How does ionizing radiation affect amyloidogenesis in plants?

PURPOSE: Ionizing radiation is a harsh environmental factor that could induce plant senescence. We hypothesized that radiation-related senescence remodels proteome, particularly by triggering the accumulation of prion-like proteins in plant tissues. The object of this study, pea (Pisum sativum L.), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species. MATERIALS AND METHODS: Pea seeds were irradiated in the dose range 5-50 Gy of X-rays. Afterward, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated 3-day-old seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled the detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to predict putative prion-like proteins. RESULTS: We showed a reduced germination rate but higher plant height and faster appearance of reproductive organs in the irradiated at dose of 50 Gy group compared with the control; furthermore, we demonstrated more ß-sheets and amyloid aggregates in the roots of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 45 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, the discovery proteomics provided deep data on novel aspects of plant stress biology. CONCLUSION: Our data hinted that protein accumulation stimulated seedlings' growth as well as accelerated ontogenesis and, eventually, senescence, primarily through translation and RNA processing. The increased abundance of primary metabolism-related proteins indicates more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies.

Application study of infrared free-electron lasers towards the development of amyloidosis therapy.

Amyloidosis is known to be caused by the deposition of amyloid fibrils into various biological tissues; effective treatments for the disease are little established today. An infrared free-electron laser (IR-FEL) is an accelerator-based picosecond-pulse laser having tunable infrared wavelengths. In the current study, the irradiation effect of an IR-FEL was tested on an 11-residue peptide (NFLNCYVSGFH) fibril from ß2-microglobulin (ß2M) with the aim of applying IR-FELs to amyloidosis therapy. Infrared microspectroscopy (IRM) and scanning electron microscopy showed that a fibril of ß2M peptide was clearly dissociated by IR-FEL at 6.1 µm (amide I) accompanied by a decrease of the ß-sheet and an increase of the α-helix. No dissociative process was recognized at 6.5 µm (amide II) as well as at 5.0 µm (non-specific wavelength). Equilibrium molecular dynamics simulations indicated that the α-helix can exist stably and the probability of forming interchain hydrogen bonds associated with the internal asparagine residue (N4) is notably reduced compared with other amino acids after the ß-sheet is dissociated by amide I specific irradiation. This result implies that N4 plays a key role for recombination of hydrogen bonds in the dissociation of the ß2M fibril. In addition, the ß-sheet was disrupted at temperatures higher than 340 K while the α-helix did not appear even though the fibril was heated up to 363 K as revealed by IRM. The current study gives solid evidence for the laser-mediated conversion from ß-sheet to α-helix in amyloid fibrils at the molecular level.

Dissolution of a fibrous peptide by terahertz free electron laser.

Fibrous peptides such as amyloid fibrils have various roles in biological system, e.g., as causal factor of serious amyloidosis in human and as functional regulator of cell formation in bacteria and eukaryotes. In addition, the fiber-type format is promising as biocompatible scaffold. Therefore, the dissolution method of peptide fibril is potentially useful at many scenes in medical and material fields: as reductive way of pathogenic amyloid, as modification technique of cell structure, and as fabrication tool of biomaterials. However, the fibril structure is generally difficult to be dissociated due to its rigid stacked conformation. Here, we propose a physical engineering technology using terahertz free electron laser (FEL) at far-infrared wavelengths from 70 to 80 µm. Infrared microscopy analysis of the irradiated fibril of calcitonin peptide as a model showed that ß-sheet was decreased, and α-helix, turn, and others were increased, compared to those of the fibril before the FEL irradiation. Interestingly, the dissociative effect by the far-infrared laser was remarkable than that by the mid-infrared laser tuned to 6.1 µm that corresponds to amide I. In addition, simple heating at 363 K deformed the fibril state but increased the amount of ß-sheet, which was contrast with the action by the FEL, and scanning-electron microscopy and Congo-red staining revealed that the fibril was collapsed power-dependently within a range from 25 to 900 mJ energies supplied with the FEL at 74 µm. It can be considered that irradiation of intense terahertz wave can dissociate fibrous conformation of peptide with little influence of thermal effect.

Continuous Isotropic-Nematic Transition in Amyloid Fibril Suspensions Driven by Thermophoresis.

