The Min oscillator uses MinD-dependent conformational changes in MinE to spatially regulate cytokinesis.

In E. coli, MinD recruits MinE to the membrane, leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring. How these proteins interact, however, is not clear because the MinD-binding regions of MinE are sequestered within a six-stranded ß sheet and masked by N-terminal helices. minE mutations that restore interaction between some MinD and MinE mutants were isolated. These mutations alter the MinE structure leading to release of the MinD-binding regions and the N-terminal helices that bind the membrane. Crystallization of MinD-MinE complexes revealed a four-stranded ß sheet MinE dimer with the released ß strands (MinD-binding regions) converted to α helices bound to MinD dimers. These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface.

FtsA acts through FtsW to promote cell wall synthesis during cell division in Escherichia coli.

In Escherichia coli, FtsQLB is required to recruit the essential septal peptidoglycan (sPG) synthase FtsWI to FtsA, which tethers FtsZ filaments to the membrane. The arrival of FtsN switches FtsQLB in the periplasm and FtsA in the cytoplasm from a recruitment role to active forms that synergize to activate FtsWI. Genetic evidence indicates that the active form of FtsQLB has an altered conformation with an exposed domain of FtsL that acts on FtsI to activate FtsW. However, how FtsA contributes to the activation of FtsW is not clear, as it could promote the conformational change in FtsQLB or act directly on FtsW. Here, we show that the overexpression of an activated FtsA (FtsA*) bypasses FtsQ, indicating it can compensate for FtsQ's recruitment function. Consistent with this, FtsA* also rescued FtsL and FtsB mutants deficient in FtsW recruitment. FtsA* also rescued an FtsL mutant unable to deliver the periplasmic signal from FtsN, consistent with FtsA* acting on FtsW. In support of this, an FtsW mutant was isolated that was rescued by an activated FtsQLB but not by FtsA*, indicating it was specifically defective in activation by FtsA. Our results suggest that in response to FtsN, the active form of FtsA acts on FtsW in the cytoplasm and synergizes with the active form of FtsQLB acting on FtsI in the periplasm to activate FtsWI to carry out sPG synthesis.

Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes.

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try to understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.

Fluorinated CRA13 analogues: Synthesis, in vitro evaluation, radiosynthesis, in silico and in vivo PET study.

Fluorine is a unique atom that imparts distinct properties to bioactive molecules upon incorporation. Herein, we prepare and study fluorinated derivatives of the nanomolar affine peripherally restricted dual CB1R/CB2R agonist; CRA13 and its analogs. Binding affinity evaluation relative to CRA13 proved the stronger binding affinity of compound 7c to CB1R and CB2R by 6.95 and 5.64 folds. Physicochemical properties evaluation proved compound 7c improved lipophilicity profile suggesting some enhanced BBB penetration relative to CRA13. Radiosynthesis of 18F-labeled compound 7c was conducted conveniently affording pure hot ligand. In vivo PET study investigation demonstrated efficient distribution of 18F-labeled compound 7c in peripheral tissues visualizing peripheral CB1R/CB2R generating time-activity-curves showing good standard uptake values. Despite enhanced BBB penetration and increased cannabinoid receptors binding affinity, low brain uptake of 7c was observed. In silico docking study explained the measured binding affinities of compounds 7a-d to CB1R. While most of previous efforts aimed to develop central cannabinoid PET imaging agents, 18F-labeled compound 7c might be a promising agent serving as a universal CB1R/CB2R PET imaging agents for diagnosis and therapy of various diseases correlated with peripheral cannabinoid system. It might also serve as a lead compound for development of PET imaging of peripheral and central cannabinoid systems.

MinE conformational dynamics regulate membrane binding, MinD interaction, and Min oscillation.

