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Eaf2, ELL-associated factor 2, encodes a protein that is homologous to the human EAF1, which was shown to interact with the transcriptional elongation factor MEN/ELL. During mouse embryogenesis, Eaf2 is preferentially expressed in the central nervous system and in sensory and neuroendocrine organs, including the brain, spinal cord, cranial and spinal ganglia, developing otocyst, the retina, and the pituitary. Eaf2 transcripts were also found in sites where active epithelium–mesenchymal interactions are occurring. These included the invaginating tooth buds, mammary gland anlage, submandibular glands, the lung, the pancreas, and the kidney. Other sites of expression included bladder and intestine. In the developing lens, Eaf2 transcripts were absent in the proliferating anterior lens epithelial cells but were present in the terminally differentiated primary lens fiber cells and also in nonproliferating lens fiber cells in the equatorial zone where lens epithelial cells withdraw from cell cycle and terminally differentiate into secondary lens fiber cells. This spatially restricted pattern of Eaf2 expression in the developing lens suggests that Eaf2 may play an important role in regulating lens maturation. Developmental Dynamics 228:273–280, 2003. © 2003 Wiley-Liss, Inc.

• Background: Intraocular transplantation of genetically modified cells that release a particular substance could have a major impact on the treatment of various ocular diseases. We studied the expression of the reporter gene ß-galactosidase (lacZ) in transplanted retinal pigment epithelial (RPE) cells in vivo • Methods: RPE cells from pigmented rabbits were transduced with the ß-galactosidase gene in a retroviral vector. Cells were then assayed for gene expression and transplanted subretinally into the eyes of New Zealand White rabbits. RPE cells that were transduced with a similar vector without the ß-galactosidase gene were used as controls. Rabbits were killed on days 1, 7, and 21 and the eyes processed for transmission electron microscopy • Results: Neomycin-resistant rabbit RPE cells that showed ß-galactosidase activity were generated within 2–5 weeks. After transplantation, viable RPE cells that expressed the transgene and that phagocytosed rod outer segments were observed on days 1, 7, and 21 • Conclusions: The results show that generation of genetically modified RPE cells is feasible and that the transplanted cells remain viable and continue to express the transgene in the subretinal space of the host animal for at least 21 days. Transplantation of such genetically modified RPE cells could provide a new tool for studying retinal diseases and, potentially, for correcting metabolic abnormalities in retinal degenerations and dystrophies.

A rhodopsin-related protein is preferentially expressed at high levels in retinal pigment epithelium (RPE) and in Müller cells. The putative RPE-retinal G protein-coupled receptor (RGR) was localized in light-adapted bovine retina by means of electron microscopic immunocytochemistry. In the RPE, the protein was localized to a widespread intracellular compartment. Except for the region adjacent to the basal surface, the RPE cytoplasm was labeled throughout the cell including the apical surface. In Müller cells also RGR was found in the intracellular compartment, especially in the cytoplasm in the region of the Müller cell endfeet and proximal cell processes. Subcellular fractionalion studies of bovine RPE and neural retina indicated that RGR is a membrane-bound protein. The intracellular localization of RGR is a unique variation in the subcellular distribution of seven-transmembrane-domain receptors and suggests an unconventional role for RGR in the signal transduction process.

Hormonal stimulation of Gq-protein coupled receptors triggers Ca2+mobilization from internal stores. This is followed by a Ca2+entry through the plasma membrane.DrosophilaTrp and Trpl proteins have been implicated in Ca2+entry and three mammalian homologues ofDrosophilaTrp/Trpl, hTrp1, hTrp3 and bTrp4 (also bCCE) have been cloned and expressed. Using mouse brain RNA as template, we report here the polymerase chain reaction-based cloning and functional expression of a novel Trp, mTrp6. The cDNA encodes a protein of 930 amino acids, the sequence of which is 36.8, 36.3, 43.1, 38.6, and 74.1% identical to DrosophilaTrp and Trpl, bovine Trp4, and human Trp1 and Trp3, respectively. Transient expression of mTrp6 in COS.M6 cells by transfection of the full-length mTrp6 cDNA increases Ca2+entry induced by stimulation of co-transfected M5 muscarinic acetylcholine receptor with carbachol (CCh), as seen by dual wavelength fura 2 fluorescence ratio measurements. The mTrp6-mediated increase in Ca2+entry activity was blocked by SKF-96365 and La3+. Ca2+entry activity induced by thapsigargin was similar in COS cells transfected with or without the mTrp6 cDNA. The thapsigargin-stimulated Ca2+entry could not be further stimulated by CCh in control cells but was markedly increased in mTrp6-transfected cells. Records of whole cell transmembrane currents developed in response to voltage ramps from −80 to +40 mV in control HEK cells and HEK cells stably expressing mTrp6 revealed the presence of a muscarinic receptor responsive non-selective cation conductance in Trp6 cells that was absent in control cells. Our data support the hypothesis that mTrp6 encodes an ion channel subunit that mediates Ca2+entry stimulated by a G-protein coupled receptor, but not Ca2+entry stimulated by intracellular Ca2+store depletion.

