Mechanical Modulation of ATP-binding Affinity of V1-ATPase

V₁-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F₁-ATPase (the closest relative of V₁-ATPase evolutionarily), the role of ATP binding for V₁-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V₁-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V₁-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V₁-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (k_on) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (k_off) was exponentially reduced. The angle dependence of the k_off of V₁-ATPase was significantly smaller than that of F₁-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V₁-ATPase. When V₁-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, k_on was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V₁-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions.     

Incidence of a skeletal deformity (truncated upper jaw) in an isolated population of white-spotted charr Salvelinus leucomaenis

Although dorsal and caudal fin deformities (curvature), abnormal body color patterns, and fluctuating asymmetry of meristic characters have been previously reported for white-spotted charr (Salvelinus leucomaenis), the incidence of skeletally deformed individuals in the wild are extremely rare. Here, we report on skeletally deformed individuals of white-spotted charr with extremely truncated upper jaw, in a headwater reach of the Yoneshiro River, northern Honshu, Japan. In this study, we examined the genetic diversity of deformed and non-deformed populations of white-spotted charr using polymorphic microsatellite DNA markers. We also compared individual growth histories of deformed individuals with normal individuals using a back-calculation of fish lengths at previous ages from otolith annuli. We collected 41 and 100 individuals from the below- and above-waterfall sites, respectively. Of these, two individuals from the above-waterfall site were skeletally deformed, whereas the other individuals had a normal morphology. Microsatellite DNA genetic diversities of the above-waterfall population were notably lower than those of the just below-waterfall population. Individuals of the above-waterfall population had significantly higher internal relatedness and lower standardized heterozygosity values compared with those of the below-waterfall population, indicating higher inbreeding situation of the above-waterfall individuals compared with individuals in the below-waterfall site. Back-calculation of individual growth using otoliths revealed that deformed individuals grew significantly slower than sympatric normal individuals. Although incidence of deformed individuals in the wild might be related with accumulated inbreeding as suggested for other fishes, mechanisms for their long-term persistence in the wild remain unclear.     

Characterization of Edenia gomezpompae Isolated from a Patient with Keratitis

Edenia gomezpompae, isolated from a patient suffering from keratitis, who was an agricultural worker with a 4-year history of diabetes mellitus, is described. Fungal hyphae were detected in corneal scrapings after microscopic examination and repeated culture. Isolates were identified by morphology and by sequencing the internal transcribed spacer region of ribosomal DNA. Chlamydospore characteristics of E. gomezpompae are described for the first time. The in vitro antifungal susceptibilities of the isolate were tested using five antifungal agents. With the exception of fluconazole, the other drugs (natamycin, amphotericin B, itraconazole, and voriconazole) assayed were highly effective against this fungus. The keratitis was cured after debridement of the corneal ulcer and treatment with 5 % natamycin. After 3 months, when the patient was reexamined, the corneal ulcer showed signs of healing, with no recurrence. To our knowledge, this is the first reported case of keratitis caused by E. gomezpompae, as well as being the first known case of disease in humans caused by this species.     

Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales

The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein–protein complexes, which suggested the possibility of self-assembling of STOPP. Protein–protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin.     

Contributions of phase resetting and interlimb coordination to the adaptive control of hindlimb obstacle avoidance during locomotion in rats: a simulation study

Obstacle avoidance during locomotion is essential for safe, smooth locomotion. Physiological studies regarding muscle synergy have shown that the combination of a small number of basic patterns produces the large part of muscle activities during locomotion and the addition of another pattern explains muscle activities for obstacle avoidance. Furthermore, central pattern generators in the spinal cord are thought to manage the timing to produce such basic patterns. In the present study, we investigated sensory-motor coordination for obstacle avoidance by the hindlimbs of the rat using a neuromusculoskeletal model. We constructed the musculoskeletal part of the model based on empirical anatomical data of the rat and the nervous system model based on the aforementioned physiological findings of central pattern generators and muscle synergy. To verify the dynamic simulation by the constructed model, we compared the simulation results with kinematic and electromyographic data measured during actual locomotion in rats. In addition, we incorporated sensory regulation models based on physiological evidence of phase resetting and interlimb coordination and examined their functional roles in stepping over an obstacle during locomotion. Our results show that the phase regulation based on interlimb coordination contributes to stepping over a higher obstacle and that based on phase resetting contributes to quick recovery after stepping over the obstacle. These results suggest the importance of sensory regulation in generating successful obstacle avoidance during locomotion.     

Genetic and family structure in a group of 165 common bottlenose dolphins caught off the Japanese coast

The biological and genetic structure of common bottlenose dolphins (Tursiops truncatus) that migrate seasonally near Japan remains largely unknown. We investigated the genetic and family structure in a group of 165 common bottlenose dolphins caught off the coast of Japan using mitochondrial DNA (mtDNA) and 20 microsatellite DNA markers. Phylogenetic analysis of the mtDNA control region sequences suggested that the dolphins were related more closely to oceanic types from Chinese waters than other geographic regions. The information on sex, sexual maturation and age together with the genetic markers revealed a strong likelihood for 37 familial relationships related mostly to maternity and an under‐representation of juvenile female offspring. The maternal dolphins had a similar offspring‐birth interval as the coastal types from North Atlantic Ocean, but a slightly younger first‐progeny age. The sex bias in the captured group was particularly marked towards an over‐representation of males among the young and immature dolphins, whereas the mature adults had an equal number of males and females. These results should be useful for future comparative biological, genetic and evolutionary investigations of bottlenose dolphins from the North Pacific Ocean with those from other regions.     

Photochemistry of Acetabularia rhodopsin II from a marine plant, Acetabularia acetabulum

Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986-998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P(i)) were sufficient to simulate the data. Noticeably, both P(3) and P(4) contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK(a) values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK(a) of Asp81 (Asp85(BR)) in the dark, Asp92 (Asp96(BR)) at N, Glu199 (Glu204(BR)) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.