Jesse L. Owens

Transfecting neurons and glia in the rat using pH-sensitive immunoliposomes

Immunoliposomes were constructed using antibody 5-113 (directed to an antigen on the external surface rat glial cells), the antibody Thy 1.1, and a non-immune antibody. The antibodies were conjugated to N-gluytaryl-phosphatidylethanolamine. Liposomes were constructed with these conjugated antibodies, other lipids and a beta-galactosidase plasmid under the control of the cytomegalovirus promoter. When immunoliposomes decorated with one of three different antibodies were injected into the brain or spinal cord of adult rats, the X-gal reaction product was observed in neurons, astrocytes and vascular elements. There was an increase in neuronal labeling when animals were injected with Thy 1.1 conjugated liposomes and there was an increase in glial labeling in animals injected with 5-113 liposomes. In spinal cords, the immunoliposomes appear to penetrate a substantial distance, transfecting neurons several centimeters from the site of delivery. These data suggest that immunoliposomes may provide an effective transfection system for gene delivery in the CNS.

Listeriolysin O enhances cytoplasmic delivery by Her-2 targeting liposomes

To enhance cytoplasmic delivery of liposomal contents to breast cancer cells, the authors have attached the pore-forming protein, listeriolysin O (LLO), to thermosensitive liposomes. The antibody trastuzumab (Herceptin®) was also conjugated with the outer surface of the liposomes, resulting in highly specific binding and internalization into mammary epithelial cells that overexpress the human epidermal growth factor receptor 2 (Her-2). The liposomes were preloaded with a marker fluorescent dye, and the effect of LLO on the distribution of dye within the cells was monitored using fluorescence microscopy. Owing to the thermosensitive nature of the liposomes, hyperthermia at 42°C triggered the release of the encapsulated fluorescent calcein from the endocytosed liposomes into the interior of the endosomes. LLO, when conjugated to these liposomes, subsequently formed pores in the endosomal membrane, allowing calcein to flow out of the endosomal compartment into the cytoplasm. Her-2–targeted liposomes bearing LLO delivered a 22-fold greater concentration of calcein to mammary epithelial cells that overexpress Her-2 compared to cells with normal Her-2 expression. Thus, the addition of LLO to preformed liposomes offers a method for significantly enhancing delivery of liposomal contents to the cytoplasm of targeted cells.

Beyond the wheelchair: development of motorised transport for people with severe mobility impairments in developing countries

Purpose. In developing countries, lack of over-the-road transportation reduces a vast number of disabled people to a life of begging, dependency and social isolation. Although there have been some excellent efforts to provide low-cost wheelchairs and hand-powered tricycles, little has been done to further provide transport for disabled people. Methods. To address this problem directly we devised a low-cost motorised wheelchair transporter prototype (the Skeeter) using a combination of bicycle parts, a custom built frame, and an inexpensive small engine for propulsion. Results. The Skeeter can provide independent operation and travel by a wheelchair user. It is an economically feasible solution to the lack of over-the-road transportation for people with mobility impairments in developing countries. Conclusions. The Skeeter has the potential to provide a major enhancement in the quality of life for people with disabilities in developing countries around the world.

MRF4 Gene Expression in Xenopus Embryos and Aneural Myofibers

Vertebrate embryos express the transcription factor MRF4 during skeletal muscle differentiation. Previous studies of MRF4 expression in embryonic Xenopus laevis and its response to muscle denervation in adults have led to the suggestion that its transcription may be activated in myotomes and in multinucleate myofibers through an interaction with the motor nerves. We tested this hypothesis by assaying for MRF4 gene transcripts in early neurula stage embryos, beginning before the appearance of neurons. MRF4 transcripts were detectable by reverse transcriptase‐polymerase chain reaction (RT‐PCR) from at least stage 13–14, well before the differentiation of either nerves or myocytes. We also tested the nerve‐dependence of MRF4 gene expression in multinucleate myofibers by comparing transcript levels between interhyoideus muscles in normal larvae and muscles whose motor innervation had been prevented through surgical removal of the brain before cranial motor axon outgrowth. RT‐PCR demonstrated similar MRF4 transcript levels in the aneural muscles and controls. These results fail to support the hypothesis that MRF4 gene expression is triggered or is significantly up‐regulated in myogenic cells by signals from motor axons.

