Reapplication of the Pavlik Harness for Treatment of Developmental Dysplasia of the Hip After Initial Pavlik Harness Failure.

OBJECTIVE: The Pavlik harness (PH) has been widely used as the standard treatment for infants with developmental dysplasia of the hip (DDH). When the initial application of the PH fails, alternative treatments, such as closed reduction, open reduction, and reapplication of the PH will be considered. Compared with other treatments, reapplication of the PH offers certain advantages, including simplicity and reduced physical, and psychological stress, on both infants and caregivers. This study aims to investigate the effectiveness of reapplying the PH in patients with DDH. METHODS: This study included patients with DDH (complete dislocation) who were treated by reapplication of PH between 1988 and 2012. Patients who were able to follow-up for more than 5 years were included. We examined the reduction rate and several factors to identify indicators associated with successful reduction during reapplication, including age, sex, side of hip dislocation, and the presence of the Ortolani sign. At the final follow-up, hip development was assessed using the Severin classification, whereas avascular necrosis (AVN) was evaluated using the Kalamchi classification and the Salter criteria. RESULTS: A total of 56 patients (48 females and 8 males) and 57 hips were included in this study. The mean age at first and second application of PH was 4.2 months old (range: 0.12 to 6.4), and 5.8 months old (3.0 to 11.4), respectively. The reduction rate was 49% (28 out of 57 hips). Among the successfully reduced hips, the AVN rate was 3.6% (1 out of 28 hips). The Severin classification revealed 27 hips in class I and 1 hip in class III. Statistical analysis indicated a significantly higher proportion of left hip involvement in the reduction group (85% vs 41%, χ 2 test, P < 0.001). Although not statistically significant, the rate of positive Ortolani sign tended to be higher in the reduction group (61% vs 38%, χ 2 test, P = 0.06). CONCLUSION: The reapplication method demonstrated a 49% reduction rate and a low AVN rate of 3.6% in our study. It is worth considering for patients who fail the initial PH treatment, particularly in cases of left-side dislocation and a positive Ortolani sign during the initial application.

Determining factors that maintain physical function or increase frailty using the Kihon checklist among community-dwelling older adults: a six-year longitudinal study in Agano, Japan.

BACKGROUND: A significant increase in the older adult population in Japan will significantly increase healthcare costs. This study aimed to examine the risk factors contributing to robustness transitioning to frailty in older residents. METHODS: Participants were aged 70 in 2016 and 76 in 2022. Participants were evaluated using the Kihon Checklist (KCL). RESULTS: Participants for this longitudinal study included 444 older persons who completed the KCL surveys in 2016 and 2022. The follow-up rate was 80.6%; therefore, 358 participants were included in the analysis. The median KCL score increased significantly from 2 to 2016 to 3 in 2022 (p < 0.001). The prevalence of robustness significantly decreased from 60.9 to 48.6% (p = 0.042). In a stepwise logistic regression analysis, robustness was independently associated with regular continuous walks for 15 min and a body mass index of above 18.5%. The following variables were associated with the transition to prefrailty: experiencing a fall in the past year and not going out at least once a week. For the transition to frailty, the variables were turned to family or friends for advice, experienced a fall in the past year, and felt helpless in the last two weeks. The independent factor for the transition from prefrailty to frailty was having a BMI of less than 18.5. In contrast, the independent factor for improving from frailty to robustness or prefrailty was going out at least once a week. CONCLUSIONS: We recommend maintaining continuous walking for more than 15 min, maintaining a BMI of at least 18.5, and going out more than once a week to improve being house-bounded and depressive mood, not only to prevent the transition to prefrailty or frailty but also to improve frailty.

Positioning of Bristol Stool Form Scale type 3 in constipation treatment satisfaction: A multicenter study in Japan.