The isotropic and nematic (I + N) coexistence for rod-like colloids is a signature of the first-order thermodynamics nature of this phase transition. However, in the case of amyloid fibrils, the biphasic region is too small to be experimentally detected, due to their extremely high aspect ratio. Herein, we study the thermophoretic behaviour of fluorescently labelled ß-lactoglobulin amyloid fibrils by inducing a temperature gradient across a microfluidic channel. We discover that fibrils accumulate towards the hot side of the channel at the temperature range studied, thus presenting a negative Soret coefficient. By exploiting this thermophoretic behaviour, we show that it becomes possible to induce a continuous I-N transition with the I and N phases at the extremities of the channel, starting from an initially single N phase, by generating an appropriate concentration gradient along the width of the microchannel. Accordingly, we introduce a new methodology to control liquid crystal phase transitions in anisotropic colloidal suspensions. Because the induced order-order transitions are achieved under stationary conditions, this may have important implications in both applied colloidal science, such as in separation and fractionation of colloids, as well as in fundamental soft condensed matter, by widening the accessibility of target regions in the phase diagrams.

Light-driven porphyrin modulating fibrillation of hIAPP(20-29) peptide.

The human Islet amyloid polypeptide (20-29) (hIAPP20-29) is considered to be the core fibrillating fragment of hIAPP, which is associated with the pathogenesis of Type-II diabetes mellitus. A current challenge is the discovery of an efficient way to modulate amyloid aggregation and inhibit the toxicity of its aggregates. In this work, photoexcited porphyrins are successfully used to inhibit the fibrillation of hIAPP20-29. Insights on the inhibitory mechanism are explored by the analysis of the secondary structure, the morphology and the mechanical properties of amyloid aggregates. In addition, photoexcited porphyrins displayed a retained inhibitory effect on hIAPP20-29 aggregation without irradiation. These findings may establish a new avenue to inhibit the aggregation of amyloid peptide hIAPP and enrich the current selection of modulators.

Highly Efficient Destruction of Amyloid-ß Fibrils by Femtosecond Laser-Induced Nanoexplosion of Gold Nanorods.

Alzheimer's disease (AD) is associated with the aggregation of the amyloid-beta (Aß) peptides into toxic aggregates. How to inhibit the aggregation of Aß peptides has been extensively studied over recent decades. The investigation on eliminating preformed fibrils, however, has rarely been reported. In this paper, near-infrared femtosecond (fs) laser is applied for the destruction of preformed Aß fibrils in conjunction with gold nanorods (AuNRs). Our results demonstrate that the 800 nm fs-laser irradiation can locally trigger the explosion of AuNRs due to the strong localized surface plasmon resonance effect. As a result, the majority of Aß fibrils are efficiently destroyed into small fragments by the irradiation of fs-laser with a light dose less than 75 J·cm-2. Meanwhile, significant reduction of ß-sheet structures is observed by thioflavin T (ThT) fluorescence measurements. In contrast, the destruction effect by continuous wave (cw) laser irradiation is much weaker with equivalent power density and irradiation time. Furthermore, the laser-induced destruction of fibrils by Au nanoparticles (AuNPs) is also investigated, which reveals that most of the Aß fibrils remain well under the surface explosion of spherical AuNPs. Overall, our results provide a novel design for the fast destruction of amyloid fibrils locally and biocompatibly, which may have remarkable potentials in the therapy of AD.

Picosecond pulsed infrared laser tuned to amide I band dissociates polyglutamine fibrils in cells.

Amyloid fibrils are causal substances for serious neurodegenerative disorders and amyloidosis. Among them, polyglutamine fibrils seen in multiple polyglutamine diseases are toxic to neurons. Although much efforts have been made to explore the treatments of polyglutamine diseases, there are no effective drugs to block progression of the diseases. We recently found that a free electron laser (FEL), which has an oscillation wavelength at the amide I band (C = O stretch vibration mode) and picosecond pulse width, was effective for conversion of the fibril forms of insulin, lysozyme, and calcitonin peptide into their monomer forms. However, it is not known if that is also the case in polyglutamine fibrils in cells. We found in this study that the fibril-specific ß-sheet conformation of polyglutamine peptide was converted into nonfibril form, as evidenced by the infrared microscopy and scanning-electron microscopy after the irradiation tuned to 6.08 µm. Furthermore, irradiation at this wavelength also changed polyglutamine fibrils to their nonfibril state in cultured cells, as shown by infrared mapping image of protein secondary structure. Notably, infrared thermography analysis showed that temperature increase of the cells during the irradiation was within 1 K, excluding thermal damage of cells. These results indicate that the picosecond pulsed infrared laser can safely reduce amyloid fibril structure to the nonfibril form even in cells.

Picosecond dissociation of amyloid fibrils with infrared laser: A nonequilibrium simulation study.