Role of M2 domain residues in conductance and gating of acetylcholine receptors in developing Xenopus muscle

1 The contributions of specific residues in γ‐ and ɛ‐subunits to the developmental changes in conductance and open time of Xenopus muscle acetylcholine receptors (AChRs) were investigated. This study was directed primarily at residues in the M2 domains of γ‐ and ɛ‐subunits; however, the results of additional mutations in the extracellular region flanking M2 and in the amphipathic region between M3 and M4 are also described. 2 The M2 domains of γ‐ and ɛ‐subunits differ at only three amino acid residues, two of which are adjacent to each other and located near the narrowest part of the pore. These two residues (NI in γ, SV in ɛ) were found to be major determinants of the difference in conductance and open time of AChRs bearing γ‐ or ɛ‐subunits. 3 Mutation of N to S in the γ‐subunit converted the long open time of receptors bearing the γ‐subunit (γ‐AChRs) to the brief open time characteristic of receptors bearing an ɛ‐subunit (ɛ‐AChRs). Conversely, ɛ‐AChRs with SV mutated to NI in the ɛ‐subunit exhibited a long open time characteristic of γ‐AChRs. 4 Mutation of N to S in the γ‐subunit increased the conductance of γ‐AChRs but did not confer the full conductance of wild‐type ɛ‐AChRs. Conversely, mutation of SV to NI in the ɛ‐subunit reduced the conductance of ɛ‐AChRs, but not completely to the level of wild‐type γ‐AChRs.

Junctional acetylcholine receptor channel open time is not presynaptically regulated in developing muscle

The role of motor innervation in controlling the development of acetylcholine receptor (AChR) channel open time was tested by examining synaptic current durations in transplanted muscles of Xenopus tadpoles. The presumptive lower jaw region, which gives rise to the interhyoideus muscle, was transplanted to the tail, overlying the myotomal muscle cells. The transplanted muscles became innervated, presumably by spinal nerves which normally innervate myotomal muscle. Despite development in the presence of foreign innervation, synaptic currents in the transplanted interhyoideus were predominantly long in duration and resembled those in the normally innervated interhyoideus. They did not resemble those in the myotomal muscle, where synaptic currents are brief. The apparent lack of neural influence on development of AChR function in muscle contrasts with the evidence for presynaptic control of AChR open time in frog sympathetic ganglia. This may reflect a fundamental difference between nerve and muscle in the regulation of postsynaptic function.

Expression of nicotinic acetylcholine receptors in aneural Xenopus embryos.

During gastrulation in vertebrate embryos, the mesoderm moves inward and under the ectoderm and these two cell layers subsequently differentiate in close proximity to each other, providing an opportunity for the exchange of inductive signals. This study examines whether the activation of muscle nicotinic acetylcholine receptor (AChR) genes and the subsequent expression of receptors in Xenopus myotomal muscle are dependent on interaction between the ectoderm and the mesoderm, or their derivatives, after the onset of gastrulation. We eliminated such interaction by inducing total exogastrulation of Xenopus embryos. During exogastrulation, the mesoderm moves away from the ectoderm, and the nervous system fails to develop. Single channel recordings from the myotomal muscle of exogastrulated embryos revealed the presence of two major classes of AChRs, which could be distinguished on the basis of channel conductance. The current amplitudes, conductances, reversal potentials, and open times of these channels closely resembled those reported for the two major classes of AChR channels normally expressed in vivo. We conclude that interaction between ectoderm and mesoderm following the onset of gastrulation is not required for the future expression of the major classes of AChRs in myotomal muscle.

Adapting motorbikes for independent use by people with disability

In much of the world motorbikes are the dominant means of transportation. In the developing world motorbikes are often the only form of motorized transportation affordable to the majority of the population. Unfortunately this method of transportation has not been widely exploited anywhere in the world for use by people with severe mobility impairments, especially those who must use wheelchairs. To provide affordable transport for a wide range of mobility-impaired people I have developed an inexpensive sidecar adaptation for motorbikes, capable of transporting a wheelchair user (Figure 1). This adaptation is referred to as the SideScooter. The SideScooter can be operated independently with hand controls from the sidecar or from the motorbike seat, depending on the needs of the operator. Given the vast number of motorbikes in the world and the scarcity of independent transportation for wheelchair users this device has the potential to improve the quality of life for many disabled people in developing as well as the more affluent regions of the world.