Using a cross-linking approach, we have analyzed the function of Skp, a presumed molecular chaperone of the periplasmic space of Escherichia coli, during the biogenesis of an outer membrane protein (OmpA). Following its transmembrane translocation, OmpA interacts with Skp in close vicinity to the plasma membrane. In vitro, Skp was also found to bind strongly and specifically to pOmpA nascent chains after their release from the ribosome suggesting the ability of Skp to recognize early folding intermediates of outer membrane proteins. Pulse labeling of OmpA in spheroplasts prepared from an skp null mutant revealed a specific requirement of Skp for the release of newly translocated outer membrane proteins from the plasma membrane. ⌬skp mutant cells are viable and show only slight changes in the physiology of their outer membranes. In contrast, double mutants deficient both in Skp and the periplasmic protease DegP (HtrA) do not grow at 37°C in rich medium. We show that in the absence of an active DegP, a lack of Skp leads to the accumulation of protein aggregates in the periplasm. Collectively, our data demonstrate that Skp is a molecular chaperone involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.

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Transplantation of embryonic stem (ES) cells may provide cures for the damaged nervous system. Pre-differentiated ES or neuronal precursor cells have been investigated in various animal models of neurodegenerative diseases including traumatic brain injury (TBI). To our knowledge, no study has yet examined the effects of undifferentiated, murine ES cells on functional recovery and tumorigenity following implantation into injured rat brains. We evaluated the effect of transplantation of undifferentiated, murine embryonic cells on the recovery of motor function following lateral fluid percussion brain injury in Sprague-Dawley rats. At 3 days post-injury, animals received stereotactic injections of either embryonic stem cell suspension or injections of phosphate buffered saline without cells (control) into the injured cortex. Neurological motor function assessments were performed before injury, 72 h, 1, 3, and 6 weeks after transplantation using a Rotatrod and a Composite Neuroscore test. During this time period brain injured animals receiving ES cell transplantation showed a significant improvement in the Rotarod Test and in the Composite Neuroscore Test as compared to phosphate buffered saline (PBS)-treated animals. At 1 week post-transplantation, ES cells were detectable in 100% of transplanted animals. At 7 weeks following transplantation, EScells were detectable in only one animal. Two of 10 xenotransplanted animals revealed tumor formation over the observation period. These findings provide evidence for therapeutic potency of embryonic stem cell transplantation after TBI in rat, but also raise serious safety concerns about the use of such cells in human.

Pluripotent embryonic stem cells were shown to survive and differentiate into mature neuronal cells after implantation in experimental models of Parkinson disease and cerebral ischemia. Embryonic stem cell transplantation has also been proposed as a potential therapy for cerebral trauma, characteristic of massive loss of multiple cell types due to primary insult and secondary sequelae. Green fluorescent protein (GFP)-transfected murine embryonic stem cells were implanted into the ipsi or contralateral cortex of male Sprague-Dawley rats 72 h after fluid-percussion injury. Animals were sacrificed at day 5 or week 7 postimplantation. Brain sections were examined using conventional and fluorescent double-labelling immunohistochemistry. Five days after implantation, clusters of GFP-positive cells undergoing partial differentiation along neuronal pathway, were detected at the implantation site. However, after 7 weeks, only a few GFP-positive cells were found, indicating an extensive loss of stem cells during this time period. For the first time, we proved the observed cell loss to be mediated via phagocytosis of implanted cells by activated macrophages. Cerebral trauma, induced 3 days prior to implantation, has activated the inflammatory potential of otherwise immunologically privileged tissue. Subsequent cell implantation was accompanied by reactive astrogliosis, activation of microglia, as well as a massive invasion of macrophages into transplantation sites even if the grafts were placed into contralateral healthy hemispheres, remote from the traumatic lesion. Our results demonstrate a significant post-traumatic inflammatory response, which impairs survival and integration of implanted stem cells and has generally not been taken into account in designs of previous transplantation studies.