Physical exertion exacerbates decline in the musculature of an animal model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by loss of the protein dystrophin. In humans, DMD has early onset, causes developmental delays, muscle necrosis, loss of ambulation, and death. Current animal models have been challenged by their inability to model the early onset and severity of the disease. It remains unresolved whether increased sarcoplasmic calcium observed in dystrophic muscles follows or leads the mechanical insults caused by the muscle's disrupted contractile machinery. This knowledge has important implications for patients, as potential physiotherapeutic treatments may either help or exacerbate symptoms, depending on how dystrophic muscles differ from healthy ones. Recently we showed how burrowing dystrophic (dys-1) C. elegans recapitulate many salient phenotypes of DMD, including loss of mobility and muscle necrosis. Here, we report that dys-1 worms display early pathogenesis, including dysregulated sarcoplasmic calcium and increased lethality. Sarcoplasmic calcium dysregulation in dys-1 worms precedes overt structural phenotypes (e.g., mitochondrial, and contractile machinery damage) and can be mitigated by reducing calmodulin expression. To learn how dystrophic musculature responds to altered physical activity, we cultivated dys-1 animals in environments requiring high intensity or high frequency of muscle exertion during locomotion. We find that several muscular parameters (e.g., size) improve with increased activity. However, longevity in dystrophic animals was negatively associated with muscular exertion, regardless of effort duration. The high degree of phenotypic conservation between dystrophic worms and humans provides a unique opportunity to gain insight into the pathology of the disease as well as the initial assessment of potential treatment strategies.

First Report of Cactodera milleri in Wisconsin.

During the summer of 2005, lemon-shaped cysts and second-stage juveniles of a cyst nematode were recovered from soil at the University of Wisconsin Agricultural Research Station in Hancock, WI. Samples were collected on multiple dates from a plot (61 × 12 m) in continuous potato production for 20 years with significant weed pressure. PCR-restriction fragment length polymorphism profiles of the internal transcribed spacer (ITS) 1 region using restriction enzyme HhaI indicated Cactodera spp. (2). Morphological observations and morphometrics made on cysts, males, J2s, and eggs were consistent with Cactodera milleri Graney and Bird, 1990 (1). Host range studies were conducted in a growth chamber. Soybean, potato, and beet did not support nematode development and reproduction. Common lambsquarters (Chenopodium album), a known host of C. milleri, was an excellent host. No obvious aboveground disease symptoms were evident on lambsquarters in the growth chamber assay. This detection represents the first record of C. milleri in Wisconsin. Unless detailed morphological or molecular measurements are made, C. milleri may be easily confused with the soybean cyst nematode, Heterodera glycines. The presence of lambsquarters in fields planted with glyphosate-resistant soybeans makes the recovery of both nematode species in a single soil sample possible. References: (1) L. S. O. Graney and G. W. Bird. J. Nematol. 22:457, 1990. (2) A. L. Szalanski et al. J. Nematol. 29:255, 1997.

Incorporation of a Fluorescent Compound by Live Heterodera glycines.

The incorporation of fluorescein isothiocyanate (FITC) by J2 of Heterodera glycines, the soybean cyst nematode, and the resulting effects on fitness were determined. Live soybean cyst nematode J2 incubated in FITC fluoresced, primarily in the intestinal region, beyond auto-fluorescence. Dissection of animals, as well as fluorescence-quenching techniques, indicated that FITC was not simply bound to the cuticle. FITC was also found to cross the egg shell. Fluorescence increased in relation to FITC concentration and incubation time. Nematodes incubated in FITC remained active and did not lose their fluorescence even after two weeks at room temperature. Fluorescence of nematodes was not stable through development. Males which developed from fluorescent juveniles did not retain the stain. Both FITC and the DMF solvent reduced the hatching rate. However, those individuals that successfully hatched remained viable and able to infect roots. Incorporation of FITC was found to occur in three other genera of nematodes. Rhodamine B isothiocyanate was also found to be incorporated by H. glycines.

Insights into alpha-K toxin specificity for K+ channels revealed through mutations in noxiustoxin.

Noxiustoxin (NxTX) displays an extraordinary ability to discriminate between large conductance, calcium-activated potassium (maxi-K) channels and voltage-gated potassium (Kv1.3) channels. To identify features that contribute to this specificity, we constructed several NxTX mutants and examined their effects on whole cell current through Kv1.3 channels and on current through single maxi-K channels. Recombinant NxTX and the site-specific mutants (P10S, S14W, A25R, A25Delta) all inhibited Kv1.3 channels with Kd values of 6, 30, 0.6, 112, and 166 nM, respectively. In contrast, these same NxTX mutants had no effect on maxi-K channel activity with estimated Kd values exceeding 1 mM. To examine the role of the alpha-carbon backbone in binding specificity, we constructed four NxTX chimeras, which altered the backbone length and the alpha/beta turn. For each of these chimeras, six amino acids comprising the alpha/beta turn in iberiotoxin (IbTX) replaced the corresponding seven amino acids in NxTX (NxTX-YGSSAGA21-27-FGVDRG21-26). The chimeras differed in length of N- and C-terminal residues and in critical contact residues. In contrast to NxTX and its site-directed mutants, all of these chimeras inhibited single maxi-K channels. Under low ionic strength conditions, Kd values ranged from 0.4 to 6 microM, association rate constant values from 3 x 10(7) to 3 x 10(8) M(-1) x s(-1), and time constants for block from 5 to 20 ms. The rapid blocked times suggest that key microscopic interactions at the toxin-maxi-K channel interface may be absent. Under physiologic external ionic strength conditions, these chimera inhibited Kv1.3 channels with Kd values from 30 to 10 000 nM. These results suggest that the extraordinary specificity of NxTX for Kv1.3 over maxi-K channels is controlled, in part, by the toxin alpha-carbon backbone. These differences in the alpha-carbon backbone are likely to reflect fundamental structural differences in the external vestibules of these two channels.