Recently, mid-infrared free-electron laser technology has been developed to dissociate amyloid fibrils. Here, we present a theoretical framework for this type of experiment based on laser-induced nonequilibrium all-atom molecular dynamics simulations. We show that the fibril is destroyed due to the strong resonance between its amide I vibrational modes and the laser field. The effects of laser irradiation are determined by a balance between fibril formation and dissociation. While the overall rearrangements of the fibril finish over short time scales, the interaction between the peptides and the solvent continues over much longer times indicating that the waters play an important role in the dissociation process. Our results thus provide new insights into amyloid fibril dissociation by laser techniques and open up new venues to investigate the complex phenomena associated with amyloidogenesis.

Ultrasonication-dependent formation and degradation of α-synuclein amyloid fibrils.

Ultrasonication can be used to break the supersaturation of α-synuclein, a protein associated with Parkinson's disease, at pH7.4 above the critical concentration of fibrillation, thereby inducing the formation of amyloid fibrils. We speculated that ultrasonication could also be used to depolymerize preformed fibrils below the critical concentration. However, extensive ultrasonic irradiation transformed preformed fibrils into amorphous aggregates even above the critical concentration. Exposing preformed fibrils to the hydrophobic air-water interface of cavitation bubbles may have destabilized the fibrils and stabilized amorphous aggregates. Upon extensive ultrasonic irradiation, the accompanying decomposition of chemical structures was suggested when monitored by analytical ultracentrifugation. Amorphous aggregates produced by extensive ultrasonication showed higher cytotoxicity, suggesting that, although ultrasonication might be a useful approach for inactivating amyloid fibrils, potential cytotoxicity of amorphous aggregates should be considered.

An alternative structural isoform in amyloid-like aggregates formed from thermally denatured human γD-crystallin.

The eye lens protein γD-crystallin contributes to cataract formation in the lens. In vitro experiments show that γD-crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV-B photodamage results in its C-terminal domain forming the ß-sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet-like aggregates that contain cross-linked oligomers of the protein, according to transmission electron microscopy and SDS-PAGE. We use two-dimensional infrared spectroscopy to show that these aggregates have an amyloid-like secondary structure with extended ß-sheets, and use isotope dilution experiments to show that each protein contributes approximately one ß-strand to each ß-sheet in the aggregates. Using segmental (13) C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N-terminal and C-terminal domains contribute to ß-sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N-terminal domain into the fiber structure to intermolecular cross linking.

La neuroimagen molecular en las demencias degenerativas / Molecular neuroimaging in degenerative dementias

En el contexto de las limitaciones de la imagen estructural, los estudios de perfusión y metabolismo cerebral con SPECT y PET han aportado información relevante en el estudio del deterioro cognitivo. La introducción de radiotrazadores de amiloide cerebral ha replanteado la estrategia diagnóstica en torno a la enfermedad de Alzheimer (EA), considerada actualmente un «continuum». En este nuevo paradigma, la carga amiloide creciente se asocia al estadio preclínico y de deterioro cognitivo leve (DCL) de la enfermedad y ha podido ser objetivada en exploraciones «in vivo» gracias a la introducción del 11C-PIB y los tomógrafos PET. Las características de la imagen 11C-PIB son una elevada retención específica cortical cerebral en los casos positivos, con distribución topográfica característica y no retención en los negativos. Junto a la PET con 18F-FDG constituye la base de la neuroimagen molecular como biomarcador y se está valorando su significado pronóstico en estudios longitudinales. La PET con 11C-PIB se ha consolidado como la técnica óptima para valorar la presencia de amiloide cerebral, pero la necesidad de un ciclotrón cercano para su síntesis limita su disponibilidad, por lo que se están introduciendo trazadores análogos marcados con 18F. Nuestra experiencia en los 2 últimos años aplicando el 11C-PIB, primero en fase investigadora y después en la clínica, ha constatado la utilidad de la técnica en el campo asistencial, tanto de forma aislada como en combinación con la FDG. Así pues, la imagen de amiloide ha demostrado ser una herramienta útil en el diagnóstico diferencial de la demencia y un método prometedor de diagnóstico precoz y evaluación de tratamientos futuros(AU)