In Escherichia coli MinE induces MinC/MinD to oscillate between the ends of the cell, contributing to the precise placement of the Z ring at midcell. To do this, MinE undergoes a remarkable conformational change from a latent 6ß-stranded form that diffuses in the cytoplasm to an active 4ß-stranded form bound to the membrane and MinD. How this conformational switch occurs is not known. Here, using hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) we rule out a model in which the two forms are in rapid equilibrium. Furthermore, HDX-MS revealed that a MinE mutant (D45A/V49A), previously shown to produce an aberrant oscillation and unable to assemble a MinE ring, is more rigid than WT MinE. This mutant has a defect in interaction with MinD, suggesting it has difficulty in switching to the active form. Analysis of intragenic suppressors of this mutant suggests it has difficulty in releasing the N-terminal membrane targeting sequences (MTS). These results indicate that the dynamic association of the MTS with the ß-sheet is fine-tuned to balance MinE's need to sense MinD on the membrane with its need to diffuse in the cytoplasm, both of which are necessary for the oscillation. The results lead to models for MinE activation and MinE ring formation.

MinC and FtsZ mutant analysis provides insight into MinC/MinD-mediated Z ring disassembly.

The Min system negatively regulates the position of the Z ring, which serves as a scaffold for the divisome that mediates bacterial cytokinesis. In Escherichia coli, this system consists of MinC, which antagonizes assembly of the tubulin homologue FtsZ. MinC is recruited to the membrane by MinD and induced by MinE to oscillate between the cell poles. MinC is a dimer with each monomer consisting of functionally distinct MinCN and MinCC domains, both of which contact FtsZ. According to one model, MinCC/MinD binding to the FtsZ tail positions MinCN at the junction of two GDP-containing subunits in the filament, leading to filament breakage. Others posit that MinC sequesters FtsZ-GDP monomers or that MinCN caps the minus end of FtsZ polymers and that MinCC interferes with lateral interactions between FtsZ filaments. Here, we isolated minC mutations that impair MinCN function and analyzed FtsZ mutants resistant to MinC/MinD. Surprisingly, we found mutations in both minC and ftsZ that differentiate inhibition by MinC from inhibition by MinC/MinD. Analysis of these mutations suggests that inhibition of the Z ring by MinC alone is due to sequestration, whereas inhibition by MinC/MinD is not. In conclusion, our genetic and biochemical data support the model that MinC/MinD fragments FtsZ filaments.

Synthesis of oxidative metabolites of CRA13 and their analogs: Identification of CRA13 active metabolites and analogs thereof with selective CB2R affinity.

CRA13; a peripheral dual CB1R/CB2R agonist with clinically proven analgesic properties, infiltrates into CNS producing adverse effects due to central CB1R agonism. Such adverse effects might be circumvented by less lipophilic compounds with attenuated CB1R affinity. Metabolism produces less lipophilic metabolites that might be active metabolites. Some CRA13 oxidative metabolites and their analogues were synthesized as less lipophilic CRA13 analogues. Probing their CB1R and CB2R activity revealed the alcohol metabolite 8c as a more potent and more effective CB2R ligand with attenuated CB1R affinity relative to CRA13. Also, the alcohol analogue 8b and methyl ester 12a possessed enhanced CB2R affinity and reduced CB1R affinity. The CB2R binding affinity of alcohol analogue 8b was similar to CRA13 while that of methyl ester 12a was more potent. In silico study provided insights into the possible molecular interactions that might explain the difference in the elicited biological activity of these compounds.

Jmjd2C increases MyoD transcriptional activity through inhibiting G9a-dependent MyoD degradation.

Skeletal muscle cell differentiation requires a family of proteins called myogenic regulatory factors (MRFs) to which MyoD belongs. The activity of MyoD is under epigenetic regulation, however, the molecular mechanism by which histone KMTs and KDMs regulate MyoD transcriptional activity through methylation remains to be determined. Here we provide evidence for a unique regulatory mechanism of MyoD transcriptional activity through demethylation by Jmjd2C demethylase whose level increases during muscle differentiation. G9a decreases MyoD stability via methylation-dependent MyoD ubiquitination. Jmjd2C directly associates with MyoD in vitro and in vivo to demethylate and stabilize MyoD. The hypo-methylated MyoD due to Jmjd2C is significantly more stable than hyper-methylated MyoD by G9a. Cul4/Ddb1/Dcaf1 pathway is essential for the G9a-mediated MyoD degradation in myoblasts. By the stabilization of MyoD, Jmjd2C increases myogenic conversion of mouse embryonic fibroblasts and MyoD transcriptional activity with erasing repressive H3K9me3 level at the promoter of MyoD target genes. Collectively, Jmjd2C increases MyoD transcriptional activity to facilitate skeletal muscle differentiation by increasing MyoD stability through inhibiting G9a-dependent MyoD degradation.

