Biomedical subjects

Toru Sasaki

Publications and source records attributed to Toru Sasaki.

43 records · Page 3Linked to original sources

PubMed2002

kappa-Hefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function. Importance of the functional diad in potassium channel selectivity.

An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, kappa-hefutoxin1 and kappa-hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that kappa-hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any beta-sheets. Based on the presence of the functional diad (Tyr(5)/Lys(19)) at a distance (6.0 +/- 1.0 A) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. kappa-Hefutoxin 1 not only blocks the voltage-gated K(+)-channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of kappa-hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

Amino Acid Sequence↗

PubMed2002

Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome.

Dsk2p from Saccharomyces cerevisiae belongs to the class of proteins that contain a ubiquitin-like (UbL) domain at the N terminus together with a ubiquitin-associated (UBA) domain at the C terminus. We show here that the C-terminal UBA domain of Dsk2p binds to K48-linked polyubiquitin chains, and the N-terminal UbL domain of Dsk2p interacts with the proteasome. Overexpression of Dsk2p caused the accumulation of large amounts of polyubiquitin, and extragenic suppressors of the Dsk2p-mediated lethality proved to be temperature-sensitive mutations in two proteasome subunits, rpn1 and pre2. K48-linked ubiquitin-dependent degradation was impaired by disruption of the DSK2 gene. These results indicate that Dsk2p is K48-linked polyubiquitin-binding protein and also interacts with the proteasome. We discuss a possible role of adaptor function of Dsk2p via its UbL and UBA domains in the ubiquitin-proteasome pathway.

Binding Sites↗

PubMed2002

Demonstration of competition between endogenous dopamine and [11C]raclopride binding in in vitro brain slices using a dynamic autoradiography technique.

To elucidate the mechanism of in vivo binding competition between radioligand and endogenously released transmitter, we examined the influence of depolarization-induced dopamine (DA) release on [11C]raclopride-specific binding to D2 receptors in slices of living brain tissues using dynamic positron autoradiography. Rat brain slices were incubated in a chamber with [11C]raclopride in oxygenated medium at 34 degrees C for 150 min. Two-dimensional images of radioactivity in the slices were recorded on a storage phosphor screen and dynamic changes were measured. When the brain slices were exposed to the depolarization agents (25 mM K+, 50 mM K+, and 20 microM veratridine), the percentage inhibition of striatal [11C]raclopride-specific binding was 22 +/- 4%, 44 +/- 8% and 54 +/- 7% of the control, respectively. The percentage inhibition of [11C]raclopride-specific binding during each depolarization treatment agreed proportionally with the amount of DA released into the medium. However, preexposure of brain slices to the same depolarization treatment (50 mM K+) did not affect the [11C]raclopride-specific binding, suggesting that the reduction in receptor density and/or affinity was not involved in the decrease of [11C]raclopride-specific binding. [11C]Raclopride-specific binding decreased dose-dependently in the presence of exogenously added DA (range 0.005-3mM). The synaptic DA concentration during each depolarization treatment estimated using three different methods. These results suggest that the decrease of [11C]raclopride-specific binding to striatal slices following evoked DA release is due to competition between endogenous DA and raclopride. It is unlikely that changes in D(2) receptor density or in affinity of the receptors for raclopride are involved. These results provide supportive evidence for in vivo binding competition between radioligand and endogenous neurotransmitter.

Animals↗

PubMed2002

Assessment of choline uptake for the synthesis and release of acetylcholine in brain slices by a dynamic autoradiographic technique using [11C]choline.

The uptake of choline for the synthesis and release of acetylcholine was investigated in brain slices by dynamic positron autoradiography using [11C]choline. Brain slices (330 microm) were incubated with [11C]choline in oxygenated Krebs-Ringer medium at 34 degrees C and serial two-dimensional time-resolved images of the uptake and release of radioactivity were recorded on Storage Phosphor screens. [11C]choline uptake increased with the period of incubation and was 1.9 times higher in the striatum than cerebral cortex. The uptake in the striatum was significantly diminished by hemicholinium-3 (HC-3), an inhibitor of high-affinity choline uptake. Pretreatment of brain slices with 50 mM K(+) for 20 min enhanced the uptake in striatum. The uptake of [11C]choline in brain slices was saturable using nonlabeled choline. Two uptake systems, a high-affinity and a low-affinity system, were confirmed to exist by kinetic analysis using Lineweaver-Burk plots. The 11C radioactivity that had accumulated in the striatum disappeared on treatment with veratridine, a depolarization agent, in the presence of HC-3. This pattern of disappearance was consistent with that of the appearance of unlabeled and labeled acetylcholine in the medium. These results indicate that this method is useful for obtaining information regarding the uptake of choline for the synthesis and release of acetylcholine in live brain tissues.

