F+ RNA coliphages (FRNA) are used to source-track fecal contamination and as surrogates for enteric pathogen persistence in the environment. However, the environmental persistence of FRNA is not clearly understood and necessitates the evaluation of the survival of prototype and environmental isolates of FRNA representing all four genogroups in surface waters from the central coast of California. Water temperature played a significant role in persistence–all prototype and environmental strains survived significantly longer at 10°C compared to 25°C. Similarly, the availability of host bacterium was found to be critical in FRNA survival. In the absence of E. coli F_amp, all prototypes of FRNA disappeared rapidly with a D-value (days for one log reduction) of <1.2 d from water samples incubated at 25°C; the longest surviving prototype was SP. However, in the presence of the host, the order of persistence at 25°C was QB>MS2>SP>GA and at 10°C it was QB = MS2>GA>SP. Significant differences in survival were observed between prototypes and environmental isolates of FRNA. While most environmental isolates disappeared rapidly at 25°C and in the absence of the host, members of genogroups GIII and GI persisted longer with the host compared to members of GII and GIV. Consequentially, FRNA based source tracking methods can be used to detect phages from recent fecal contamination along with those that persist longer in the environment as a result of cooler temperatures and increased host presence.     

Dissociation of Prion Protein Amyloid Seeding from Transmission of a Spongiform Encephalopathy

Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar “prion-like” mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer''s disease. We investigated if misfolding and aggregation of corrupted prion protein (PrP^TSE) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrP^TSE from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrP^TSE. In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrP^TSE is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrP^TSE in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.     

THE INFLUENCE OF ENVIRONMENTAL TEMPERATURE ON THE UTILIZATION OF FOOD ENERGY IN BABY CHICKS

1. An optimum of environmental temperature is to be expected for the utilization of food energy in warm blooded animals if their food intake is determined by their appetite. 2. Baby chicks were kept in groups of five chicks in a climatic cabinet at environmental temperatures of 21°, 27°, 32°, 38°, and 40°C. during the period of 6 to 15 days of age. The intake of qualitatively complete food was determined by their appetite. Food intake, excretion, and respiratory exchange were measured. Control chicks from the same hatch as the experimental groups were raised in a brooder and were given the same food as the experimental chicks. The basal metabolism of each experimental group was determined from 24 to 36 hours without food at the age of 16 days. 3. The daily rate of growth increased with decreasing environmental temperature from 2.74 gm. at 40°C. to 4.88 gm. at 21°C. This was 4.2 to 6.5 per cent of their body weight. 4. The amount of food consumed increased in proportion to the decrease in temperature. 5. The availability of the food, used for birds instead of the digestibility and defined as See PDF for Structure showed an optimum at 38°C. 6. The CO₂ production increased from 2.95 liters CO₂ per day per chick at 40°C. to 6.25 liters at 21°C. Per unit of the 3/4 power of the body weight, 23.0 liters CO₂ per kilo^3/4 was produced at 40°C. and 43.4 liters per kilo^3/4 at 21°C. The CO₂ production per unit of 3/4 power of the weight increased at an average rate of approximately 1 per cent per day increase in age. The R.Q. was, on the average, 1.04 during the day and 0.92 during the night. 7. The net energy is calculated on the basis of C and N balances. A maximum of 11.8 Cal. net energy per chick per day was found at 32°C. At 21°C. only 6.9 Cal. net per day per chick was produced and at 40°C. an average of 6.7 Cal. 8. The composition of the gained body substance changed according to the environmental temperature. The protein stored per gram increase in body weight varied from 0.217 to 0.266 gm. protein and seemed unrelated to the temperature. The amount of fat per gram gain in weight dropped from a maximum of 0.153 gm. at 32°C. to 0.012 gm. at 21°C. and an average of 0.107 gm. at 40°C. The energy content per gram of gain in weight had its maximum of 2.95 Cal. per gm. at 38°C. and its minimum of 1.41 Cal. per gm. at 21°C. at which temperature the largest amount of water (0.763 gm. per gm. increase in body weight) was stored. 9. The basal metabolism increased from an average of 60 Cal. per kilo^3/4 at an environmental temperature of 40°C. to 128 Cal. per kilo^3/4 at 21°C. No indication of a critical temperature was found. 10. The partial efficiency, i.e. the increase in net energy per unit of the corresponding increase in food energy, seemed dependent on the environmental temperature, reaching a maximum of 72 per cent of the available energy at 38°C. and decreasing to 57 per cent at 21°C. and to an average of 60 per cent at 40°C. 11. The total efficiency, i.e. the total net energy produced per unit of food energy taken in, was maximum (34 per cent of the available energy) at 32°C., dropped to 16 per cent at 21°C., and to an average of 29 per cent at 40°C.     