Fbxo25 controls Tbx5 and Nkx2-5 transcriptional activity to regulate cardiomyocyte development.

The ubiquitin-proteasome system (UPS) plays an important role in protein quality control, cellular signalings, and cell differentiation through the regulated turnover of key transcription factors in cardiac tissue. However, the molecular mechanism underlying Fbxo25-mediated ubiquitination of cardiac transcription factors remains elusive. We report that an Fbxo25-mediated SCF ubiquitination pathway regulates the protein levels and activities of Tbx5 and Nkx2-5 based on our studies using MG132, proteasome inhibitor, and the temperature sensitive ubiquitin system in ts20 cells. Our data indicate that Fbxo25 directly interacts with Tbx5 and Nkx2-5 in vitro and in vivo. In support of our findings, a dominant-negative mutant of Fbxo25, Fbxo251-236, prevents Tbx5 degradation and increases Tbx5 transcriptional activity in a Tbx5 responsive luciferase assay. Therefore, Fbxo25 facilitates Tbx5 degradation in an SCF-dependent manner. In addition, the silencing of endogenous Fbxo25 increases Tbx5 and Nkx2-5 mRNA levels and suppresses mESC-derived cardiomyocyte differentiation. Likewise, the exogenous expression of FBXO25 downregulates NKX2-5 level in human ESC-derived cardiomyocytes. In myocardial infarction model, Fbxo25 mRNA decreases, whereas the mRNA and protein levels of Tbx5 and Nkx2-5 increase. The protein levels of Tbx5 and Nkx2-5 are regulated negatively by Fbxo25-mediated SCF ubiquitination pathway. Thus, our findings reveal a novel mechanism for regulation of SCFFbox25-dependent Nkx2-5 and Tbx5 ubiquitination in cardiac development and provide a new insight into the regulatory mechanism of Nkx2-5 and Tbx5 transcriptional activity.

Oligomerization of FtsZ converts the FtsZ tail motif (conserved carboxy-terminal peptide) into a multivalent ligand with high avidity for partners ZipA and SlmA.

A short conserved motif located at the carboxy terminus of FtsZ, referred to here as the CCTP (conserved carboxy-terminal peptide), is required for the interaction of FtsZ with many of its partners. In Escherichia coli interaction of FtsZ with its membrane anchors, ZipA and FtsA, as well as the spatial regulators of Z-ring formation, MinC and SlmA, requires the CCTP. ZipA interacts with FtsZ with high affinity and interacts with the CCTP with low affinity, but the reason for this difference is not clear. In this study, we show that this difference is due to the oligomerization of FtsZ converting the CCTP to a multivalent ligand that binds multiple ZipAs bound to a surface with high avidity. Artificial dimerization of the CCTP is sufficient to increase the affinity for ZipA in vitro. Similar principles apply to the interaction of FtsZ with SlmA. Although done in vitro, these results have implications for the recruitment of FtsZ to the membrane in vivo, the interaction of FtsZ with spatial regulators and the reconstitution of FtsZ systems in vitro.

MinC/MinD copolymers are not required for Min function.

In Escherichia coli, precise placement of the cytokinetic Z ring at midcell requires the concerted action of the three Min proteins. MinD activates MinC, an inhibitor of FtsZ, at least in part, by recruiting it to the membrane and targeting it to the Z ring, while MinE stimulates the MinD ATPase inducing an oscillation that directs MinC/MinD activity away from midcell. Recently, MinC and MinD were shown to form copolymers of alternating dimers of MinC and MinD, and it was suggested that these copolymers are the active form of MinC/MinD. Here, we use MinD mutants defective in binding MinC to generate heterodimers with wild-type MinD that are unable to form MinC/MinD copolymers. Similarly, MinC mutants defective in binding to MinD were used to generate heterodimers with wild-type MinC that are unable to form copolymers. Such heterodimers are active and in the case of MinC were shown to mediate spatial regulation of the Z ring demonstrating that MinC/MinD copolymer formation is not required. Our results are consistent with a model in which a membrane anchored MinC/MinD complex is targeted to the Z ring through the conserved carboxy tail of FtsZ leading to breakage of FtsZ filaments.