Ca2+release from its internal stores as a result of activation of phospholipase C is accompanied by Ca2+influx from the extracellular space. Ca2+influx channels may be formed of proteins homologous toDrosophilaTrp. At least six non-allelic Trpgenes are present in the mouse genome. Full-length human, bovine, mouse, and rat cDNAs for Trp1, 3, 4, 6 have been cloned. Expression of these genes in various mammalian cells has provided evidence that Trp proteins form plasma membrane Ca2+-permeant channels that can be activated by an agonist that activates phospholipase C, by inositol 1,4,5-trisphosphate, and/or store depletion. We have stably expressed human Trp3 (hTrp3) in human embryonic kidney (HEK)293 cells. Measurement of intracellular Ca2+concentrations in Fura2-loaded cells showed that cell lines expressing hTrp3 have significantly higher basal and agonist-stimulated influxes of Ca2+, Mn2+, Ba2+, and Sr2+than control cells. The increase in Ca2+entry attributable to the expression of hTrp3 obtained upon store depletion by thapsigargin was much lower than that obtained by stimulation with agonists acting via a Gq-coupled receptor. Addition of agonists to thapsigargin-treated Trp3 cells resulted in a further increase in the entry of divalent cations. The increased cation entry in Trp3 cells was blocked by high concentrations of SKF 96365, verapamil, La3+, Ni2+, and Gd3+. The Trp3-mediated Ca2+influx activated by agonists was inhibited by a phospholipase C inhibitor, U73122. We propose that expression of hTrp3 in these cells forms a non-selective cation channel that opens after the activation of phospholipase C but not after store depletion. In addition, a subpopulation of the expressed hTrp3 may form heteromultimeric channels with endogenous proteins that are sensitive to store depletion.

An unresolved bottleneck in bacterial artificial chromosome (BAC) transgenesis is low efficiency generation of founder mice because of suboptimal quality of the manipulated BAC DNA. Using mini-gel electrophoresis and electro-elution that circumvents CsCl2centrifugation, column chromatography, and resin purifications, we have used RECOCHIP, a commercially available dialysis cassette for the purification of BAC DNA that generates transgenic founders with up to 80% efficiency.

Abstract: The human RGR gene encodes an opsin protein (retinal G protein-coupled receptor), which is expressed in Müller cells and the retinal pigment epithelium and is thought to play a role in the visual process. To investigate a possible linkage of the RGR gene to retinal dystrophies, the locus of the gene was mapped on human metaphase chromosomes. Genomic and cDNA fragments of the human RGR gene were used as probes for fluorescence in situ hybridization. Analysis of the fluorescence signals on high-resolution banded chromosomes showed that the RGR gene is localized to human chromosome 10q23. This result now provides for the rapid analysis of this gene with respect to inherited diseases of the retina.

Hair plays important roles, ranging from the conservation of body heat to the preservation of psychological well-being. Hair loss or alopecia affects millions worldwide, but methods that can be used to regrow hair are lacking. We report that quiescent (telogen) hair follicles can be stimulated to initiate anagen and hair growth by small molecules that activate autophagy, including the metabolites α-ketoglutarate (α-KG) and α-ketobutyrate (α-KB), and the prescription drugs rapamycin and metformin, which impinge on mTOR and AMPK signaling. Stimulation of hair growth by these agents is blocked by specific autophagy inhibitors, suggesting a mechanistic link between autophagy and hair regeneration. Consistently, increased autophagy is detected upon anagen entry during the natural hair follicle cycle, and oral α-KB prevents hair loss in aged mice. Our finding that anagen can be pharmacologically activated in telogen skin when natural anagen-inducing signal(s) are absent has implications for the treatment of hair loss patients.

Recruitment of intracellular vesicles containing the large GTPase CRACR2A to the immunological synapse mediates T cell receptor signaling.

Two members of the Gαifamily of G proteins form complexes with EGFR and the adaptor protein Gab1 to mediate activation of Akt.