BACKGROUND AND AIM: Many patients are not satisfied with chronic constipation (CC) treatments. The aim of this study was to identify factors linked to CC treatment satisfaction or dissatisfaction. METHODS: Our study population included patients who received CC treatment at a clinic or hospital. CC was diagnosed by a physician based on the patient's complaint. Treatment satisfaction was evaluated using the 28th question of the Patient Assessment of Constipation Quality of Life questionnaire. RESULTS: We conducted this study at 28 facilities. We included 167 patients (mean age 66.7 ± 15.2 years, male:female ratio is 1:3.07). Sixty-eight (40.7%) of patients were satisfied with their constipation treatment. Treatment dissatisfaction of CC was significantly associated with frequency of bowel movement <3/week (odds ratio [OR] = 0.376, 95% confidence interval [CI]: 0.156-0.904, P = 0.029) or Bristol Stool Form Scale (BSFS) type 3 (OR = 0.401, 95% CI: 0.170-0.946, P = 0.037). CONCLUSIONS: Our study showed that CC patients with BSFS type3 were not satisfied with constipation treatment. In general, BSFS types 3-5 are defined as normal stools. Therefore, BSFS type 3 may be set as a treatment goal even though the patient is not satisfied. The pathophysiology of CC differs by region and patient background. Therefore, parameters used to define successful treatment will be different by patient or region. We should reconsider the positioning of BSFS type 3 to improve treatment satisfaction for CC.

Atomic force microscopy analysis of SasA-KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria.

The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC-binding EnvZ-like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA3mer ) and occasionally as a hexamer (SasA6mer ) in vitro. Previously, the molecular mass of the SasA-KaiCDD complex, where KaiCDD is a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel-filtration chromatography to be larger than 670 kDa. This value disagrees with the theoretical estimation of 480 kDa for a SasA3mer -KaiC hexamer (KaiC6mer ) complex with a 1:1 molecular ratio. To clarify the structure of the SasA-KaiC complex, we analyzed KaiCDD with 0.1 mmol/L ATP and 5 mmol/L MgCl2 (Mg-ATP), SasA and a mixture containing SasA and KaiCDD6mer with Mg-ATP by atomic force microscopy (AFM). KaiCDD images were classified into two types with height distribution corresponding to KaiCDD monomer (KaiCDD1mer ) and KaiCDD6mer , respectively. SasA images were classified into two types with height corresponding to SasA3mer and SasA6mer , respectively. The AFM images of the SasA-KaiCDD mixture indicated not only KaiCDD1mer , KaiCDD6mer , SasA3mer and SasA6mer , but also wider area "islands," suggesting the presence of a polymerized form of the SasA-KaiCDD complex.

Cooperative Binding of KaiB to the KaiC Hexamer Ensures Accurate Circadian Clock Oscillation in Cyanobacteria.

The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data have identified KaiB-KaiC interaction sites; however, KaiB mutations distal from the binding surfaces can impair KaiB-KaiC interaction and the circadian rhythm. Reportedly, KaiB and KaiC exclusively form a complex in a 6:6 stoichiometry, indicating that KaiB-KaiC hexamer binding shows strong positive cooperativity. Here, mutational analysis was used to investigate the functional significance of this cooperative interaction. Results demonstrate that electrostatic complementarity between KaiB protomers promotes their cooperative assembly, which is indispensable for accurate rhythm generation. SasA does not exhibit such electrostatic complementarity and noncooperatively binds to KaiC. Thus, the findings explain KaiB distal mutation effects, providing mechanistic insights into clock protein interplay.

Circadian oscillations of KaiA-KaiC and KaiB-KaiC complex formations in an in vitro reconstituted KaiABC clock oscillator.

The circadian clock is an endogenous biological mechanism that generates autonomous daily cycles in physiological activities. The phosphorylation levels of KaiC oscillated with a period of 24 h in an ATP-dependent clock oscillator reconstituted in vitro from KaiA, KaiB and KaiC. We examined the complex formations of KaiA and KaiB with KaiC in the KaiABC clock oscillator by fluorescence correlation spectrometry (FCS) analysis. The formation of KaiB-containing protein complex(es) oscillated in a circadian manner, with a single peak at 12 h and single trough at 24 h in the circadian cycle, whereas that of KaiA-containing protein complex(es) oscillated with two peaks at 12 and 24 h. FCS and surface plasmon resonance analyses showed that the binding affinity of KaiA for a mutant KaiC with Ala substitutions at the two phosphorylation sites considered to mimic the nonphosphorylated form of KaiC (np-KaiC) was higher than that for a mutant KaiC with Asp substitutions at the two phosphorylation sites considered to mimic the completely phosphorylated form of KaiC (cp-KaiC). The results from the study suggest that a KaiA-KaiB-cp-KaiC ternary complex and a KaiA-np-KaiC complex were formed at 12 and 24 h, respectively.

A first report of East Asian students' perception of progress testing: a focus group study.