Synthesis of novel flavone derivatives possessing substituted benzamides and their biological evaluation against human cancer cells.

Baicalein is a well-known flavone derivative that possesses diverse biological properties, such as anticancer, antioxidant and anti-inflammatory activities. Numerous baicalein derivatives, including 5,6,7-trimethoxyflavone, have been synthesized with the aim of enhancing its inherent biological activities. In the present work, new flavones, possessing an N-aroylamine-substituent on the B-ring, were synthesized to improve the cytotoxicity of baicalein and 5,6,7-trimethoxyflavone against human cancer cell lines. The majority of the flavones synthesized exhibited greater cytotoxicity than baicalein and 5,6,7-trimethoxyflavone against HepG2 and MCF-7 cells. Among them, compounds 5n, possessing a 3-methoxybenzoylamino group, exhibited great cytotoxic effects on HepG2 (GI50=7.06µM) and MCF-7 (GI50=7.67µM) cells. In contrast, N-aroylamine-substituted 5-hydroxy-6,7-dimethoxyflavone derivatives showed greater cytotoxicity against MCF-7 than HepG2 cells, indicating that the replacement of a 5-methoxy group on the A-ring with a 5-hydroxy group has a marked influence on the cytotoxicity profile.

Mechanistic studies on the Rh(III)-mediated amido transfer process leading to robust C-H amination with a new type of amidating reagent.

Mechanistic investigations on the Cp*Rh(III)-catalyzed direct C-H amination reaction led us to reveal the new utility of 1,4,2-dioxazol-5-one and its derivatives as highly efficient amino sources. Stepwise analysis on the C-N bond-forming process showed that competitive binding of rhodium metal center to amidating reagent or substrate is closely related to the reaction efficiency. In this line, 1,4,2-dioxazol-5-ones were observed to have a strong affinity to the cationic Rh(III) giving rise to dramatically improved amidation efficiency when compared to azides. Kinetics and computational studies suggested that the high amidating reactivity of 1,4,2-dioxazol-5-one can also be attributed to the low activation energy of an imido-insertion process in addition to the high coordination ability. While the characterization of a cationic Cp*Rh(III) complex bearing an amidating reagent was achieved, its facile conversion to an amido-inserted rhodacycle allowed for a clear picture on the C-H amidation process. The newly developed amidating reagent of 1,4,2-dioxazol-5-ones was applicable to a broad range of substrates with high functional group tolerance, releasing carbon dioxide as a single byproduct. Additional attractive features of this amino source, such as they are more convenient to prepare, store, and use when compared to the corresponding azides, take a step closer toward an ideal C-H amination protocol.

Whole-exome sequencing reveals diverse modes of inheritance in sporadic mild to moderate sensorineural hearing loss in a pediatric population.

PURPOSE: This study was designed to delineate genetic contributions, if any, to sporadic forms of mild to moderate sensorineural hearing loss (SNHL) not related to GJB2 mutations (DFNB1) in a pediatric population. METHODS: We recruited 11 non-DFNB1 simplex cases of mild to moderate SNHL in children. We applied whole-exome sequencing to all 11 probands. We used a filtering strategy assuming that de novo variants of known autosomal dominant (AD) deafness genes, biallelic mutations in autosomal recessive (AR) genes, monoallelic mutations in X chromosome genes for males, and digenic inheritance could be associated. Candidate variants first were prioritized with allele frequency in public databases and confirmed by a phase or a segregation test in each family. Additional information from the literature or public databases was used to identify strong candidate variants. RESULTS: Strong candidate variants were detected in 5 of 11 probands (45.4%). A diverse mode of inheritance implicated the sporadic occurrence of the phenotype. AR mutations in OTOGL and SERPINB6 and digenic inheritance involving two deafness genes, GPR98 and PDZ7, were detected. A de novo AD mutation also was detected in TECTA and MYH14. No syndromic feature was detected in individuals with GPR98/PDZ7 or MYH14 variants in our cohort at this moment. CONCLUSION: Mild to moderate pediatric SNHL, even if sporadic, features a strong genetic etiology and can manifest via diverse modes of inheritance. In addition, a multidisciplinary approach should be used for a correct diagnosis.

High-level expression of Hsp90ß is associated with poor survival in resectable non-small-cell lung cancer patients.

