Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.     

Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis

Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.     

Protease-Dead Separase Is Dominant Negative in the C. elegans Embryo

Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We created a transgenic C. elegans line that expresses protease-dead separase in embryos to further characterize separase function. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos, not previously reported in other systems. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage.     

Demonstrating the Potential for Dynamic Auditory Stimulation to Contribute to Motion Sickness

Auditory cues can create the illusion of self-motion (vection) in the absence of visual or physical stimulation. The present study aimed to determine whether auditory cues alone can also elicit motion sickness and how auditory cues contribute to motion sickness when added to visual motion stimuli. Twenty participants were seated in front of a curved projection display and were exposed to a virtual scene that constantly rotated around the participant''s vertical axis. The virtual scene contained either visual-only, auditory-only, or a combination of corresponding visual and auditory cues. All participants performed all three conditions in a counterbalanced order. Participants tilted their heads alternately towards the right or left shoulder in all conditions during stimulus exposure in order to create pseudo-Coriolis effects and to maximize the likelihood for motion sickness. Measurements of motion sickness (onset, severity), vection (latency, strength, duration), and postural steadiness (center of pressure) were recorded. Results showed that adding auditory cues to the visual stimuli did not, on average, affect motion sickness and postural steadiness, but it did reduce vection onset times and increased vection strength compared to pure visual or pure auditory stimulation. Eighteen of the 20 participants reported at least slight motion sickness in the two conditions including visual stimuli. More interestingly, six participants also reported slight motion sickness during pure auditory stimulation and two of the six participants stopped the pure auditory test session due to motion sickness. The present study is the first to demonstrate that motion sickness may be caused by pure auditory stimulation, which we refer to as “auditorily induced motion sickness”.     

The RAD6 DNA repair pathway in Saccharomyces cerevisiae: What does it do,and how does it do it?

The RAD6 pathway of budding yeast, Saccharomyces cerevisiae, is responsible for a substantial fraction of this organism's resistance to DNA damage, and also for induced mutagenesis. The pathway appears to incorporate two different recovery processes, both regulated by RAD6. The error-prone recovery prcess accounts for only a small amount of RAD6-dependent resistance, but probably all induced mutagenesis. The underlying mechanism, for error-prone recovery is very likely to be translesion synthesis. The error-free recovery process accounts for most of RAD6-dependent resistace, but its mechanism is less clear; it may entail error-free bypass by template switching and/or DNA gap filling by recombination. RAD6 regulates these activities by ubiquitinateins, and the roles they play in error-free and error-prone recovery, have not yet been established.