Functional Electrical Stimulation of Intrinsic Laryngeal Muscles under Varying Loads in Exercising Horses

Bilateral vocal fold paralysis (BVCP) is a life threatening condition and appears to be a good candidate for therapy using functional electrical stimulation (FES). Developing a working FES system has been technically difficult due to the inaccessible location and small size of the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle. A naturally-occurring disease in horses shares many functional and etiological features with BVCP. In this study, the feasibility of FES for equine vocal fold paralysis was explored by testing arytenoid abduction evoked by electrical stimulation of the PCA muscle. Rheobase and chronaxie were determined for innervated PCA muscle. We then tested the hypothesis that direct muscle stimulation can maintain airway patency during strenuous exercise in horses with induced transient conduction block of the laryngeal motor nerve. Six adult horses were instrumented with a single bipolar intra-muscular electrode in the left PCA muscle. Rheobase and chronaxie were within the normal range for innervated muscle at 0.55±0.38 v and 0.38±0.19 ms respectively. Intramuscular stimulation of the PCA muscle significantly improved arytenoid abduction at all levels of exercise intensity and there was no significant difference between the level of abduction achieved with stimulation and control values under moderate loads. The equine larynx may provide a useful model for the study of bilateral fold paralysis

Vector for Pop-In/Pop-Out Gene Replacement in Pichia pastoris

Gene replacement in yeast is often accomplished by using a counterselectable marker such as URA3. Although ura3 strains of Pichia pastoris have been generated, these strains are inconvenient to work with because they grow slowly, even in the presence of uracil. To overcome this limitation, we have developed an alternative counterselectable marker that can be used in any P. pastoris strain. This marker is the T-urf13 gene from the mitochondrial genome of male-sterile maize. Previous work showed that expression of a mitochondrially targeted form of T-urf13 in Saccharomyces cerevisiae rendered the cells sensitive to the insecticide methomyl, and similar results have now been obtained with P. pastoris. We have incorporated T-urf13 into a vector that also contains an ARG4 marker for positive selection. The resulting plasmid allows for pop-in/pop-out gene replacement in P. pastoris

Tomographic Evidence for Continuous Turnover of Golgi Cisternae in Pichia pastoris

The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure–function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking. The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding “matrix.” Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these “free-floating” TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack

Sec16 is a Determinant of Transitional ER Organization

SummaryBackground: Proteins are exported from the ER at transitional ER (tER) sites, which produce COPII vesicles. However, little is known about how COPII components are concentrated at tER sites. The budding yeast Pichia pastoris contains discrete tER sites and is, therefore, an ideal system for studying tER organization.Results: We show that the integrity of tER sites in P. pastoris requires the peripheral membrane protein Sec16. P. pastoris Sec16 is an order of magnitude less abundant than a COPII-coat protein at tER sites and seems to show a saturable association with these sites. A temperature-sensitive mutation in Sec16 causes tER fragmentation at elevated temperature. This effect is specific because when COPII assembly is inhibited with a dominant-negative form of the Sar1 GTPase, tER sites remain intact. The tER fragmentation in the sec16 mutant is accompanied by disruption of Golgi stacks.Conclusions: Our data suggest that Sec16 helps to organize patches of COPII-coat proteins into clusters that represent tER sites. The Golgi disruption that occurs in the sec16 mutant provides evidence that Golgi structure in budding yeasts depends on tER organization

Strength-duration curve for innervated equine posterior cricoarytenoid muscle at rest.

<p>Data shown are mean and standard deviation for six horses.</p

Method measuring arytenoid abduction.

<p>Briefly a line is drawn connecting the dorsal- and ventral-most points of the <i>rima glottidis.</i> This line is then extended dorsally for a distance of one third of the dorsoventral height of the <i>rima glottidis</i>. A tangential line to the arytenoid cartilages is drawn, and the angle between the dorsoventral line and the tangential line is measured.</p

Titration of pulse duration and stimulation frequency with left abduction of the left arytenoid for horses with induced motor conduction block at 90% HRmax demonstrating significantly more abduction with longer pulse duration (p = 0.0037) and higher frequency (p<0.002).

<p>Linear contrast (*) indicates increased abduction at 49 Hz with pulse duration of 4.27 ms versus 0.427 ms (p = 0.03).</p

Illustration to demonstrate position of vidoendoscope in nasopharynx during resting and exercising endoscopy.

<p>Endoscope secured to horse's halter (not shown).</p