Thomas A. Griffin

Molecular Phenotypes in Cultured Maple Syrup Urine Disease Cells

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.

Stimulated secretion of pro-opiomelanocortin-related peptides in hypothalamic cells

UNLABELLED Although it has been suggested pro-opiomelanocortin (POMC) related peptides in brain may be neurotransmitters or neuromodulators, little is known about their secretion from neurons because it is difficult to study neurosecretion with an in vivo model. To demonstrate the possibility that POMC peptides may be neuroregulators which can be secreted in response to specific stimuli, we studied the secretion of immunoreactive (IR-) adrenocorticotropin (ACTH) and IR-beta-endorphin from dissociated hypothalamic cells during potassium-induced depolarization. Significant increments (p less than 0.025) in secretion of IR-ACTH (267%) and IR-beta-endorphin (88-172%) over basal secretion were stimulated by 60 mM KCl in the presence of calcium. CONCLUSION Stimulated secretion of POMC peptides from hypothalamic cells by potassium and calcium follows classical neurosecretory mechanisms and suggests these neuropeptides could be neuroregulators in brain.