No abstract

GLUT1 is essential for human brain development and function, as evidenced by the severe epileptic encephalopathy observed in children with GLUT1 deficiency syndrome resulting from inherited loss-of-function mutations in the gene encoding this facilitative glucose transporter. To further elucidate the pathophysiology of this disorder, the zebrafish orthologue of human GLUT1 was identified, and expression of this gene was abrogated during early embryonic development, resulting in a phenotype of aberrant brain organogenesis consistent with the observed expression of Glut1 in the embryonic tectum and specifically rescued by human GLUT1 mRNA. Affected embryos displayed impaired glucose uptake concomitant with increased neural cell apoptosis and subsequent ventricle enlargement, trigeminal ganglion cell loss, and abnormal hindbrain architecture. Strikingly, inhibiting expression of the zebrafish orthologue of the proapoptotic protein Bad resulted in complete rescue of this phenotype, and this occurred even in the absence of restoration of apparent glucose uptake. Taken together, these studies describe a tractable system for elucidating the cellular and molecular mechanisms of Glut1 deficiency and provide compelling in vivo genetic evidence directly linking nutrient availability and activation of mitochondria-dependent apoptotic mechanisms during embryonic brain development.The cellular uptake of glucose is dependent in part upon the GLUT family of polytopic membrane transporters that facilitate the passive diffusion of this essential nutrient across membranes (1). GLUT1, the prototypic member of this protein family, is highly expressed in erythrocytes and the central nervous system and is believed to play an essential role in the homeostasis of brain glucose in the developing human infant (2). In support of this concept, infants with GLUT1 deficiency syndrome, a rare genetic disorder resulting from inherited heterozygous loss-of-function mutations in the gene encoding GLUT1, develop seizures, acquired microcephaly and profound developmental delay in association with profound hypoglycorrhachia (3). Despite considerable study of these patients, the neurochemical and neuropathologic consequences of GLUT1 deficiency during development are not well understood, and treatment of affected patients remains challenging.Although abundant in the extracellular milieu, the cellular uptake of glucose is precisely regulated by specific growth factors and signaling pathways (4). Cell culture studies reveal that inhibition of cellular glucose uptake dramatically increases apoptosis under conditions of growth factor restriction and that cell survival under such circumstances is dependent upon the regulation of glucose uptake and metabolism by the proto-oncogene Akt (5-7). A biochemical link between glucose homeostasis and apoptosis was further suggested by studies demonstrating an interaction between the proapoptotic protein, Bad, and the glycolytic enzyme, glucokinase (8). In support of these findings, a recent study now demonstrates a...

Purpose In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu. Experimental design Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by 1-Step Human Coupled IVT (HCIVT) compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine. Results HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than 10 times higher than RRL, in both western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array. Conclusion and clinical relevance HCIVT is a robust IVTT system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer.

The effects of immunoglobulin G (IgG4. Ca2P currents in controls were unaffected by addition of nifedipine but were reduced by 37 % upon addition of w-conotoxin GVIA. In LEMS animals, however, the currents were depressed by 43 % by nifedipine but were unaffected by w-conotoxin GVIA. Thus, LEMS is associated with reduced Ca2+ currents and a shift to dihydropyridine sensitivity.

The pre-synaptic protein, a-synuclein, has been associated with the pathogenesis of Parkinson's disease. The present study indicates that a-synuclein, but not its mutants (A53T, A30P), can protect CNS dopaminergic cells from the parkinsonism-inducing drug 1-methyl-4-phenylpyridinium (MPP + ), whereas it cannot protect from the dopaminergic toxin, 6-hydroxydopamine, hydrogen-peroxide, or the b-amyloid peptide, A-b. Protection from MPP + was directly correlated with the preservation of mitochondrial function. Specifically, a-synuclein rescued cells from MPP + mediated decreases in mitochondrial dehydrogenase activity and loss of ATP levels by utilizing ketosis. It also prevented toxin-induced activation of the creatine kinase/creatine phosphate system. Similarly, a-synuclein protected cells from the complex I inhibitor rotenone and 3-nitroproprionic acid, a complex II inhibitor. Wildtype a-synuclein-mediated neuroprotection and subsequent alterations in energy were not found in dbcAMP-differentiated cells. These results suggest that the normal physiological role for a-synuclein may change during development.