AIMS: The aim of this study was to investigate the expression of Hsp90ß and GRP94, and elucidate the clinical significance of their expression, in patients with resectable non-small-cell lung cancer (NSCLC). METHODS AND RESULTS: Surgical tissue specimens were obtained from 208 patients with NSCLC who underwent surgical resection. The expression levels of Hsp90ß and GRP94 were assessed with tissue microarrays and immunohistochemistry. No correlations were observed between Hsp90ß or GRP94 expression and several clinicopathological factors. The high-Hsp90ß group [median overall survival (OS) 20.4 months; 95% confidence interval (CI) 0.000-40.864] showed a significant decrease in OS as compared with the low-Hsp90ß group (median OS not reached; P = 0.003). In contrast to the Hsp90ß analysis, the GRP94 analysis did not show a difference in OS. Moreover, in subgroup analyses of patients with squamous cell carcinoma histology, OS (P = 0.012) and relapse-free survival (P = 0.044) were significantly worse in the high-Hsp90ß group than in the low-Hsp90ß group. Multivariate analysis suggested that old age [hazard ratio (HR) 1.568; 95% CI 1.019-2.412; P = 0.041], advanced disease (HR 2.066; 95% CI 1.218-3.502; P = 0.007) and high Hsp90ß expression (HR 1.802; 95% CI 1.061-3.060; P = 0.029) were independent poor prognostic factors for OS. CONCLUSIONS: Hsp90ß expression might be a useful marker of poor OS, although further large prospective studies are warranted to validate our findings.

Efficacy and safety of deferasirox estimated by serum ferritin and labile plasma iron levels in patients with aplastic anemia, myelodysplastic syndrome, or acute myeloid leukemia with transfusional iron overload.

BACKGROUND: Patients receiving red blood cell (RBC) transfusions are at risk of iron overload, which can cause significant organ damage and is an important cause of morbidity and mortality. STUDY DESIGN AND METHODS: This study was an open-label, single-arm, prospective clinical study to evaluate the efficacy and safety of deferasirox (DFX) in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML). Patients with serum ferritin levels of at least 1000 ng/mL and ongoing transfusion requirements were enrolled. DFX was administered for up to 1 year. A total of 100 patients were enrolled. RESULTS: Serum ferritin levels decreased significantly following treatment (from 2000 to 1650 ng/mL, p = 0.004). The median absolute reduction in serum ferritin levels was -65 ng/mL in AA (p = 0.037), -647 ng/mL in lower-risk MDS (MDS-LR; p = 0.007), and -552 ng/mL in higher-risk MDS (MDS-HR)/AML (p = 0.482). Mean labile plasma iron (LPI) levels decreased from 0.24 µmol/L at baseline to 0.03 µmol/L at 1 year in all patients (p = 0.036). The mean LPI reduction in each group was -0.17 µmol/L in AA, -0.21 µmol/L in MDS-LR, and -0.30 µmol/L in MDS-HR/AML. Gastrointestinal disorders were commonly observed among groups (16.0%). DFX was temporarily skipped for adverse events in seven patients (7.0%) and was permanently discontinued in 11 patients (11.0%). CONCLUSION: DFX reduced serum ferritin and LPI levels in patients with transfusional iron overload. Despite the relatively high percentage of gastrointestinal side effects, DFX was tolerable in all subgroups.

Characteristics of the perception for unilateral facial nerve palsy.

Patients with facial nerve palsy (FNP) are actually evaluated by other people rather than doctors or the patients themselves. This study was performed to investigate the characteristics of the perception of unilateral FNP in Korean people. A questionnaire using photographs of four patients with four different grades (House-Brackmann) of FNP was given to two hundred people with no FNP. Subjects of each gender, ranging from 20 to 69 years of age, participated. The questionnaire, showing facial expressions of resting, smiling, whistling, eye closing, and frowning, consisted of questions concerning the identification and the involved side of FNP, the unnatural areas of the face, and the unnaturalness of the facial expressions. The overall identification rate of FNP was 75.0%. The identification rate increased according to the increase in the grade of the patient's FNP (p < .001). The overall detection rate of the involved side was 54.5%, and that rate decreased with increasing subject age (p < .001). The area of the most unnatural facial expression was reported to be the mouth, followed by the eyes and cheeks. The most unnatural facial expression was also reported to be smiling, followed by eye closing and whistling. There was no difference in the identification rate of FNP according to education level. However, the overall detection rate of the involved side was higher in the high-education group (p < .001). The detection rate for the involved side of FNP was lower than the rate of identification of FNP and was significantly low in the middle-aged/elderly and low-education level groups.

