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Objective: Stilbene derivative, cis-3, 4', 5-trimethoxy-3'-aminostilbene (stilbene 5c), is highly potent to induce cell death in ovarian cancer cells. This study is to investigate its mechanism to induce cell death., Methods: UCI101 ovarian cancer cells were used for this study. Cell death was analyzed by Alamar blue staining. Cell cycle was analyzed by flow cytometry after PI staining. Mitochondrial potential and reactive oxygen species were determined by MitoTracker green and DCF-DA, respectively. Immunofluorescent staining was done with tubulin antibody following by confocal microscope examination. Cell lysates were collected after treatment with stilbene 5c for Western blotting analysis of various cell cycle regulators and signal transduction mediators., Results: Stilbene-treated cells die in both cell cycle-dependent and -independent pathways. Low concentration (30 nM) induces cell death without cell cycle arrest. This process involves disruption of mitochondrial potential and production of ROS by a Bcl-2-independent pathway. Higher concentration of stilbene 5c arrests cell cycle in G(2)/M phase, which is supported by dephosphorylation of Cdc2 and Cdc25C, and transiently elevation of spindle checkpoint BubR1. Although phosphorylation of Chk1 and Chk2 both increases after treatment, loss of Chk1 suppresses, whereas loss of Chk2 enhances, stilbene 5c-induced cell death. Phosphorylation of Akt and Stat3, but not MAPK, is suppressed after stilbene 5c treatment., Conclusion: These studies provide a mechanistic insight in using stilbenes in ovarian cancer. Stilbenes could be potentially useful agents for ovarian cancer therapy and induce cell death through mitochondrial damage and cell cycle arrest.

Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.

By reducing the progressive degeneration and disconnection of axons following spinal cord injury the functional outcome should improve. After direct transection of dorsal column sensory axons, neurotrophin-3 (NT-3) treatment can reduce degeneration and promote regeneration of the proximal stumps. Here, we tested in adult rats whether NT-3 infusion at the site of a moderate T9 spinal cord contusion would rescue sensory connections to the gracile nucleus in the medulla. Sensory projections were anterogradely traced bilaterally with injections of cholera toxin B (CTB) into the sciatic nerve 3 days before analysis. Seven days after the contusion plus intrathecal (subarachnoid) vehicle infusion as a control, the CTB-positive innervation of the gracile nucleus was reduced to approximately 25% of sham-operated rats. Intrathecal infusion of 10 microg/day of NT-3 did not affect this reduced innervation. To ensure good tissue penetration and high concentrations of NT-3 early after the injury, other rats received intraparenchymal infusions of vehicle or NT-3 near the injury site starting 2 days before until 7 days after the injury. This NT-3 treatment also did not affect the reduced innervation. This suggests that local NT-3 treatments cannot protect sensory axons from secondary degeneration after a contusive spinal cord injury. These results are likely because TrkC is not present in axons of the dorsal columns or gracile nucleus, or in other dorsal column cell types, even after the contusion. Together with published results, our data suggest that NT-3 is a peripherally--but not centrally--derived neurotrophic factor for sensory neurons.

Testing the effects of drugs that stimulate endogenous neurogenesis in different species is important for the development of neural repair strategies in humans. We have previously shown in adult rats that a 14-day intracerebroventricular infusion of the D3 preferential agonist 7-hydroxydipropyl-amino-tetraline (7-OH-DPAT) increases BrdU labeling of neural precursors in the subventricular zone of the anterior lateral ventricle (SVZ). Here, we show that such a treatment failed to affect neurogenesis in C57Bl/6 and FVB mice, even at a high dose or when infused into the neostriatum. We confirmed that such a treatment was effective in adult rats. Moreover, D3 receptor inhibition or genetic knockout failed to affect the neurogenesis in mice. These results raise the possibilities that neurogenesis is not regulated by D3 receptors in all species and, therefore, that D3 agonists like pramipexole may not be useful to harness endogenous neurogenesis in cell replacement strategies for Parkinson's disease.