Subcritical Water Hydrolysis Effectively Reduces the In Vitro Seeding Activity of PrPSc but Fails to Inactivate the Infectivity of Bovine Spongiform Encephalopathy Prions

The global outbreak of bovine spongiform encephalopathy (BSE) has been attributed to the recycling of contaminated meat and bone meals (MBMs) as feed supplements. The use of MBMs has been prohibited in many countries; however, the development of a method for inactivating BSE prions could enable the efficient and safe use of these products as an organic resource. Subcritical water (SCW), which is water heated under pressure to maintain a liquid state at temperatures below the critical temperature (374°C), exhibits strong hydrolytic activity against organic compounds. In this study, we examined the residual in vitro seeding activity of protease-resistant prion protein (PrP^Sc) and the infectivity of BSE prions after SCW treatments. Spinal cord homogenates prepared from BSE-infected cows were treated with SCW at 230–280°C for 5–7.5 min and used to intracerebrally inoculate transgenic mice overexpressing bovine prion protein. Serial protein misfolding cyclic amplification (sPMCA) analysis detected no PrP^Sc in the SCW-treated homogenates, and the mice treated with these samples survived for more than 700 days without any signs of disease. However, sPMCA analyses detected PrP^Sc accumulation in the brains of all inoculated mice. Furthermore, secondary passage mice, which inoculated with brain homogenates derived from a western blotting (WB)-positive primary passage mouse, died after an average of 240 days, similar to mice inoculated with untreated BSE-infected spinal cord homogenates. The PrP^Sc accumulation and vacuolation typically observed in the brains of BSE-infected mice were confirmed in these secondary passage mice, suggesting that the BSE prions maintained their infectivity after SCW treatment. One late-onset case, as well as asymptomatic but sPMCA-positive cases, were also recognized in secondary passage mice inoculated with brain homogenates from WB-negative but sPMCA-positive primary passage mice. These results indicated that SCW-mediated hydrolysis was insufficient to eliminate the infectivity of BSE prions under the conditions tested.     

Miscanthus Establishment and Overwintering in the Midwest USA: A Regional Modeling Study of Crop Residue Management on Critical Minimum Soil Temperatures

Miscanthus is an intriguing cellulosic bioenergy feedstock because its aboveground productivity is high for low amounts of agrochemical inputs, but soil temperatures below −3.5°C could threaten successful cultivation in temperate regions. We used a combination of observed soil temperatures and the Agro-IBIS model to investigate how strategic residue management could reduce the risk of rhizome threatening soil temperatures. This objective was addressed using a historical (1978–2007) reconstruction of extreme minimum 10 cm soil temperatures experienced across the Midwest US and model sensitivity studies that quantified the impact of crop residue on soil temperatures. At observation sites and for simulations that had bare soil, two critical soil temperature thresholds (50% rhizome winterkill at −3.5°C and −6.0°C for different Miscanthus genotypes) were reached at rhizome planting depth (10 cm) over large geographic areas. The coldest average annual extreme 10 cm soil temperatures were between −8°C to −11°C across North Dakota, South Dakota, and Minnesota. Large portions of the region experienced 10 cm soil temperatures below −3.5°C in 75% or greater for all years, and portions of North and South Dakota, Minnesota, and Wisconsin experienced soil temperatures below −6.0°C in 50–60% of all years. For simulated management options that established varied thicknesses (1–5 cm) of miscanthus straw following harvest, extreme minimum soil temperatures increased by 2.5°C to 6°C compared to bare soil, with the greatest warming associated with thicker residue layers. While the likelihood of 10 cm soil temperatures reaching −3.5°C was greatly reduced with 2–5 cm of surface residue, portions of the Dakotas, Nebraska, Minnesota, and Wisconsin still experienced temperatures colder than −3.5°C in 50–80% of all years. Nonetheless, strategic residue management could help increase the likelihood of overwintering of miscanthus rhizomes in the first few years after establishment, although low productivity and biomass availability during these early stages could hamper such efforts.     