A prospective, open-label, multicenter study of the clinical efficacy of extended-release hydromorphone in treating cancer pain inadequately controlled by other analgesics.

PURPOSE: The objective of this study was to evaluate whether extended-release hydromorphone (osmotic-controlled release oral delivery system [OROS] hydromorphone) treatment provided pain relief in cancer patients whose pain was inadequately controlled by other analgesics. METHODS: In this prospective, open-label, multicenter trial, patients who have sustained cancer pain with other analgesics were enrolled. After the baseline evaluation (visit 1), OROS hydromorphone was administered. Two evaluations (visits 2 and 3) were made: 29 ± 7 and 57 ± 7 days later, respectively. The primary end point was the pain intensity difference (PID) at visit 3 relative to visit 1 (expressed as percent PID). RESULTS: In total, 879 patients were screened and 432 completed all three visits. Of the 874 full analysis set patients, 343 (39.2 %) improved by more than 30 % PID. Of the 432 per-protocol patients, 282 (65.3 %) improved by more than 30 % PID. At visits 2 and 3, the degree of sleep disturbance, the number of awakenings, and the degree of sleep satisfaction were significantly better than at visit 1 (all P < 0.0001 for both visit 1-visit 2 and visit 1-visit 3). However, this pain relief was not associated with improved quality of life (P = 0.326 and P = 0.055 for visit 1-visit 2 and visit 1-visit 3, respectively). CONCLUSIONS: This study suggested that active pain management using the strong opioid OROS hydromorphone was beneficial in the management of cancer pain that was not controlled by other analgesics.

Analysis of impact noise induced by hitting of titanium head golf driver.

The hitting of titanium head golf driver against golf ball creates a short duration, high frequency impact noise. We analyzed the spectra of these impact noises and evaluated the auditory hazards from exposure to the noises. Noises made by 10 titanium head golf drivers with five maximum hits were collected, and the spectra of the pure impact sounds were studied using a noise analysis program. The noise was measured at 1.7 m (position A) and 3.4 m (position B) from the hitting point in front of the hitter and at 3.4 m (position C) behind the hitting point. Average time duration was measured and auditory risk units (ARUs) at position A were calculated using the Auditory Hazard Assessment Algorithm for Humans. The average peak levels at position A were 119.9 dBA at the sound pressure level (SPL) peak and 100.0 dBA at the overall octave level. The average peak levels (SPL and overall octave level) at position B were 111.6 and 96.5 dBA, respectively, and at position C were 111.5 and 96.7 dBA, respectively. The average time duration and ARUs measured at position A were 120.6 ms and 194.9 units, respectively. Although impact noises made by titanium head golf drivers showed relatively low ARUs, individuals enjoying golf frequently may be susceptible to hearing loss due to the repeated exposure of this intense impact noise with short duration and high frequency. Unprotected exposure to impact noises should be limited to prevent cochleovestibular disorders.

Mechanism of the asymmetric activation of the MinD ATPase by MinE.

MinD is a component of the Min system involved in the spatial regulation of cell division. It is an ATPase in the MinD/ParA/Mrp deviant Walker A motif family which is within the P loop GTPase superfamily. Its ATPase activity is stimulated by MinE; however, the mechanism of this activation is unclear. MinD forms a symmetric dimer with two binding sites for MinE; however, a recent model suggested that MinE occupying one site was sufficient for ATP hydrolysis. By generating heterodimers with one binding site for MinE we show that one binding site is sufficient for stimulation of the MinD ATPase. Furthermore, comparison of structures of MinD and related proteins led us to examine the role of N45 in the switch I region. An asparagine at this position is conserved in four of the deviant Walker A motif subfamilies (MinD, chromosomal ParAs, Get3 and FleN) and we find that N45 in MinD is essential for MinE-stimulated ATPase activity and suggest that it is a key residue affected by MinE binding.