The most widely used immunosuppressant in neural transplantation is cyclosporine- A (CsA). However, CsA has significant toxic effects when administered systemically. Tacrolimus (FK506), is a more potent immunosuppressant than CsA and can be prepared in lipid micelles (LTAC). This liposomal preparation allows for the administration of tacrolimus to the site of transplantation, possibly reducing the systemic side effects of immunosuppression. In this study we investigated the ability of LTAC to promote graft survival in hemiparkinsonian rats implanted with fetal mouse xenografts when LTAC is administered systemically to the host, when added to the donor cell suspension, or in combination. Rats with unilateral 6-hydroxydopamine lesions were transplanted with 800,000 fetal mouse ventral mesencephalic (VM) cells and were randomly divided into four groups. Group 1 was not immunosuppressed; Group 2 received daily systemic injections of LTAC; Group 3 received LTAC within the cell suspensions of mouse VM cells; and Group 4 received LTAC in the cell suspensions along with daily systemic administration of LTAC. Transplanted rats were assessed for rotational behavior 3 and 6 weeks posttransplantation. Cell survival was assessed using tyrosine hydroxylase (TH) immunohistochemistry. A significant reduction in rotational scores was observed only in the group of animals receiving the combination of LTAC-treated donor cells and systemic LTAC administration. This functional improvement correlated with a significantly greater survival of TH-immunoreactive cells in this group of animals. The other groups had poor cell survival and no significant functional improvement. We conclude that a combination of systemic immunosuppression and treatment of the cell suspension with LTAC may be a superior strategy to optimize xenograft survival. This strategy may have important implications for clinical neural transplantation., ((c)2001 Elsevier Science.)

The clinical findings on neural transplantation for Parkinson's disease (PD) reported thus far are promising but many issues must be addressed before neural transplantation can be considered a routine therapeutic option for PD. The future of neural transplantation for the treatment of neurological disorders may rest in the discovery of a suitable alternative cell type for fetal tissue. One such alternative may be neurons derived from a human teratocarcinoma (hNT). hNT neurons have been shown to survive and integrate within the host brain following transplantation and provide functional recovery in animal models of stroke and Huntington's disease. In this study, we describe the transplantation of hNT neurons in the substantia nigra (SN) and striatum of the rat model for PD. Twenty-seven rats were grafted with one of three hNT neuronal products; hNT neurons, hNT-DA neurons, or lithium chloride (LiCl) pretreated hNT-DA neurons. Robust hNT grafts could be seen with anti-neural cell adhesion molecule and anti-neuron-specific enolase immunostaining. Immunostaining for tyrosine hydroxylase (TH) expression revealed no TH-immunoreactive (THir) neurons in any animals with hNT neuronal grafts. THir cells were observed in 43% of animals with hNT-DA neuronal grafts and all animals with LiCl pretreated hNT-DA neuronal grafts (100%). The number of THir neurons in these animals was low and not sufficient to produce significant functional recovery. In summary, this study has demonstrated that hNT neurons survive transplantation and express TH in the striatum and SN. Although hNT neurons are promising as an alternative to fetal tissue and may have potential clinical applications in the future, further improvements in enhancing TH expression are needed., (Copyright 2000 Academic Press.)

The effectiveness of a standard ESR broadening agent, potassium trioxalatochromiate (CrOx), for use with the spin-label tempone, was compared to that of diethylenetriaminepentaacetic acid (DTPA) containing an ion (Gd, Cr, Mn, Fe) with a large magnetic moment. Signal attenuation, line broadening, toxicity, and cell membrane permeability were compared. As a broadening agent, CrOx was most effective, followed by Fe-DTPA. CrOx proved mildly toxic while Gd-DTPA and Fe-DTPA were virtually non-toxic. The human red blood cell membrane was tested for permeability to Fe- and Gd-DTPA and found to be impermeable to both. In situations where toxicity to cells is critical, the DTPA chelates, particularly Fe-DTPA, may prove an acceptable substitute for CrOx.