The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) Steroid hormone receptors belong to a structurally and functionally related group of intracellular proteins, known as the nuclear receptor or steroid/thyroid hormone receptor superfamily, that serve as ligand-activated transcriptional regulators (1). Binding of the cognate hormone to steroid receptors causes a conformational change that allows the receptors to dissociate from an inhibitory complex of proteins, bind as dimers to specific regulatory sequences (enhancer elements) that are associated with the target genes regulated by the hormone, and modulate the transcription of the target genes. DNA binding by hormone-activated steroid receptors has been shown to cause chromatin remodeling, but the mechanism of transcriptional regulation is also believed to involve some type of direct or indirect interaction of the DNA-bound receptor with the transcription machinery.Like other nuclear receptors, steroid receptors are composed of three major functional domains: an N-terminal transcriptional activation domain (AD), a central DNA binding domain (DBD), and a C-terminal hormone binding domain (HBD) (1, 2). In spite of this nomenclature, both the Nterminal AD and the HBD contribute to the transcriptional activation function of steroid receptors (3, 4). In the absence of the HBD, the N-terminal AD, called AF-1, can function in a hormone-independent manner in mammalian cells. In contrast, the transcriptional activation function of the HBD, called AF-2, is hormone dependent. Although each isolated AD has some activity in mammalian cells, these two together appear to function synergistically.The mechanism by which DNA-bound steroid receptors can activate transcription initiation from associated promoters is still unknown. It is proposed that DNA-binding transcriptional activator proteins, including the steroid receptors, stimulate the efficiency of transcription initiation by RNA polymerase II by either directly or indirectly affecting the assembly of basal transcription factors into a preinitiation complex (1, 5). In addition to RNA polymerase II, the preinitiation complex consists of seven basal transcription factors, namely TFIIA, TFIIB, TATA-box binding protein (a subunit of TFIID), TFIIE, TFIIF, TFIIH, and TFIIJ. This complex alone can initiate transcription at a basal rate from TATA-containing promoters, whereas additional TFIID subunits are required for TATA-less promoters and for enhancer-activated transcription. Some of the basal transcription factors may be the targets for regulation by DNA-binding transcriptional activator proteins. Although examples of direct interaction between basal transcription factors and some DNA-binding transcriptional activators have been reported, in most cases the DNAbound transcriptional activator proteins require ...

Blood vessels in the central nervous system (CNS) form a specialized and critical structure, the blood-brain barrier (BBB). We present a resource to understand the molecular mechanisms that regulate BBB function in health and dysfunction during disease. Using endothelial cell enrichment and RNA sequencing, we analyzed the gene expression of endothelial cells in mice, comparing brain endothelial cells to peripheral endothelial cells. We also assessed the regulation of CNS Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are involved in both injury and repair mechanisms in the CNS. Pharmacological blockade of MMPs, limited to the first several days after spinal cord injury, improves locomotor recovery. This beneficial response is, however, lost when treatment is extended beyond the acutely injured cord to include wound healing and tissue remodeling. This suggests that some MMPs play a beneficial role in wound healing. To test this hypothesis, we investigated the role of MMP-2, which is actively expressed during wound healing, in white matter sparing and axonal plasticity, the formation of a glial scar, and locomotor recovery after spinal cord injury. MMP-2 increased between 7 and 14 d after injury, where it was immunolocalized in reactive astrocytes bordering the lesion epicenter. There was reduced white matter sparing and fewer serotonergic fibers, caudal to the lesion in injured MMP-2 null animals. MMP-2 deficiency also resulted in increased immunoreactivity to chondroitin sulfate proteoglycans and a more extensive astrocytic scar. Most importantly, locomotion in an open field, performance on a rotarod, and grid walking were significantly impaired in injured MMP-2 null mice. Our findings suggest that MMP-2 promotes functional recovery after injury by regulating the formation of a glial scar and white matter sparing and/or axonal plasticity. Thus, strategies exploiting MMPs as therapeutic targets must balance these beneficial effects during wound healing with their adverse interactions in the acutely injured spinal cord.

The immune response that accompanies spinal cord injury contributes to both injury and reparative processes. It is this duality that is the focus of this review. Here we consider the complex cellular and molecular immune responses that lead to the infiltration of leukocytes and glial activation, promote oxidative stress and tissue damage, influence wound healing, and subsequently modulate locomotor recovery. Immunomodulatory strategies to improve outcomes are gaining momentum as ongoing research carefully dissects those pathways, which likely mediate cell injury from those, which favor recovery processes. Current therapeutic strategies address divergent approaches including early immunoblockade and vaccination with immune cells to prevent early tissue damage and support a wound-healing environment that favors plasticity. Despite these advances, there remain basic questions regarding how inflammatory cells interact in the injured spinal cord. Such questions likely arise as a result of our limited understanding of immune cell/neural interactions in a dynamic environment that culminates in progressive cell injury, demyelination, and regenerative failure.

It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2′-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testicular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy.

[6909][6910][6911][6912][6913][6914][6915][6916] 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.The hepatitis B virus (HBV) encodes a gene that produces a 154-amino-acid polypeptide, X. X has been shown to be essential for viral replication in animal hosts (7, 69), and it has also been implicated in the development of HBV-mediated carcinogenesis since X is able to induce liver tumors in certain transgenic mice strains (26,29,52). Although the exact role of X in the life cycle of the virus or in carcinogenesis is not known, it has been well established that X is a potent transactivator of a diverse number of viral and cellular promoters. X has been shown to activate RNA polymerase III (pol III)-dependent promoters (3, 58) and RNA pol II-dependent promoters which contain recognition sequences for AP-1, AP-2, ATF/CREB, c/EBP, NF-B, serum response factor, and a variety of acidic activator proteins (for reviews, see 43 and 68).There are at least two distinct mechanisms by which X appears to activate gene expression. For certain promoters, X may interact directly with the transcriptional machinery to augment RNA synthesis. Although X is not able to bind to DNA directly, it has been shown to bind to the CREB/ATF family of transcription factors and to enhance the affinity of the factor for its DNA recognition sequence (66) or alter its specificity (37). In addition, X has been shown to interact with the RPB5 subunit of the RNA polymerases (8, 33), the tumor suppressor p53 (15, 59), transcription factor IIB (TFIIB) (23,33), TFIIH (23,41), and the TATA-binding prot...

Neuropathic pain and bladder dysfunction represent significant quality-of-life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhances GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that 6 months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals show improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury.

The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with an X gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (