Glucose transporters are required to bring glucose into cells, where it is an essential energy source and precursor in protein and lipid synthesis. These transporters are involved in important common diseases such as cancer and diabetes. Here, we report the crystal structure of the Staphylococcus epidermidis glucose/H + symporter in an inward-facing conformation at 3.2-Å resolution. The Staphylococcus epidermidis glucose/H + symporter is homologous to human glucose transporters, is very specific and has high avidity for glucose, and is inhibited by the human glucose transport inhibitors cytochalasin B, phloretin, and forskolin. On the basis of the crystal structure in conjunction with mutagenesis and functional studies, we propose a mechanism for glucose/H + symport and discuss the symport mechanism versus facilitated diffusion.major facilitator superfamily | membrane protein | GLUT | sugar transporter | solute-carrier 2A
In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75NTR . During retinal injury, endogenous NGF, TrkA, and p75 NTR are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75 NTR affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75 NTR agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75 NTR action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-␣ and ␣ 2 -macroglobulin. Antagonists of p75 NTR inhibit TNF-␣ and ␣ 2 -macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.Neuropathic diseases of the retina that involve the death of retinal ganglion cells (RGCs) 4 are irreversible. This is because RGCs are neurons whose fibers and axons make up the optic nerve (ON) and relay visual input from the retina to the cerebral cortex.Commonly used animal models of neuropathy that cause RGC death include ON axotomy and glaucoma. ON axotomy is an acute model of trauma where the optic nerve is completely severed, causing rapid death of the RGCs (ϳ90% within 2 weeks). Glaucoma is a chronic and progressive optic nerve neuropathy often concomitant with elevated intraocular pressure (IOP) (1). The etiology of RGC death in glaucoma remains unknown.One mechanism is the deprivation of survival signals that neurotrophins provide by acting through the TrkA and TrkB receptors expressed in RGCs (2, 3). Indeed, activation of TrkA (4) or TrkB (5) directly activate pro-survival signals during glaucoma and rescues RGCs from death during ON axotomy or glaucoma. However, it seems paradoxical that whereas TrkA activity is protective, neither endogenous nerve growth factor (NGF) (up-regulated in glaucoma (6)) nor exogenous NGF applied as a drug afford effective RGC neuroprotection during ON axotomy or glaucoma (4, 7).A second mechanism of RGC death in glaucoma is the increased production of tumor necrosis factor-␣ (TNF-␣) (8-10) and ␣ 2 -macroglobulin (␣ 2 M) (11). These neurotoxic factors are produced by activated microglia (12), which express the neurotrophin receptor p75 NTR (7). Indeed, the p75 NTR receptor has been implicated in the acute release of TNF-␣ during acute toxicity leading to RGC death within a few hours after intravitreal injection of glutamate (13) or after activatio...
Nerve growth factor (NGF) is a neurotrophin that induces neuritogenic and trophic signals by binding to
The role of the NH2-terminal region of nerve growth factor (NGF) was studied with an NGF delta 9/13 deletion mutant, overexpressed in a baculovirus system, and mouse NGF truncated at Met-9 by cleavage with CNBr (des-(1-9)-NGF). Structural studies have been performed on the purified proteins, in addition to biological activity assessment, in order to determine effects of such modifications on global conformation and stability. The activity of NGF delta 9/13 was reduced below detectable levels, and the activity of the des-(1-9)-NGF form was decreased by at least a 50-fold in a PC12 bioassay. Competitive binding of NGF delta 9/13 to low affinity receptors on PC12 cells was not impaired; however, the mutant was not capable of competing for the cold chase-stable, high affinity binding of NGF to the cells. The binding of NGF delta 9/13 to Sf21 cells ectopically expressing the TrkA NGF receptor was also abolished. Thus, deletion of residues 9-13 significantly altered the binding affinity for the high affinity receptors on PC12 cells and for the TrkA receptor, but not for the low affinity receptor. Neither the secondary structure, determined by circular dichroism, nor the conformational stability determined by equilibrium denaturation of NGF delta 9/13 was altered as compared with wild type NGF. Slight conformational and stability perturbations of des-(1-9)-NGF were revealed by the same analysis; however, these changes were found to reflect the influence of the formic acid treatment, not the truncation of 9 residues. Our results support the conclusion that the NH2-terminal domain encompassing residues 1-9 and 9-13 is essential for maintaining the binding capability of NGF for high affinity TrkA receptors. Moreover, conformational and stability data show that the functional results of these modifications of the NH2-terminal region are directly due to receptor binding and not to secondary effects of improper folding or other indirect structural changes.
Mumie, a semihard black resin formed by long-term humification, is believed to have therapeutic properties. Although mumie has been used in folk medicine since ancient times, there is little information available concerning the physicochemical properties of its constituents and the mechanisms of its therapeutic efficacy. For this study crude mumie was fractionated into fulvic acid (FA), humic acid (HA), humin, hymatomelanic acid, and two low molecular weight fractions (LMW1 and LMW2). The FA fraction was divided into five subfractions, FA1-FA5. The mumie fractions were characterized by IR, UV-vis, and fluorescence spectroscopy. Total carbohydrate content in the fractions was analyzed using the phenol reaction method. The relative content of polar groups and nonpolar hydrocarbon fragments in the mumie fractions correlated well with solubility in an aqueous medium. Biological characterization was performed using only the FA fractions. FA1 and FA2 enhanced the production of reactive oxygen species (ROS) and nitric oxide in murine peritoneal macrophages, as determined with the use of 2',7'-dichlorofluorescin diacetate and Griess reagent, respectively. The enchancement of ROS and nitric oxide production correlated with the level of total carbohydrates in the fractions. Murine splenic lymphocytes treated with FA1 showed a dose-dependent increase in [(3)H]thymidine uptake. These findings suggest that FA derived from mumie has immunomodulatory activity.
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