BACKGROUND: Progress testing (PT) is used in Western countries to evaluate students' level of functional knowledge, and to enhance meaning-oriented and self-directed learning. However, the use of PT has not been investigated in East Asia, where reproduction-oriented and teacher-centered learning styles prevail. Here, we explored the applicability of PT by focusing on student perceptions. METHODS: Twenty-four students from Years 2, 3, and 5 at Jichi Medical University in Japan attended a pilot PT session preceded by a brief introduction of its concept and procedures. Variations in obtained test scores were analyzed by year, and student perceptions of PT were explored using focus groups. RESULTS: Formula scores (mean ± standard deviation) in Years 2, 3, and 5 were 12.63 ± 3.53, 35.88 ± 14.53, and 71.00 ± 18.31, respectively. Qualitative descriptive analysis of focus group data showed that students disfavored testing of medical knowledge without tangible goals, but instead favored repetitive assessment of knowledge that had been learned and was tested on a unit basis in the past in order to achieve deep learning. Further, students of all school years considered that post-test explanatory lectures by teachers were necessary. CONCLUSIONS: East Asian students' perceptions indicated that, in addition to their intensive memorization within narrow test domains compartmentalized by end-of-unit tests, the concept of PT was suitable for repetitive memorization, as it helped them to integrate their knowledge and to increase their understanding. Post-test explanatory lectures might lessen their dislike of the intangible goals of PT, but at the expense of delaying the development of self-directed learning. Key issues for the optimization of PT in East Asia may include administration of PT after completed end-of-unit tests and a gradual change in feedback methodology over school years from test-oriented post-test lectures to the provision of literature references only, as a means of enhancing test self-review and self-directed learning.

Site-directed spin labeling-electron spin resonance mapping of the residues of cyanobacterial clock protein KaiA that are affected by KaiA-KaiC interaction.

The cyanobacterial clock proteins KaiA, KaiB and KaiC interact with each other to generate circadian oscillations. We have identified the residues of the KaiA homodimer affected through association with hexameric KaiC (KaiC6mer) using a spin-label-tagged KaiA C-terminal domain protein (KaiAc) and performing electron spin resonance (ESR) analysis. Cys substitution and/or the attachment of a spin label to residues located at the bottom area of the KaiAc concave surface, a KaiC-binding groove, hindered the association of KaiAc with KaiC6mer, suggesting that the groove likely mediates the interaction with KaiC6mer. The residues affected by KaiC6mer association were concentrated in the three areas: the concave surface, a lobe-like structure (a mobile lobe near the concave surface) and a region adjacent to both the concave surface and the mobile lobe. The distance between the two E254, D255, L258 and R252 residues located on the mobile lobe decreased after KaiC association, suggesting that the two mobile lobes approach each other during the interaction. Analyzing the molecular dynamics of KaiAc showed that these structural changes suggested by ESR analysis were possible. Furthermore, the analyses identified three asymmetries in KaiAc dynamic structures, which gave us a possible explanation of an asymmetric association of KaiAc with KaiC6mer.

The K346T mutant of GnT-III bearing weak in vitro and potent intracellular activity.

BACKGROUND: N-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific N-glycan branch, bisecting GlcNAc, in the Golgi apparatus. Bisecting GlcNAc is a key residue that suppresses N-glycan maturation and is associated with the pathogenesis of cancer and Alzheimer's disease. However, it remains unclear how GnT-III recognizes its substrates and how GnT-III activity is regulated in cells. METHODS: Using AlphaFold2 and structural comparisons, we predicted the key amino acid residues in GnT-III that interact with substrates in the catalytic pocket. We also performed in vitro activity assay, lectin blotting analysis and N-glycomic analysis using point mutants to assess their activity. RESULTS: Our data suggested that E320 of human GnT-III is the catalytic center. More interestingly, we found a unique mutant, K346T, that exhibited lower in vitro activity and higher intracellular activity than wild-type GnT-III. The enzyme assays using various substrates showed that the substrate specificity of K346T was unchanged, whereas cycloheximide chase experiments revealed that the K346T mutant has a slightly shorter half-life, suggesting that the mutant is unstable possibly due to a partial misfolding. Furthermore, TurboID-based proximity labeling showed that the localization of the K346T mutant is shifted slightly to the cis side of the Golgi, probably allowing for prior action to competing galactosyltransferases. CONCLUSIONS: The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity. GENERAL SIGNIFICANCE: Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.

