No abstract

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS) where aggregation of copper/zinc superoxide dismutase (SOD1) is implicated in pathogenesis. We report here that fully metallated (holo) SOD1 under physiologically relevant solution conditions can undergo changes in metallation and/or dimerization over time and form aggregates that do not exhibit classical characteristics of amyloid. The relevance of the observed aggregation to disease is demonstrated by structural and tinctorial analyses, including the novel observation of binding of an anti-SOD1 antibody that specifically recognizes aggregates in ALS patients and mice models. ALS-associated SOD1 mutations can promote aggregation but are not essential. The SOD1 aggregation is characterized by a lag phase, which is diminished by self-or cross-seeding and by heterogeneous nucleation. We interpret these findings in terms of an expanded aggregation mechanism consistent with other in vitro and in vivo findings that point to multiple pathways for the formation of toxic aggregates by different forms of SOD1. Amyotrophic lateral sclerosis (ALS)2 is a devastating, rapidly progressive, and invariably fatal neurodegenerative disease, characterized by motor neuron degeneration and paralysis (1). Approximately 10% of ALS cases are familial (fALS), the remaining cases being sporadic (sALS). The familial and sporadic diseases are clinically indistinguishable and so have been proposed to share common disease mechanisms (1). Mutations in copper/zinc superoxide dismutase (SOD1) account for ϳ20% of fALS and represent a major known cause of the disease. It is generally accepted that fALS-linked SOD1 mutations result in a toxic gain of function rather than a loss of function (1). Much attention has focused on toxic protein aggregation as causing ALS, analogous to pathogenic protein aggregation in other neurodegenerative diseases, including Alzheimer, Huntington, Parkinson, and prion diseases (1-5). SOD1 is present in aggregates in motor neurons of SOD1-linked fALS patients (3, 6) and mice models (3, 7-9) and in some sALS patients (4, 5, 10).Mature SOD1 is a homodimeric protein, with each ␤-barrel subunit containing one catalytic copper ion, one structural zinc ion, one intrasubunit disulfide bond, and two free cysteines (11) (see Fig. 1). More than 147 mainly missense mutations throughout the SOD1 structure have been associated with fALS. Aggregation has been reported previously only for immature (metal and/or disulfide deficient) or aberrant forms of SOD1, often under highly destabilizing conditions, which favor aggregation in general (12-19). The relevance to human disease of such aggregation is not known, nor is it known what forms of SOD1 may give rise to or be present in aggregates in patients (20). Mature (holo) SOD1 is a major form of SOD1 in cells for wild type and most mutant SOD1s (11). Although this form has been proposed to be incompatible with aggregation because of its very high stability (15, 21), several studies have rep...

A battery-operated, atmospheric pressure, self-igniting, planar geometry Ar-H(2) microplasma for elemental analysis of liquid microsamples is described. The inexpensive microplasma device (MPD) fabricated for this work was a hybrid plastic-quartz structure that was formed on chips with an area (roughly) equal to that of a small-sized postage stamp (MPD footprint, 12.5-mm width by 38-mm length). Plastic substrates were chosen due to their low cost, for rapid prototyping purposes, and for a speedy microplasma device evaluation. To enhance portability, the microplasma was operated from an 18-V rechargeable battery. To facilitate portability even further, it was demonstrated that the battery can be recharged by a portable solar panel. The battery-supplied dc voltage was converted to a high-voltage ac. The ~750-μm (diameter) and 12-mm (long) Ar-H(2) (3% H(2)) microplasma was formed by applying the high-voltage ac between two needle electrodes. Spectral interference from the electrode materials or from the plastic substrate was not observed. Operating conditions were found to be key to igniting and sustaining a microplasma that was simply "warm" to the touch (thus alleviating the need for cooling or other thermal management) and that had a stable background emission. A small-sized (900 μL internal volume) electrothermal vaporization system (40-W max power) was used for microsample introduction. Microplasma background emission in the spectral region between 200 and 850 nm obtained using a portable fiber-optic spectrometer is reported and the effect of the operating conditions is described. Analyte emission from microliter volumes of dilute single-element standard solutions of Cd, Cu, K, Li, Mg, Mn, Na, Pb, and Zn is documented. The majority of spectral lines observed for the elements tested were from neutral atoms. The relative lack of emission from ion lines simplified the spectra, thus facilitating the use of a portable spectrometer. Despite the relative spectral simplicity, some spectral interference effects were noted when running a multi-element solution. An example of how interference in the spectral domain can be resolved in the time domain using selective thermal vaporization is provided. Analytical utility and performance characteristics are reported; for example, K concentrations in diluted (~30 times) bottled water were determined to be 4.1 ± 1.0 μg/mL (4 μg/mL was the stated concentration), precision was about 25%, and the estimated detection limits were in the picogram range (or in nanograms per milliliter in relative units).

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 degrees C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO(2), H(2), L-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 +/- 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 +/- 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe(2+). The enzyme was thermostable with a half-life of about 10 h at 70 degrees C, and its catalytic activity increased along with the rise of temperature up to 95 degrees C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K (m) values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.

Because of their desirable characteristics, for example small size, lightness, low power and gas consumption, and potential for portability, miniaturized plasma sources are receiving significant attention in the scientific literature. To take advantage of these characteristics we micromachined and fabricated new, planar-geometry, self-igniting, atmospheric-pressure microplasma devices (MPDs) on chips. These microplasmas required such low power for their operation they could be operated from a re-chargeable battery (of the type used in cordless power-tools). Despite their advantages, most miniaturized plasma sources reported in the literature have not performed well with liquid samples; analysis of powders or solids that can be converted to a powder (and processed and used as slurries) is even more difficult. To address these shortcomings we coupled an electrothermal, mini-in-torch vaporization (mini-ITV) "dry" sample-introduction system to the low-power planar microplasma devices we developed. In this preliminary investigation, absolute detection limits obtained from microsamples of single-element liquid standards and optical emission spectrometry with photomultiplier-tube detection and a spectral bandpass similar to that of portable, commercially available fiber-optic spectrometers were in the low-pg to ng range, for example 2 pg (for K) to 25 ng (for Pb). Mini-ITV also enabled (as far as we are aware, for the first time) measurement of analyte emission from microsamples of powdered solids (as slurries). In addition to the 3% H2 in Ar mixtures, the ac-operated microplasmas were sustained by use of a variety of electrode materials and different plasma-support gases (e.g. Ar, He and 3% H2 in He) thus indicating fabrication versatility and operational flexibility. Such flexibility has the potential to enable microplasmas to be tailored to analytical problems, and this is demonstrated by using a He MPD and chlorine emission measurements (837.594 nm) from gaseous microsamples as an example.