Hiromi Morimoto

Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain

By integrating convergent protein engineering and rational inhibitor design, we have developed an in vivo conditional protein knockout and/or manipulation technology. This method is based on the creation of a specific interaction interface between a modified protein domain and sensitized inhibitors. By introducing this system into genetically modified mice, we can readily manipulate the activity of a targeted protein, such as α-Ca2+/calmodulin-dependent protein kinase II (αCAMKII), on the time scale of minutes in specific brain subregions of freely behaving mice. With this inducible and region-specific protein knockout technique, we analyzed the temporal stages of memory consolidation process and revealed the first postlearning week as the critical time window during which a precise level of CaMKII reactivation is essential for the consolidation of long-term memories in the brain.

Maze training alters brain weights and cortical RNA/DNA ratios

In order to test whether training leads to anatomical and chemical changes in the brain, individual rats were given self-paced trials in mazes, traversing the maze in order to get from a food station to a water station. In 30 days of this training, during which they had no social interaction, the rats developed significant increases in weight and RNA/DNA of standard samples of cerebral cortex, as compared with littermate rats in either of two control conditions: (a) rats confined to small individual cages ( N = 70 per condition); (b) rats that traversed the empty maze box with no maze barriers present ( N = 29 per condition). Whereas the rats who faced maze problems decreased average transit times through the maze on successive trials, the rats that traversed the empty box showed no regular trend in running times over trials. The cerebral effects of maze experience versus control conditions were similar in pattern but were smaller in magnitude than effects of experience in a social group in a multisensory complex environment. This clear evidence of cerebral changes as consequences of maze training adds further support to the indications that similar cerebral changes resulting from enriched experience are due to learning rather than to other factors. The changes that follow training or enriched experience can be linked with other evidence concerning the roles of RNA and of protein synthesis in the formation of long-term memory traces. The U.S. Governments right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged.

Spectrophotometric analysis of RNA and DNA using cetyltrimethylammonium bromide

Abstract A versatile procedure is described for the analysis of RNA and DNA in brain using cetyltrimethylammonium bromide as the initial precipitant. Optimal conditions are described for the precipitation, hydrolysis, and effective separation of the RNA and DNA fractions from contaminating protein. The RNA and DNA fraction can now be accurately estimated by uv absorbance without a two wavelength correction. This method has also been used for the analysis of other mammalian organs and for mammalian cells obtained from tissue culture. The method may also be used for the simultaneous determination of radioactivity in nucleic acids. The orcinol reaction is shown to give high values for brain RNA.

Characterization of the adipokinetic hormone receptor from the fat body of Manduca sexta

A tritium labeled Manduca sexta adipokinetic hormone (M-AKH) was synthesized (pE-L-T-[p3H]F-T-S-S-W-G-NH2) (specific activity 27 Ci/mmol) which was fully active in a bioassay. It was used in a filtration based binding assay to characterize the M-AKH receptor from the fat body of M. sexta. Membrane fractions were prepared from fat body and optimal binding conditions were determined. A Kd of 7.10(-10) M was determined and the receptor concentration estimated to be 0.5 pmol/mg membrane protein. No receptor binding was found when membranes were prepared from brain, heart or flight muscle of M. sexta or from fat body of the cockroach Blaberus discoidalis. However, specific binding was found with membrane preparations from the pterothoracic ganglion of M. sexta. The membranes from the ganglion had a much smaller number of binding sites than the fat body membranes, however, the binding was specific and observed in each experiment.

Probing a putative dantrolene-binding site on the cardiac ryanodine receptor

Dantrolene is an inhibitor of intracellular Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). Direct photoaffinity labelling experiments using [3H]azidodantrolene and synthetic domain peptides have demonstrated that this drug targets amino acids 590-609 [termed DP1 (domain peptide 1)] of RyR1 (ryanodine receptor 1), the skeletal muscle RyR isoform. Although the identical sequence exists in the cardiac isoform, RyR2 (residues 601-620), specific labelling of RyR2 by dantrolene has not been demonstrated, even though some functional studies show protective effects of dantrolene on heart function. Here we test whether dantrolene-active domains exist within RyR2 and if so, whether this domain can be modulated. We show that elongated DP1 sequences from RyR1 (DP1-2s; residues 590-628) and RyR2 (DP1-2c; residues 601-639) can be specifically photolabelled by [3H]azidodantrolene. Monoclonal anti-RyR1 antibody, whose epitope is the DP1 region, can recognize RyR1 but not RyR2 in Western blot and immunoprecipitation assays, yet it recognizes both DP1-2c and DP1-2s. This suggests that although the RyR2 sequence has an intrinsic capacity to bind dantrolene in vitro, this site may be poorly accessible in the native channel protein. To examine whether it is possible to modulate this site, we measured binding of [3H]dantrolene to cardiac SR as a function of free Ca2+. We found that > or =10 mM EGTA increased [3H]dantrolene binding to RyR2 by approximately 2-fold. The data suggest that the dantrolene-binding site on RyR2 is conformationally sensitive. This site may be a potential therapeutic target in cardiovascular diseases sensitive to dysfunctional intracellular Ca2+ release.

Primary tritium and deuterium isotope effects on chemical shifts of compounds having an intramolecular hydrogen bond

The primary deuterium and tritium isotope effects, δ(XH) − δ(XD/T), were measured for 55 compounds having one or more intramolecular hydrogen bonds. The primary isotope effects were measured at various temperatures. For compounds displaying tautomerism the primary isotope effects are found to have contributions from both intrinsic and equilibrium isotope effects. The primary tritium isotope effect, PΔ(1H,3H), and the primary deuterium isotope effect, PΔ (1H,2H), are shown to be related by

Analogs of Manduca Adipokinetic Hormone Tested in a Bioassay and in a Receptor-Binding Assay

^{\rm P}\Delta (^{1}\hbox{H},^{3}\hbox{H}) =1.4 ^{\rm P}\Delta (^{1}\hbox{H},^{2}\hbox{H})

The Biochemistry Of the Particulate Methane Monooxygenase

This finding is valid for both tautomeric compounds and compounds with localized hydrogen bonds. Large negative primary tritium and deuterium isotope effects were observed for compounds displaying tautomerism and having sulfur as donor or acceptor. These isotope effects show a strong temperature dependence, which is related to the change in equilibrium due to isotope substitution. For the compounds with localized hydrogen bonds, the primary deuterium and tritium isotope effects correlated with the two bond deuterium isotope effect on 13C chemical shifts. The primary deuterium and tritium isotope effects are therefore a measure of the hydrogen bond strength for compounds with localized hydrogen bonds. Copyright