Biomedical subjects

Akio Yamazaki

Publications and source records attributed to Akio Yamazaki.

8 recordsLinked to original sources

PubMed2003

A critical role for ATP in the stimulation of retinal guanylyl cyclase by guanylyl cyclase-activating proteins.

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 +/- 0.20 mM), GCAP2 enhanced the retGC activity 10-13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3-4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 +/- 2.7 nM). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nM). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nM). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.

Adenine↗

PubMed2003

Fibrin glue and bioabsorbable felt patch for intraoperative intractable air leaks.

OBJECTIVE: We worked to devise a new way to prevent postoperative persistent air leaks in high-risk pulmonary surgery patients. METHODS: From November 1993 to June 2002, 60 patients with difficult to control intraoperative pulmonary air leakage were treated using bioabsorbable polyglycolide felt patches soaked in fibrin glue to cover the leakage site. RESULTS: After application, the felt patch adhered tightly to the lung surface without peeling off, enabling good leakage closure with only 2 ml of fibrin glue used. Air leakage was controlled successfully in 52 (86.7%) of the 60. Four of the 8 patients in whom this method failed to stop air leakage also developed mild pyothorax, with 2 requiring a second operation by video-assisted thoracic surgery. Leakage was eventually controlled in all patients, with no postoperative deaths relating to air leakage. CONCLUSIONS: Fibrin-glue-soaked bioabsorbable felt patches effectively seal intraoperative intractable air leaks. Felt patch use may increase the risk of postoperative infection. It should be considered for use on patients with fistulas that cannot be controlled by direct closure or otherwise intraoperatively and who may potentially develop uncontrollable air leakage postoperatively.

Absorbable Implants↗

PubMed2003

Neural network training with global optimization techniques.

This paper presents an approach of using Simulated Annealing and Tabu Search for the simultaneous optimization of neural network architectures and weights. The problem considered is the odor recognition in an artificial nose. Both methods have produced networks with high classification performance and low complexity. Generalization has been improved by using the backpropagation algorithm for fine tuning. The combination of simple and traditional search methods has shown to be very suitable for generating compact and efficient networks.

Algorithms↗

PubMed2002

Binding of cGMP to GAF domains in amphibian rod photoreceptor cGMP phosphodiesterase (PDE). Identification of GAF domains in PDE alphabeta subunits and distinct domains in the PDE gamma subunit involved in stimulation of cGMP binding to GAF domains.

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Palphabeta) and two identical regulatory subunits (Pgamma). Biochemical studies have shown that amphibian Palphabeta has high affinity, cGMP-specific, non-catalytic binding sites and that Pgamma stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pgamma-depleted membrane-bound PDE (20-40% Pgamma still present), a single type of cGMP-binding site with a relatively low affinity (K(d) approximately 100 nm) was observed, and addition of Pgamma increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pgamma reduced its ability to stimulate cGMP binding. Among these, the site involved in Pgamma phosphorylation by Cdk5 (positions 20-23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pgamma inhibition of the cGMP hydrolytic activity of Palphabeta. In addition, the Pgamma concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Palphabeta heterodimer contains two spatially and functionally distinct types of Pgamma-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Palphabeta-Pgamma interaction in which Pgamma, by binding to one of the two sites in Palphabeta, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.

Amino Acid Sequence↗

PubMed2002

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit (Pgamma) of retinal cgmp phosphodiesterase (PDE6): its implications in phototransduction.

Cyclic GMP phosphodiesterase (PDE6) is a key enzyme in vertebrate retinal phototransduction. After GTP/GDP exchange on the a subunit of transducin (Talpha) by illuminated rhodopsin, the GTP-bound form Talpha (GTP/Talpha) interacts with the regulatory subunit (Pgamma) of PDE6 to activate cGMP hydrolytic activity. The regulatory mechanism of PDE6 has been believed to be a typical G protein-mediated signal transduction process. We found that cyclin-dependent protein kinase 5 (Cdk5) phosphorylates Pgamma complexed with GTP/Talpha in vitro and in vivo. Phosphorylated Py dissociates from GTP/Talpha without GTP hydrolysis and interacts effectively with catalytic subunits of PDE6 to inhibit the enzyme activity. These observations provide new twists to the current model of retinal phototransduction. In this article, in addition to the details of Py phosphorylation by Cdk5, we review previous studies implying the Pgamma phosphorylation and the turnoff of PDE6 without GTP hydrolysis and indicate the direction for future studies of Py phosphorylation, including the possible involvement of Ca2+/Ca2+-binding proteins.

Amino Acid Motifs↗

PubMed2002

Measurement of membrane binding between recoverin, a calcium-myristoyl switch protein, and lipid bilayers by AFM-based force spectroscopy.

Myristoyl switch is a feature of several peripheral membrane proteins involved in signal transduction pathways. This unique molecular property is best illustrated by the "Ca(2+)-myristoyl switch" of recoverin, which is a Ca(2+)-binding protein present in retinal rod cells of vertebrates. In this transduction pathway, the Ca(2+)-myristoyl switch acts as a calcium sensor involved in cell recovery from photoactivation. Ca(2+) binding by recoverin induces the extrusion of its myristoyl group to the solvent, which leads to its translocation from cytosol to rod disk membranes. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the extent of membrane binding of recoverin in the absence and presence of calcium, and to quantify this force of binding. An adhesion force of 48 +/- 5 pN was measured between recoverin and supported phospholipid bilayers in the presence of Ca(2+). However, no binding was observed in the absence of Ca(2+). Experiments with nonmyristoylated recoverin confirmed these observations. Our results are consistent with previously measured extraction forces of lipids from membranes.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms.

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2002

A novel role of RGS9: inhibition of retinal guanylyl cyclase.

Cyclic GMP plays a key role in retinal phototransduction and its photoreceptor concentration is precisely controlled by the cooperative action of cGMP phosphodiesterase (PDE) and retinal guanylyl cyclase (retGC). However, studies of the relationship between these two systems have focused only on a Ca2+-mediated, indirect connection. This article summarizes our studies strongly suggesting that RGS9-1 is directly involved in the cooperative action of PDE and retGC, and that this ingenious mechanism plays an important role in tuning of cGMP concentration in photoreceptors.

Animals↗