The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.

The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB(1-94)), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB(1-94) interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB(1-94) also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB(1-94) showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB(1-94).

Phase-dependent generation and transmission of time information by the KaiABC circadian clock oscillator through SasA-KaiC interaction in cyanobacteria.

Circadian clocks allow organisms to predict environmental changes of the day/night cycle. In the cyanobacterial circadian clock machinery, the phosphorylation level and ATPase activity of the clock protein KaiC oscillate with a period of approximately 24 h. The time information is transmitted from KaiC to the histidine kinase SasA through the SasA autophosphorylation-enhancing activity of KaiC, ultimately resulting in genome-wide transcription cycles. Here, we showed that SasA derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 has the domain structure of an orthodox histidine kinase and that its C-terminal domain, which contains a phosphorylation site at His160, is responsible for the autophosphorylation activity and the temperature- and phosphorylation state-dependent trimerization / hexamerization activity of SasA. SasA and KaiC associate through their N-terminal domains with an affinity that depends on their phosphorylation states. Furthermore, the SasA autophosphorylation-enhancing activity of KaiC requires the C-terminal ATPase catalytic site and depends on its phosphorylation state. We show that the phosphotransfer activity of SasA is essential for the generation of normal circadian gene expression in cyanobacterial cells. Numerical simulations suggest that circadian time information (free phosphorylated SasA) is released mainly by unphosphorylated KaiC during the late subjective night.

Implications for better nursing practice: psychological aspects of patients undergoing post-operative wound care.

AIMS AND OBJECTIVES: To understand the psychological aspects in patients undergoing post-operative wound care and to gain insights for improving nursing practice. BACKGROUND: Very few studies have examined education on or practice of wound care with a view towards the patient's psychology. DESIGN: Descriptive exploratory qualitative study. METHODS: Four patients who had undergone open surgery of the upper gastrointestinal tract were interviewed using a semi-structured format to gain an understanding of their feelings and opinions with regard to wound care. Interview transcripts were analysed using an inductive coding approach. RESULTS: Fifteen categories of responses were finally identified from the data. Patients wanted nursing staff to observe their wound more often so that patients could recognise improvement, to have better knowledge of the patient's disease and condition, to explain the patient's situation more completely and to appropriately answer questions. Patients also said that they felt more comfortable in posing questions or concerns regarding their condition to nursing staff than to their surgeons and did so while the wounds were being taken care of by nurses. CONCLUSIONS: These findings suggested the importance of nursing staff to fully understand and to be ready to share feelings regarding a patient's postoperative condition and to have skills in properly explaining the importance of each procedure or steps in treatments that a patient must undergo. The present study also indicates that it is imperative for nursing staff to learn methods to build relationships with patients so that they can express their feelings of fear or anxiety freely to nurses. RELEVANCE TO CLINICAL PRACTICE: It is not possible to develop nursing practice without understanding psychological aspects of patients undergoing postoperative wound care.

Allocation of absorbed light energy in PSII to thermal dissipations in the presence or absence of PsbS subunits of rice.

The thermal dissipation (TD) of absorbed light energy in PSII is considered to be an important photoprotection process in photosynthesis. A major portion of TD has been visualized through the analysis of Chl fluorescence as energy quenching (qE) which depends on the presence of the PsbS subunit. Although the physiological importance of qE-associated TD (qE-TD) has been widely accepted, it is not yet clear how much of the absorbed light energy is dissipated through a qE-associated mechanism. In this study, the fates of absorbed light energy in PSII with regard to different TD processes, including qE-TD, were quantitatively estimated by the typical energy allocation models using transgenic rice in which psbS genes were silenced by RNA interference (RNAi). The silencing of psbS genes resulted in a decrease in the light-inducible portion of TD, whereas the allocation of energy to electron transport did not change over a wide range of light intensities. The allocation models indicate that the energy allocated to qE-TD under saturating light is 30-50%. We also showed that a large portion of absorbed light energy is thermally dissipated in manners that are independent of qE. The nature of such dissipations is discussed.

Direct interaction between KaiA and KaiB revealed by a site-directed spin labeling electron spin resonance analysis.