THE TEMPERATURE CHARACTERISTIC OF RESPIRATION OF AZOTOBACTER

The temperature characteristic of respiration of Azotobacter vinelandii possesses a constant value of 19,330 ± 165 over the temperature range 20–30°C. This value is independent of pH, oxygen tension, age of culture, and other factors within the limits studied. The optimum temperature of respiration is 34–35°C., with limits at about 10° and 50°C.     

BOUND WATER IN MUSCLE

1. The amount of free unfrozen water, i.e. water acting as normal solvent, in frog''s muscle at temperatures below the initial freezing-point has been calculated from the vapour pressure isotherm of the muscle. 2. Significant amounts of free water are present at –20°C. The total amount of unfrozen water at –20°C. cannot, therefore, be taken as a measure of the bound water in muscle. 3. The calculated values of free water, when compared with experimentally determined values of total unfrozen water, indicate that the amount of bound water in muscle at various temperatures is small. 4. A temperature considerably below –20°C., roughly between –40° and –60°C., is required to freeze completely the free water in muscle.     

Red-Backed Vole Brain Promotes Highly Efficient In Vitro Amplification of Abnormal Prion Protein from Macaque and Human Brains Infected with Variant Creutzfeldt-Jakob Disease Agent

Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrP^TSE) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP^TSE in tissues and blood. Macaque vCJD PrP^TSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrP^TSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP^TSE. Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP^TSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP^TSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP^TSE was more permissive than human PrP^TSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP^TSE from brains of humans and macaques with vCJD. PrP^TSE signals were reproducibly detected by Western blot in dilutions through 10^-12 of vCJD-infected 10% brain homogenates. This is the first report showing PrP^TSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrP^TSE in vCJD-infected human and macaque blood.     

NMR Structure of the Human Prion Protein with the Pathological Q212P Mutation Reveals Unique Structural Features

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrP^C) conformer, denoted as infectious scrapie isoform or PrP^Sc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrP^Sc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β₂–α₂ loop region. This structure might provide new insights into the early events of conformational transition of PrP^C into PrP^Sc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β₂–α₂ loop and α₃ helix.     

A Model for Sclerotinia sclerotiorum Infection and Disease Development in Lettuce,Based on the Effects of Temperature,Relative Humidity and Ascospore Density

The plant pathogen Sclerotinia sclerotiorum can cause serious losses on lettuce crops worldwide and as for most other susceptible crops, control relies on the application of fungicides, which target airborne ascospores. However, the efficacy of this approach depends on accurate timing of these sprays, which could be improved by an understanding of the environmental conditions that are conducive to infection. A mathematical model for S. sclerotiorum infection and disease development on lettuce is presented here for the first time, based on quantifying the effects of temperature, relative humidity (RH) and ascospore density in multiple controlled environment experiments. It was observed that disease can develop on lettuce plants inoculated with dry ascospores in the absence of apparent leaf wetness (required for spore germination). To explain this, the model conceptualises an infection court area containing microsites (in leaf axils and close to the stem base) where conditions are conducive to infection, the size of which is modified by ambient RH. The model indicated that minimum, maximum and optimum temperatures for ascospore germination were 0.0, 29.9 and 21.7°C respectively and that maximum rates of disease development occurred at spore densities >87 spores cm⁻². Disease development was much more rapid at 80–100% RH at 20°C, compared to 50–70% RH and resulted in a greater proportion of lettuce plants infected. Disease development was also more rapid at 15–27°C compared to 5–10°C (85% RH). The model was validated by a further series of independent controlled environment experiments where both RH and temperature were varied and generally simulated the pattern of disease development well. The implications of the results in terms of Sclerotinia disease forecasting are discussed.     

