Molecular cloning of an isoform of Doc2 having two C2-like domains

We previously isolated a novel protein having two C2-like domains known to interact with Ca2+ and phospholipid, and named Doc2 (Double C2). Doc2 is predominantly expressed in brain and is implicated in Ca(2+)-dependent neurotransmitter release. We have isolated here an isoform of Doc2 and named the original one Doc2 alpha and the new one Doc2 beta. Doc2 beta alsp has two C2-like domains and is 61% identical to Doc2 alpha at the amino acid level. In contrast to Doc2 alpha, the Doc2 beta mRNA is expressed ubiquitously. These results indicate that there are at least two isoforms of Doc2, and suggest that Doc2 beta is involved in Ca(2+)-dependent intracellular vesicle trafficking in various types of cells.     

Properties and structure of spermidine acetyltransferase in Escherichia coli

Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion.     

The role of repeat transurethral resection in stage A1 carcinoma of the prostate

In order to evaluate the significance of repeat transurethral resection (TUR) in differentiating stage A1 prostatic adenocarcinoma from those with stage A2, we performed repeat TUR in 34 patients with an initial diagnosis of stage A1 prostatic adenocarcinoma. It was found that residual adenocarcinoma was present in five cases (14.7%), but the diagnosis was changed from stage A1 to stage A2 in only one case (2.9%). In one patient with final diagnosis of stage A1 carcinoma, bone metastases were detected seven months after the repeat TUR. It was concluded that repeat TUR for stage A1 prostatic adenocarcinoma did not yield clinically significant information.     

Imipramine-induced inactivation of a cytochrome P450 2D enzyme in rat liver microsomes: in relation to covalent binding of its reactive intermediate

Preincubation of microsomes from male Wistar rats with imipramine (IMI) in the presence of NADPH caused a time-dependent loss of bunitrolol 4-hydroxylase activity, indicating that the CYP2D enzyme is inactivated during IMI metabolism, which has also been observed after in vivo administration of IMI. A similar effect was obtained when desipramine, an N-demethylated metabolite of IMI, was used as an inhibitor, whereas 2-hydroxy-IMI had no effect on the activity. Thus, it seems likely that the inactivation of the CYP2D enzyme is related to 2-hydroxylation process of IMI. Incubation of microsomes with [3H]IMI in the presence of NADPH resulted in covalent binding of a 3H-labeled material to microsomal protein. Formation rates of the reactive metabolites covalently bound to protein followed Michaelis-Menten kinetics, and the K(m) value (1.1 microM) was close to that for microsomal IMI 2-hydroxylation. The metabolism-dependent covalent binding of [3H]IMI was lower in Dark Agouti rats, which is an animal model of CYP2D deficiency, than in Wistar rats. The binding was inhibited by propranolol and quinidine, a substrate and an inhibitor of CYP2D, respectively, and by an antibody against CYP2D. Similar strain difference (Dark Agouti < Wistar) and inhibitory effects by the compounds and the antibody were observed in IMI 2-hydroxylase but not in N-demethylase activity. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of microsomal protein incubated with [3H]IMI and NADPH showed that the binding was prominent at the molecular mass of approximately 50 kDa, which would be consistent with the P450 protein being a target for the binding. Furthermore, proteins to which [3H]IMI metabolites covalently bound were immunoprecipitated with the anti-CYP2D antibody. These results suggest that IMI is biotransformed into a chemically reactive metabolite (probably arene-oxide) through its 2-hydroxylation step by the CYP2D enzyme in rat liver microsomes, and the metabolite binds covalently to the enzyme itself, resulting in the inactivation.     

Rules for robust generation of accurate reduced-order models forhigh-speed coupled interconnections

Recently, robust algorithms have been established fur passive order reduction of electrical models of complex interconnection networks. However, very little is known about the way the order of the reduced model should be chosen to ensure accuracy in subsequent transient simulation studies, in this paper, a rule is derived for the selection of the order of the reduced model for interconnections modeled as transmission lines. It is shown that pulse rise time, interconnection length, and physical properties impact the order of the reduced model. The proposed rule is validated through numerical studies involving both analytic and numerical results from the frequency- and time-domain response of multiconductor transmission line circuits     

The role of dispersed particles in strengthening and fracture mechanisms in a Mo-ZrC alloy processed by mechanical alloying

The tensile properties of a ZrC particle-dispersed Mo, which was processed by spark plasma sintering with mechanically alloyed powder, were investigated at room temperature and at elevated temperatures of 1170 to 1970 K. The Mo-ZrC alloy showed much higher strength at room temperature than a fully recrystallized pure Mo. The high strength of Mo-ZrC is mainly attributed to a very small grain size (about 3 μm). The main role of the ZrC particle is not to increase strength due to the particle-dislocation interaction, but to limit grain growth during sintering and to attain the very small grain size. The elongation at room temperature of Mo-ZrC was much lower than that of pure Mo. This is probably related to the higher interstitial contents. However, Mo-ZrC showed a large elongation of 180 pct at 1970 K and 6.7×10⁻⁴ s⁻¹. It was suggested that the ZrC particles stabilized the fine-grained microstructure yet provided no cavitation sites at 1970 K; as a result, the large elongation was attained.