In cyanobacteria, three clock proteins, KaiA, KaiB and KaiC, play essential roles in generating circadian oscillations. The interactions of these proteins change during the circadian cycle. Here, we demonstrated direct interaction between KaiA and KaiB using electron spin resonance spectroscopy. We prepared cystein (Cys)-substituted mutants of Thermosynechococcus elongatus KaiB, labeled specifically their Cys residues with spin labels and measured the ESR spectra of the labeled KaiB. We found that KaiB labeled at the 64th residue showed spectral changes in the presence of KaiA, but not in the presence of KaiC or bovine serum albumin as a negative control. KaiB labeled at the 101st residue showed no such spectral changes even in the presence of KaiA. The results suggest that KaiB interacts with KaiA in the vicinity of the 64th residue of KaiB. Further analysis demonstrated that the C-terminal clock-oscillator domain of KaiA is responsible for this interaction.

Drehmann sign and femoro-acetabular impingement in SCFE.

BACKGROUND: Drehmann sign is a characteristic clinical feature in slipped capital femoral epiphysis (SCFE). The presence of SCFE indicates an anatomic change of the proximal femur, which induces obligatory hip external rotation with hip flexion. In contrast, a cam-type femoro-acetabular impingement (FAI) is well known as sequelae of SCFE. The purpose of this study was to clarify the relationship between Drehmann sign and radiologic FAI. METHODS: We studied 92 hips of 80 SCFE patients who had been treated with in situ fixation. The occurrence rate of Drehmann sign was analyzed according to the degree of remodeling (the Jones classification) and the radiologic α-angle measured in each class at the final follow-up. At a mean 12.2 years after the final follow-up, the patients' present condition was clinically investigated with a questionnaire using a part of the Harris Hip Rating Scale (HHRS). In addition, 3-dimensional computed tomography analysis was performed to clarify the anatomic relationship between the femoral head and the acetabulum during testing for Drehmann sign. RESULTS: Among the 92 hips in the study, 60 were well remodeled (Jones type A), 24 were type B, and 8 were type C, with 6.5 years of mean follow-up. The mean of the modified α-angles for the 3 groups (A, B, and C) were 61.8, 84.7, and 119.4, respectively (P < 0.05); 25%, 75%, and 100% of the hips in the 3 groups, respectively, exhibited Drehmann sign. The set of hips (n = 41) with a positive Drehmann sign had a mean α-angle of 85.6 versus 63.0 degrees for the set of hips (n = 51) with a negative Drehmann sign (P < 0.05). Seven (13.5%) of 52 patients responding to the questionnaire reported hip pain and/or limp in the positive Drehmann sign group, but no patient in the negative sign group complained of either. Three-dimensional computed tomography delineated FAI at 2 different positions during testing for Drehmann sign. CONCLUSIONS: Drehmann sign is highly valuable for clinically evaluating the existence of FAI and for following up with observation or realignment to prevent early osteoarthritis.

Immunogenicity of BNT162b2 mRNA COVID-19 Vaccine in Patients with Cardiovascular Disease.

Concern has been raised about the effectiveness of the coronavirus disease 2019 (COVID-19) vaccine in the population of patients with various comorbidities such as heart disease. We investigated the humoral response to the BNT162b2 mRNA COVID-19 vaccine in patients with cardiovascular disease (CVD). We measured IgG against severe acute respiratory syndrome coronavirus 2 spike receptor-binding domain (RBD-IgG) in 85 CVD patients and 179 healthcare workers (HCWs). Blood samples were collected from patients and HCWs three times: (1) before the first dose of vaccination, (2) two weeks after the first dose of vaccination, and (3) two weeks after the second dose of vaccination. Patients with CVD showed a significantly inferior serological response to the BNT162b2 mRNA COVID-19 vaccine at 14 days after the prime dose compared to HCWs (21% vs. 95%, p < 0.001). Median RBD-IgG titers of patients with CVD at 14 days after the second dose were significantly lower than those of HCWs (137.2 U/mL (80.6-200.4 U/mL) vs. 176.2 U/mL (123.9-260.0 U/mL), p < 0.001). In multivariable analyses, CVD is significantly associated with seropositivity after first vaccination and RBD-IgG titers after second vaccination. CVD patients may have a poor humoral response to the BNT162b2 mRNA COVID-19 vaccine, need to be closely monitored, and require earlier revaccination to ensure stronger immunity and protection against infection.

Kynurenine 3-monooxygenase deficiency induces depression-like behavior via enhanced antagonism of α7 nicotinic acetylcholine receptors by kynurenic acid.

