Synthesis,in vitro and in vivo studies of Gd-DTPA-XDA-D1-Glc(OH) complex as a new potential MRI contrast agent

A new type of dendritic molecules Gd-DTPA-XDA-D1-Glc(OH), which work as a functionalized ligand coordinating gadolinium(III) ion at the center of their frameworks with two glucose moieties on the molecular surfaces, were readily synthesized with high yield. The structures were established by IR, ¹H, ¹³C NMR, and mass spectral studies. Its bio-distribution patterns were evaluated on rats.     

Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation,catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.     

Magnesium gating of cardiac gap junction channels

We aimed to study kinetics of modulation by intracellular Mg²⁺ of cardiac gap junction (Mg²⁺ gate). Paired myocytes of guinea-pig ventricle were superfused with solutions containing various concentrations of Mg²⁺. In order to rapidly apply Mg²⁺ to one aspect of the gap junction, the non-junctional membrane of one of the pair was perforated at nearly the connecting site by pulses of nitrogen laser beam. The gap junction conductance (G_j) was measured by clamping the membrane potential of the other cell using two-electrode voltage clamp method. The laser perforation immediately increased G_j, followed by slow G_j change with time constant of 3.5 s at 10 mM Mg²⁺. Mg²⁺ more than 1.0 mM attenuated dose-dependently the gap junction conductance and lower Mg²⁺ (0.6 mM) increased G_j with a Hill coefficient of 3.4 and a half-maximum effective concentration of 0.6 mM. The time course of G_j changes was fitted by single exponential function, and the relationship between the reciprocal of time constant and Mg²⁺ concentration was almost linear. Based on the experimental data, a mathematical model of Mg²⁺ gate with one open state and three closed states well reproduced experimental results. One-dimensional cable model of thirty ventricular myocytes connected to the Mg²⁺ gate model suggested a pivotal role of the Mg²⁺ gate of gap junction under pathological conditions.     

Protein Collective Motions Coupled to Ligand Migration in Myoglobin

Ligand migration processes inside myoglobin and protein dynamics coupled to the migration were theoretically investigated with molecular dynamics simulations. Based on a linear response theory, we identified protein motions coupled to the transient migration of ligand, carbon monoxide (CO), through channels. The result indicates that the coupled protein motions involve collective motions extended over the entire protein correlated with local gating motions at the channels. Protein motions, coupled to opening of a channel from the distal pocket to a neighboring xenon site, were found to share the collective motion with experimentally observed protein motions coupled to a doming motion of the heme Fe atom upon photodissociation of the ligand. Analysis based on generalized Langevin dynamics elucidated slow and diffusive features of the protein response motions. Remarkably small transmission coefficients for rates of the CO migrations through myoglobin were found, suggesting that the CO migration dynamics are characterized as motions governed by the protein dynamics involving the collective motions, rather than as thermally activated transitions across energy barriers of well-structured channels.     

Characterization of thermostable native alkaline phosphatase from an aerobic hyperthermophilic archaeon, <Emphasis Type="Italic">Aeropyrum pernix</Emphasis> K1

This paper reports the characterization of an alkaline phosphatase (AP) from an aerobic hyperthermophilic Archaeon Aeropyrum pernix K1. The native AP was purified into homogeneity. The enzyme is predicted as a homodimeric structure with a native molecular mass of about 75 kDa and monomer of about 40 kDa. Apparent optimum pH and temperature were estimated at 10.0 and above 95°C, respectively. Magnesium ion increased both the stability and the activity of the enzyme. A. pernix AP has been demonstrated as a very thermostable AP, retaining about 76% of its activity after being incubated at 90°C for 5.5 h and 67% of its activity after being incubated at 100°C for 2.5 h, respectively, under the presence of Mg(II). Enzyme activity was increased in addition of exogenous Mg(II), Ca(II), Zn(II), and Co(II).     

Striated muscle twitchin of bivalves has "catchability", the ability to bind thick filaments tightly to thin filaments, representing the catch state

Catch muscles are found in some invertebrates which can maintain high passive tension with little energy expenditure for long periods after their active contraction. Twitchin in the catch muscles has the ability to facilitate the tight binding of thick filaments to thin filaments, which is the structural basis of the catch tension. We defined this ability as catchability and assessed the catchability of twitchins purified from striated muscles of an oyster (Crassostrea gigas) and a scallop (Mimachlamys nobilis), by using an in vitro catch assay where the binding of filaments could be directly visualized under a light microscope. We found that both twitchins had catchability, even though these muscles are not considered to be catch muscles in physiological experiments. In addition, these muscles contained water-soluble factors regulating the binding of the catch, probably protein kinase A and protein phosphatase 2B. These findings suggest that not only bivalve smooth muscles but also striated muscles have a system that regulates their relaxation rate through the catchability of twitchin, at least at the molecular level.     

Global structure analysis of acid-unfolded myoglobin with consideration to effects of intermolecular coulomb repulsion on solution X-ray scattering

To obtain information on the global structure of protein in the acid-unfolded (AU) state, the structure of apomyoglobin (apoMb) was analyzed by using the solution X-ray scattering (SXS) method. SXS profiles were obtained over a wide range of protein concentrations, 1-18 mg mL-1, under strongly acidic conditions. From analysis of the SXS profile extrapolated to a zero protein concentration, the mean square radius, Rsq, of AU-apoMb at 20 mM HCl was estimated to be 4.81 +/- 0.31 nm. This estimate is more than 1.3 nm larger than those of 3.0-3.5 nm reported thus far. The difference originates from the fact that effects of Coulomb repulsive forces acting between AU-apoMb molecules have not been correctly taken into account in the conventional analysis. In fact, even at a low protein concentration of 1 mg mL-1 close to the limit of measurement in the present SXS method, the solution condition applicable to estimating accurately structural parameters of AU-apoMb is very limited. At HCl concentrations lower than 10 mM, the scattering intensity at a small scattering vector decreases remarkably through the effect of intermolecular repulsive forces and the forward scattering intensity is significantly lower than the estimate from the partial specific volume of protein. On the other hand, at HCl concentrations higher than 50 mM, some compact molten-globule-like structures emerge. As a result, the intermediate concentration of 20 mM HCl is the best choice of the solution condition for determining Rsq of AU-apoMb. The effect of intermolecular Coulomb repulsion on the SXS profile of AU-apoMb is at its maximum for forward scattering and decreases monotonously with an increase in the scattering angle to be virtually negligible at K approximately 0.63 nm(-1). Whereas urea-denatured apoMb shows a SXS profile typical of Gaussian chains, the intrinsic SXS profile of AU-apoMb differs significantly from those of Gaussian chains.     

Delta-opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-epsilon-mediated phosphorylation of connexin 43

The aim of this study was to examine the hypothesis that delta-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the delta-opioid receptor activation. A delta-opioid receptor agonist, [D-Ala(2),D-Leu(5)]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-epsilon inhibitor [PKC-epsilon translocation inhibitory peptide (PKC-epsilon-TIP)] but was not affected by a PKC-delta inhibitor (rottlerin). After DADLE infusion, PKC-epsilon, but not PKC-delta, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser(368)) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-epsilon-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-epsilon activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-epsilon plays a crucial role in delta-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic delta-opioid receptor activation.