Nuclear Magnetic Resonance Studies of Sodium Ions in Isolated Frog Muscle and Liver

Nuclear magnetic resonance (NMR) was used to determine Na⁺ complexing in muscle and liver (at 23°C) from bullfrogs (Rana catesbeiana) and to study the influence of temperature on Na⁺ complexing in muscle from leopard frogs (Rana pipiens). The Na⁺ complexed in muscle and liver was found to be 36.6 ± 4.6% and 66.1 ± 3.5% respectively. A temperature decrease from +34°C to -2°C results in a 20% decrease in the mobility of the free Na⁺ in the fresh muscle. This 20% decrease in mobility results in about 50% of the free Na⁺ at 34°C being complexed at the lower temperature.     

Biofilm Production Potential of Salmonella Serovars Isolated from Chickens in North West Province,South Africa

Bacterial biofilms have recently gained considerable interest in the food production and medical industries due to their ability to resist destruction by disinfectants and other antimicrobials. Biofilms are extracellular polymer matrices that may enhance the survival of pathogens even when exposed to environmental stress. The effect of incubation temperatures (25°C, 37°C, and 40°C) and Salmonella serotype on biofilm-forming potentials was evaluated. Previously typed Salmonella serotypes (55) isolated from the gut of chickens were accessed for biofilms formation using a standard assay. Salmonella Typhimurium ATCC 14028^TM and Salmonella Enteritidis ATCC 13076^TM (positive controls), Escherichia coli (internal control) and un-inoculated Luria Bertani (LB) broth (negative control) were used. The isolates formed no biofilm (11.86–13.56%), weak (11.86–45.76%), moderate (18.64–20.34%), strong biofilms (23.73–54.24%) across the various temperatures investigated. Serotypes, Salmonella Heidelberg and Salmonella Weltevreden were the strongest biofilm formers at temperatures (25°C, 37°C, and 40°C, respectively). The potential of a large proportion (80%) of Salmonella serotypes to form biofilms increased with increasing incubation temperatures but decreased at 40°C. Findings indicate that average temperature favours biofilm formation by Salmonella serotypes. However, the influence of incubation temperature on biofilm formation was greater when compared to serotype. A positive correlation exists between Salmonella biofilm formed at 25°C, 37°C and 40°C (p ≥ 0.01). The ability of Salmonella species to form biofilms at 25°C and 37°C suggests that these serotypes may present severe challenges to food-processing and hospital facilities.Key words: Salmonella, biofilm, biofilm production potential, crystal violet microtitre     

The Structural Architecture of an Infectious Mammalian Prion Using Electron Cryomicroscopy

The structure of the infectious prion protein (PrP^Sc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrP^Sc replicates by converting the non-infectious, cellular prion protein (PrP^C) into the misfolded, infectious conformer through an unknown mechanism. PrP^Sc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 ų, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.     

ON THE TEMPERATURE CHARACTERISTICS FOR FREQUENCY OF BREATHING MOVEMENTS IN INBRED STRAINS OF MICE AND IN THEIR HYBRID OFFSPRING. I

Young mice of a selected line of the dilute brown strain of mice exhibit over the range 15–25°C. (body temperature) a relation of frequency of breathing movements to temperature such that when fitted by the Arrhenius equation the data give a value for the constant µ of 24,000± calories or, less frequently, 28,000±. Young mice of an inbred albino strain show over the range 15–20°C. a value of µ = 34,000±, or, less frequently, 14,000±, with a critical temperature at about 20°C. and a value of µ = 14,000± above 20°C. The F₁ hybrids of these two strains, and the backcross generations to either parent strain, exhibit only those four values of the temperature characteristic observed in the parent strains and none other. One may therefore speak of the inheritance of the value of the constant µ, but the inheritance shows in this instance no Mendelian behavior. Furthermore there appears to be inherited the occurrence (or absence) of a critical temperature at 20°C. These experiments indicate the "biological reality" of the temperature characteristics.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.