In the context of the limitations of structural imaging, brain perfusion and metabolism using SPECT and PET have provided relevant information for the study of cognitive decline. The introduction of the radiotracers for cerebral amyloid imaging has changed the diagnostic strategy regarding Alzheimer's disease, which is currently considered to be a «continuum.» According to this new paradigm, the increasing amyloid load would be associated to the preclinical phase and mild cognitive impairment. It has been possible to observe «in vivo» images using 11C-PIB and PET scans. The characteristics of the 11C-PIB image include specific high brain cortical area retention in the positive cases with typical distribution pattern and no retention in the negative cases. This, in combination with 18F-FDG PET, is the basis of molecular neuroimaging as a biomarker. At present, its prognostic value is being evaluated in longitudinal studies. 11C-PIB-PET has become the reference radiotracer to evaluate the presence of cerebral amyloid. However, its availability is limited due to the need for a nearby cyclotron. Therefore, 18F labeled radiotracers are being introduced. Our experience in the last two years with 11C-PIB, first in the research phase and then as being clinically applied, has shown the utility of the technique in the clinical field, either alone or in combination with FDG. Thus, amyloid image is a useful tool for the differential diagnosis of dementia and it is a potentially useful method for early diagnosis and evaluation of future treatments(AU)

Amyloid-like fibril elongation follows michaelis-menten kinetics.

A number of proteins can aggregate into amyloid-like fibrils. It was noted that fibril elongation has similarities to an enzymatic reaction, where monomers or oligomers would play a role of substrate and nuclei/fibrils would play a role of enzyme. The question is how similar these processes really are. We obtained experimental data on insulin amyloid-like fibril elongation at the conditions where other processes which may impact kinetics of fibril formation are minor and fitted it using Michaelis-Menten equation. The correlation of the fit is very good and repeatable. It speaks in favour of enzyme-like model of fibril elongation. In addition, obtained [Formula: see text] and [Formula: see text] values at different conditions may help in better understanding influence of environmental factors on the process of fibril elongation.

Magnetic-responsive hybrids of Fe3O4 nanoparticles with ß-lactoglobulin amyloid fibrils and nanoclusters.

We report on the synthesis and magnetic-responsive behavior of hybrids formed by dispersing negatively charged iron oxide (Fe3O4) magnetic nanoparticles in positively charged ß-lactoglobulin protein solutions at acidic pH, followed by heating at high temperatures. Depending on the pH used, different hybrid aggregates can be obtained, such as nanoparticle-modified amyloid fibrils (pH 3) and spherical nanoclusters (pH 4.5). We investigate the effect of magnetic fields of varying strengths (0-5 T) on the alignment of these Fe3O4-modified amyloid fibrils and spherical nanoclusters using a combination of scattering, birefringence and microscopic techniques and we find a strong alignment of the hybrids upon increasing the intensity of the magnetic field, which we quantify via 2D and 3D order parameters. We also demonstrate the possibility of controlling magnetically the sol-gel behavior of these hybrids: addition of salt (NaCl, 150 mM) to a solution containing nanoparticles modified with ß-lactoglobulin amyloid fibrils (2 wt % fibrils modified with 0.6 wt % Fe3O4 nanoparticles) induces first the formation of a reversible gel, which can then be converted back to solution upon application of a moderate magnetic field of 1.1 T. These hybrids offer a new appealing functional colloidal system in which the aggregation, orientational order and rheological behavior can be efficiently controlled in a purely noninvasive way by external magnetic fields of weak intensity.

Effect of mid-infrared free-electron laser irradiation on refolding of amyloid-like fibrils of lysozyme into native form.

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610-1,630 cm(-1), characteristic of a ß-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ß-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620 cm(-1) (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70-90 % of the native form. Both irradiations at 1,535 cm(-1)(amide II band) and 1,240 cm(-1) (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000 cm(-1) afforded only about 60 % recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.

670 nm laser light and EGCG complementarily reduce amyloid-ß aggregates in human neuroblastoma cells: basis for treatment of Alzheimer's disease?

OBJECTIVE: The aim of the present study is to present the results of in vitro experiments with possible relevance in the treatment of Alzheimer's disease (AD). BACKGROUND DATA: Despite intensive research efforts, there is no treatment for AD. One root cause of AD is the extra- and intracellular deposition of amyloid-beta (Aß) fibrils in the brain. Recently, it was shown that extracellular Aß can enter brain cells, resulting in neurotoxicity. METHODS: After internalization of Aß(42) into human neuroblastoma (SH-EP) cells, they were irradiated with moderately intense 670-nm laser light (1000 Wm(-2)) and/or treated with epigallocatechin gallate (EGCG). RESULTS: In irradiated cells, Aß(42) aggregate amounts were significantly lower than in nonirradiated cells. Likewise, in EGCG-treated cells, Aß(42) aggregate amounts were significantly lower than in non-EGCG-treated cells. Except for the cells simultaneously laden with Aß(42) and EGCG, there was a significant increase in cell numbers in response to laser irradiation. EGCG alone had no effect on cell proliferation. Laser irradiation significantly increased ATP levels in Aß(42)-free cells, when compared to nonirradiated cells. Laser-induced clearance of Aß(42) aggregates occurred at the expense of cellular ATP. CONCLUSIONS: Irradiation with moderate levels of 670-nm light and EGCG supplementation complementarily reduces Aß aggregates in SH-EP cells. Transcranial penetration of moderate levels of red to near-infrared (NIR) light has already been amply exploited in the treatment of patients with acute stroke; the blood-brain barrier (BBB) penetration of EGCG has been demonstrated in animals. We hope that our approach will inspire a practical therapy for AD.

