Approach to studying the structure-activity relationship of native peptides. I. Structural organization of the opioid peptide Tyr-Glu-Gly-Phe-Met-Arg-Gly-Leu and its artificial analogs]

Theoretical conformational analysis was carried out for the octapeptide Tyr1-Gly2-Gly3-Phe4-Met5-Arg6-Gly7-Leu8. Possible structure of the opioid peptide under physiological conditions may be described by a set of low-energy conformations belonging to 14 different forms of the backbone. The solution of the "reverse conformational problem" for the opioid peptide enables one to predict the modified amino acid sequences (Ala2, D-Ala2, Ala3, D-Ala3, Ala7, D-Ala7, MeMet5, MeArg6-analogues) which may assume one of the low-energy states of the native hormone. The influence of the solute was not taken into account in our calculations.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demonstrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.     

Pyruvate dehydrogenase E1α phosphorylation is induced by glucose but does not control metabolism-secretion coupling in INS-1E clonal β-cells

Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.     

Human 75-kDa DNA-pairing protein is identical to the pro-oncoprotein TLS/FUS and is able to promote D-loop formation

Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single- and double-stranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA double-strand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/TLS require Mg(2+) and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.