Acetylcholine↗

PubMed2002

Total middle ear reconstructive surgery for the radicalized ear.

OBJECTIVE: To evaluate the efficacy of total middle ear reconstructive surgery (TMRS) for patients with open cavity problems. STUDY DESIGN: Retrospective study of patients treated between 1994 and 1997. SETTING: Tertiary care, referral medical center. PATIENTS: Fifty-nine consecutive patients (62 ears) with open cavity problems (draining ears) who underwent TMRS. MAIN OUTCOME MEASURES: Postoperative states of the ears and complications were evaluated in two groups, with and without persistent purulent otorrhea at the time of surgery. RESULTS: In the noninfected group, ears without otorrhea were maintained in 12 (100%) of 12 ears in the short term and 10 (83%) of 12 ears in the long term. In the infected group, the proportions were 40 (80%) of 50 ears and 35 (73%) of 48 ears, for the short and long terms, respectively. Hollowing and/or retraction of the reconstructed canal wall was observed in 1 (8%) of 12 ears of the noninfected group and in 15 (31%) of 48 ears of the infected group after long-term follow-up. Postoperative complications were encountered in 9 ears (14.5%) in the infected group only. CONCLUSIONS: Total middle ear reconstructive surgery is considered useful for the management of persistent discharge from radicalized cavities. It was found important to minimize infection at the time of surgery to achieve satisfactory results.

Adult↗

PubMed2002

PF1163A, a novel antifungal agent, inhibit ergosterol biosynthesis at C-4 sterol methyl oxidase.

PF1163A and B are a pair of antifungal agents isolated from a fermentation broth of Penicillium sp. PF1163A inhibited ergosterol synthesis in Saccharomyces cerevisiae, resulting in an accumulation of 4,4-dimethylzymosterol and a decrease of ergosterol. The ERG25 strain overexpressing the ERG25 gene was resistant to PF1163A. ERG25p is a C-4 sterol methyl oxidase known to be essential for the viability of yeast and fungi because of the known role of ERG25 gene disruption in S. cerevisiae-led lethality. ERG25p is the enzyme responsible for the first step in the removal of the two methyl groups at the C-4 position of sterol. From the results obtained here, we conclude that PF1163A is a novel natural antifungal that inhibits C-4 sterol methyl oxidase.

Antifungal Agents↗

PubMedDate not supplied

Optimal dose-finding study of bi-weekly paclitaxel in unresectable advanced or recurrent gastric cancer.

BACKGROUND: Tri-weekly and weekly regimens of paclitaxel have been reported to be effective in unresectable advanced or recurrent gastric cancer. The present study was conducted to determine the optimal dose of bi-weekly paclitaxel and evaluate safety and antitumor effect. PATIENTS AND METHODS: The study included patients with a histologically confirmed diagnosis of advanced or recurrent gastric cancer. Paclitaxel was intravenously infused for 1 h bi-weekly. One cycle consisted of four weeks and at least two cycles were performed. Dose levels 1, 2, 3, 4 and 5 were 100, 120, 140, 160 and 180 mg/m2, respectively. RESULTS: Of the 21 patients enrolled, 18 patients (85.7%) had received prior treatment. The maximum tolerated dose was 160 mg/m2 (level 4) and dose-limiting toxicities included grade 3 fatigue, anorexia and neuropathy and grade 4 neutropenia and allergic reaction. The recommended dose was designated as to be 140 mg/m2 (level 3). At this dose level, only one patient experienced a dose-limiting toxicity of neutropenia. No patient had grade 3 or higher non-hematological toxicity. The response rate was 12.5% (2/16) and 14 patients (87.5%) had partial response or stable disease. The median number of treatment courses was four (up to 13 courses). The median survival time was 222 days and the time to progression was 76 days. CONCLUSION: In advanced or recurrent gastric cancer, bi-weekly paclitaxel may be safely administered at a recommended dose of 140 mg/m2. A phase II study is now underway.

Adult↗