Tryptophan (TRP) is metabolized via the kynurenine (KYN) pathway, which is related to the pathogenesis of major depressive disorder (MDD). Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the metabolism of KYN to 3-hydroxykynurenine. In rodents, KMO deficiency induces a depression-like behavior and increases the levels of kynurenic acid (KA), a KYN metabolite formed by kynurenine aminotransferases (KATs). KA antagonizes α7 nicotinic acetylcholine receptor (α7nAChR). Here, we investigated the involvement of KA in depression-like behavior in KMO knockout (KO) mice. KYN, KA, and anthranilic acid but not TRP or 3-hydroxyanthranilic acid were elevated in the prefrontal cortex of KMO KO mice. The mRNA levels of KAT1 and α7nAChR but not KAT2-4, α4nAChR, or ß2nAChR were elevated in the prefrontal cortex of KMO KO mice. Nicotine blocked increase in locomotor activity, decrease in social interaction time, and prolonged immobility in a forced swimming test, but it did not decrease sucrose preference in the KMO KO mice. Methyllycaconitine (an α7nAChR antagonist) antagonized the effect of nicotine on decreased social interaction time and prolonged immobility in the forced swimming test, but not increased locomotor activity. Galantamine (an α7nAChR allosteric agonist) blocked the increased locomotor activity and prolonged immobility in the forced swimming test, but not the decreased social interaction time in the KMO KO mice. In conclusion, elevation of KA levels contributes to depression-like behaviors in KMO KO mice by α7nAChR antagonism. The ameliorating effects of nicotine and galantamine on depression-like behaviors in KMO KO mice are associated with the activation of α7nAChR.

Functionally important structural elements of the cyanobacterial clock-related protein Pex.

Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the alpha3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the alpha3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its alpha3 helix is probably important to its stability.

ATPase activity and its temperature compensation of the cyanobacterial clock protein KaiC.

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria. KaiC is a homohexamer; its subunit contains duplicated halves, each with a set of ATPase motifs. Here, using highly purified KaiC preparations of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 produced in Escherichia coli, we found that the N- and C-terminal domains of KaiC had extremely weak ATPase activity. ATPase activity showed temperature compensation in wild-type KaiC, but not in KaiC(S431A/T432A), a mutant that lacks two phosphorylation sites. We concluded that KaiC phosphorylation is involved in the ATPase temperature-compensation mechanism-which is probably critical to the stability of the circadian clock in cyanobacteria-and we hypothesized the following temperature-compensation mechanism: (i) The C-terminal phosphorylation sites of a KaiC hexamer subunit are phosphorylated by the C-terminal domain of an adjacent KaiC subunit; (ii) the phosphorylation suppresses the ATPase activity of the C-terminal domain; and (iii) the phosphorylated KaiC spontaneously dephosphorylates, resulting in the recover of ATPase activity.

Glucocorticoid inhibits bone regeneration after osteonecrosis of the femoral head in aged female rats.

Idiopathic osteonecrosis of the femoral head (ION) is a painful disease of the hip, the pathogenic mechanism of which is still unclear. The most common extraneous factor is steroid treatment. Steroids have inhibiting effects on bone formation and resorption. When bone regenerative treatments are indicated for ION patients who are exposed to steroids, we cannot ignore the effects of corticosteroid itself on bone healing. The aim of this study was to investigate the effects of glucocorticoid on bone regeneration after osteonecrosis of the femoral head in a rat model. Osteonecrosis was induced surgically on the left femoral heads of aged female rats (about 6 months old) on day 0. Methylprednisolone sodium succinate (MPSS) or normal saline was administrated at a dose of 100 mg/kg/day from day 7 to 11. Femoral heads were analyzed histologically. There were no pathological findings in the right femoral heads of the MPSS-treated and saline-treated rats, as control for the contralateral injury. The newly formed bone volume and the osteoclast number were significantly smaller in the MPSS-treated group. The normal bone marrow was regenerated in the saline-treated group, whereas most of the bone marrow space still contained fibroblast-like spindle-shaped cells in the MPSS-treated group on day 42. Alkaline phosphatase-positive cells were only seen in the areas around the regenerated bone marrow cavities. Thus, MPSS inhibits bone formation by suppressing osteoblast proliferation and resorption by suppressing the recruitment of osteoclast precursors. These findings may be useful when designing treatments for ION patients exposed to steroids.