Expression and characterization of a recombinant Drosophila tyramine-β-hydroxylase in silkworm infected with recombinant baculovirus

The insect nervous system contains biogenic amines such octopamine (OA), which is synthesized from tyramine (TA) by catalysis of tyramine-β-hydroxylase (TβH). In this study, the Drosophila 70 kDa tyramine-β-hydroxylase (DmTβH) protein was purified after the recombinant nucleopolyhedrovirus isolated from Bombyx mori (BmNPV) containing the TβH gene was injected into the hemocoel of the fifth instar larvae from the d17 B. mori strain. Western blot analysis revealed an immunoreactive band with a molecular mass of 70 kDa. The products formed by incubating the enzyme reaction mixture were separated and detected by reverse phase high-performance liquid chromatography. The optimum pH, temperature, and incubation time for the conversion of TA to OA were 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3H)-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.     

Influence of Structural Symmetry on Protein Dynamics

Structural symmetry in homooligomeric proteins has intrigued many researchers over the past several decades. However, the implication of protein symmetry is still not well understood. In this study, we performed molecular dynamics (MD) simulations of two forms of trp RNA binding attenuation protein (TRAP), the wild-type 11-mer and an engineered 12-mer, having two different levels of circular symmetry. The results of the simulations showed that the inter-subunit fluctuations in the 11-mer TRAP were significantly smaller than the fluctuations in the 12-mer TRAP while the internal fluctuations were larger in the 11-mer than in the 12-mer. These differences in thermal fluctuations were interpreted by normal mode analysis and group theory. For the 12-mer TRAP, the wave nodes of the normal modes existed at the flexible interface between the subunits, while the 11-mer TRAP had its nodes within the subunits. The principal components derived from the MD simulations showed similar mode structures. These results demonstrated that the structural symmetry was an important determinant of protein dynamics in circularly symmetric homooligomeric proteins.     

Attempt at cloning high-quality goldfish breed 'Ranchu' by fin-cultured cell nuclear transplantation

The viability of ornamental fish culture relies on the maintenance of high-quality breeds. To improve the profitability of culture operations we attempted to produce cloned fish from the somatic nucleus of the high-quality Japanese goldfish (Carassius auratus auratus) breed 'Ranchu'. We transplanted the nucleus of a cultured fin-cell from an adult Ranchu into the non-enucleated egg of the original goldfish breed 'Wakin'. Of the 2323 eggs we treated, 802 underwent cleavage, 321 reached the blastula stage, and 51 reached the gastrula stage. Two of the gastrulas developed until the hatching stage. A considerable number of nuclear transplants retained only the donor nucleus. Some of these had only a 2n nucleus derived from the same donor fish. Our results provide insights into the process of somatic cell nuclear transplantation in teleosts, and the cloning of Ranchu.     

Protein structural change upon ligand binding correlates with enzymatic reaction mechanism

Overall structural changes of enzymes in response to ligand binding were investigated by database analysis of 62 non-redundant enzymes whose ligand-unbound and ligand-bound forms were available in the Protein Data Bank. The results of analysis indicate that transferases often undergo large rigid-body domain motions upon ligand binding, while other enzymes, most typically, hydrolases, change their structures to a small extent. It was also found that the solvent accessibility of the substrate molecule was low in transferases but high in hydrolases. These differences are explained by the enzymatic reaction mechanisms. The transferase reaction requires the catalytic groups to be insulated from the water environment, and thus transferases bury the ligand molecule inside the protein by closing the cleft. On the other hand, the hydrolase reaction involves the surrounding water molecules and occurs at the protein surface, requiring only a small structural change.     

Simulation analysis of intracellular Na+ and Cl- homeostasis during beta 1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Natural selection in the <Emphasis Type="Italic">TLR-</Emphasis>related genes in the course of primate evolution

The innate immune system constitutes the front line of host defense against pathogens. Toll-like receptors (TLRs) recognize molecules derived from pathogens and play crucial roles in the innate immune system. Here, we provide evidence that the TLR-related genes have come under natural selection pressure in the course of primate evolution. We compared the nucleotide sequences of 16 TLR-related genes, including TLRs (TLR1–10), MYD88, TILAP, TICAM1, TICAM2, MD2, and CD14, among seven primate species. Analysis of the non-synonymous/synonymous substitution ratio revealed the presence of both strictly conserved and rapidly evolving regions in the TLR-related genes. The genomic segments encoding the intracellular Toll/interleukin 1 receptor domains, which exhibited lower rates of non-synonymous substitution, have undergone purifying selection. In contrast, TLR4, which carried a high proportion of non-synonymous substitutions in the part of extracellular domain spanning 200 amino acids, was found to have been the suggestive target of positive Darwinian selection in primate evolution. However, sequence analyses from 25 primate species, including eight hominoids, six Old World monkeys, eight New World monkeys, and three prosimians, showed no evidence that the pressure of positive Darwinian selection has shaped the pattern of sequence variations in TLR4 among New World monkeys and prosimians. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simultaneous determination of prednisolone, prednisone, cortisol, and cortisone in plasma by GC-MS: estimating unbound prednisolone concentration in patients with nephrotic syndrome during oral prednisolone therapy

Individual variability of the pharmacokinetics of prednisolone based on the unbound concentration in plasma is of significant clinical consideration. The unbound concentrations of prednisolone were measured in 10 patients with nephrotic syndrome, two patients with systemic lupus erythematosus, and one patient with dermatomyositis by examining protein bindings of prednisolone on one or more occasions during prednisolone treatment. In this study, plasma concentrations of prednisolone, prednisone, cortisol, and cortisone were simultaneously analyzed by GC-MS by using stable isotope-labeled internal standards. Equilibrium dialysis was employed to accurately estimate the unbound fractions of prednisolone in plasma. The unbound fraction of prednisolone changed depending on plasma total prednisolone concentration and plasma albumin concentration. The unbound fraction of prednisolone (Y) is calculated: Y=(-0.0101x' + 0.0736) x + 10.23, where x' is the plasma albumin concentration and x is the total prednisolone concentration. The estimated concentrations of unbound prednisolone by using the above equation were in good agreement with the measured concentrations of unbound prednisolone. Since the protein binding of prednisolone did not change in the presence of prednisone (114.0 ng/ml), it appeared that prednisone produced from the therapeutic dose of prednisolone did not affect the unbound fraction of prednisolone.