Ultrasonication-dependent acceleration of amyloid fibril formation.

Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With ß(2)-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.

Photo-induced inhibition of insulin amyloid fibrillation on online laser measurement.

Protein aggregation and amyloid fibrillation can lead to several serious diseases and protein drugs ineffectiveness; thus, the detection and inhibition of these processes have been of great interest. In the present study, the inhibition of insulin amyloid fibrillation by laser irradiation was investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), far-UV circular dichroism (far-UV CD), and thioflavin T (ThT) fluorescence. During heat-induced aggregation, the size distribution of two insulin solutions obtained by online and offline dynamic light scattering were different. The laser-on insulin in the presence of 0.1M NaCl exhibited fewer fibrils than the laser-off insulin, whereas no insulin fibril under laser irradiation was observed in the absence of 0.1M NaCl for 45 h incubation. Moreover, our CD results showed that the laser-irradiated insulin solution maintained mainly an α-helical conformation, but the laser-off insulin solution formed bulk fibrils followed by a significant increase in ß-sheet content for 106 h incubation. These findings provide an inhibition method for insulin amyloid fibrillation using the laser irradiation and demonstrate that the online long-time laser measurements should be carefully used in the study of amyloid proteins because they may change the original results.

Enhancing the activity of a protein by stereospecific unfolding: conformational life cycle of insulin and its evolutionary origins.

A central tenet of molecular biology holds that the function of a protein is mediated by its structure. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biological constraints. A model is provided by insulin, well characterized at atomic resolution by x-ray crystallography. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element. A bifunctional beta-strand mediates both self-assembly (within beta-cell storage vesicles) and receptor binding (in the bloodstream). This strand is anchored by an invariant side chain (Phe(B24)); its substitution by Ala leads to an unstable but native-like analog of low activity. Substitution by d-Ala is equally destabilizing, and yet the protein diastereomer exhibits enhanced activity with segmental unfolding of the beta-strand. Corresponding photoactivable derivatives (containing l- or d-para-azido-Phe) cross-link to the insulin receptor with higher d-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is associated with accelerated fibrillation, a form of aggregation-coupled misfolding associated with cellular toxicity. Conservation of Phe(B24), enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences.

Electric birefringence study of an amyloid fibril system: the short end of the length distribution.

In this article, a system of amyloid fibrils, based on the protein beta-lactoglobulin, is studied by transient electric birefringence. Single pulses of an electric field were applied to the solution, and the initial rise and subsequent decay of birefringence analysed. The decay takes place on a range of relaxation times, and therefore contains information about the length distribution of fibrils in the system. The information can be extracted using theories of the electric polarisability of polyelectrolyte rods, since the fibrils are an example of these. Despite the long-standing complications of such theories, useful quantitative information about the system can still be obtained. Using the Fixman model of polyelectrolyte polarisability, we obtain a measurement of the short end of the length distribution which shows the fibril concentration as a function of length rising linearly from 0.02-2 microm. The short end of the length distribution was unobtainable in our previous study using rheo-optics (S.S. Rogers et al., Macromolecules 38, 2948 (2005)), but reasonable agreement between the two techniques shows they are complementary.

Microwave radiation can alter protein conformation without bulk heating.

Exposure to microwave radiation enhances the aggregation of bovine serum albumin in vitro in a time- and temperature-dependent manner. Microwave radiation also promotes amyloid fibril formation by bovine insulin at 60 degrees C. These alterations in protein conformation are not accompanied by measurable temperature changes, consistent with estimates from field modelling of the specific absorbed radiation (15-20 mW kg(-1)). Limited denaturation of cellular proteins could explain our previous observation that modest heat-shock responses are induced by microwave exposure in Caenorhabditis elegans. We also show that heat-shock responses both to heat and microwaves are suppressed after RNA interference ablating heat-shock factor function.