Characterization of rhodanine derivatives as potential disease-modifying drugs for experimental mouse osteoarthritis.

OBJECTIVE: This study was performed to characterize selected rhodanine derivatives as potential preclinical disease-modifying drugs for experimental osteoarthritis (OA) in mice. METHODS: Three rhodanine derivatives, designated rhodanine (R)-501, R-502, and R-503, were selected as candidate OA disease-modifying drugs. Their effects were evaluated by intra-articular (IA) injection in OA mouse models induced by DMM (destabilization of the medial meniscus) or adenoviral overexpression in joint tissues of hypoxia-inducible factor (HIF)-2α or zinc importer ZIP8. The regulatory mechanisms impacted by the rhodanine derivatives were examined in primary-culture chondrocytes and fibroblast-like synoviocytes (FLS). RESULTS: All three rhodanine derivatives inhibited OA development caused by DMM or overexpression of HIF-2α or ZIP8. Compared to vehicle-treated group, for example, IA injection of R-501 in DMM-operated mice reduced median OARSI grade from 3.78 (IQR 3.00-5.00) to 1.89 (IQR 0.94-2.00, P = 0.0001). R-502 and R-503 also reduced from 3.67 (IQR 2.11-4.56) to 2.00 (IQR 1.00-2.00, P = 0.0030) and 2.00 (IQR 1.83-2.67, P = 0.0378), respectively. Mechanistically, the rhodanine derivatives inhibited the nuclear localization and transcriptional activity of HIF-2α in chondrocytes and FLS. They did not bind to Zn2+ or modulate Zn2+ homeostasis in chondrocytes or FLS; instead, they inhibited the nuclear localization and transcriptional activity of the Zn2+-dependent transcription factor, MTF1. HIF-2α, ZIP8, and interleukin-1ß could upregulate matrix-degrading enzymes in chondrocytes and FLS, and the rhodanine derivatives inhibited these effects. CONCLUSION: IA administration of rhodanine derivatives significantly reduced OA pathogenesis in various mouse models, demonstrating that these derivatives have disease-modifying therapeutic potential against OA pathogenesis.

Impact of caudal block on postoperative complications in children undergoing tubularised incised plate urethroplasty for hypospadias repair: a retrospective cohort study.

This study aimed to assess the association between caudal block and postoperative complications after tubularised incised plate urethroplasty. The medical records of 388 paediatric patients who underwent urethroplasty at a tertiary medical centre were analysed retrospectively. Among the 342 patients included, 216 patients received a caudal block and 72 (21.1%) patients suffered surgical complications. The number of patients having surgical complications was significantly greater among patients who received a caudal block than among patients who did not receive a caudal block (53 (24.5%) versus 19 (15.1%), respectively, p = 0.04). Based on multivariate logistic regression analysis, duration of surgery, caudal block and hypospadias types were independent risk factors for the surgical complications. Patients with caudal block had an odds ratio of 2.1 (95% CI, 1.14-3.81, p = 0.018) for the development of postoperative complications compared with patients without caudal block. This analysis demonstrates that caudal block is associated with surgical complications after tubularised incised plate urethroplasty.

Long-term outcome of transanal irrigation for children with spina bifida.

STUDY DESIGN: Experimental, prospective study. OBJECTIVES: We evaluated the long-term clinical efficacy of transanal irrigation (TAI) and its effect on the quality of life of spina bifida children and their caregivers. SETTING: Republic of Korea. METHOD: Forty-four spina bifida pediatric patients with constipation, fecal incontinence or both, underwent a TAI program at our spina bifida clinic between December 2010 and October 2013. The children and their caregivers were evaluated using a self-administered questionnaire before TAI and at 3 months and 3 years after initiation of the program. RESULTS: Successful treatment outcome was achieved in 38 (86.4%) children after a mean follow-up duration of 33 months (range, 30-36). The mean number of fecal incontinence episodes per week, the number of diaper changes and the total time for bowel care per day before the program decreased at the latest follow-up examination from 7.3 to 0.4 (P<0.001), 1.6 to 0.2 (P<0.001) and 29.2 to 19.4 min (P=0.038), respectively. These results remained constant from short-term follow-up at 3 months to 3 years. Caregivers and children could go out more often (P=0.002), and the emotional impact of bowel care on caregivers decreased (P<0.001). The reported mean overall satisfaction with TAI was 8/10. The common adverse effect during TAI was abdominal discomfort (60.5%). CONCLUSION: We observed a sustained significant improvement in defecation symptoms and quality of life for 3 years in spina bifida children who underwent continuous TAI.

Analgesic efficacy of caudal dexamethasone combined with ropivacaine in children undergoing orchiopexy.

BACKGROUND: Epidural administration of dexamethasone might reduce postoperative pain in adults. We evaluated whether a caudal block of 0.1 mg kg(-1) dexamethasone combined with ropivacaine improves analgesic efficacy in children undergoing day-case orchiopexy. METHODS: This randomized, double-blind study included 80 children aged 6 months to 5 yr who underwent day-case, unilateral orchiopexy. Patients received either 1.5 ml kg(-1) of 0.15% ropivacaine (Group C) or 1.5 ml kg(-1) of 0.15% ropivacaine in which dexamethasone of 0.1 mg kg(-1) was mixed (Group D) for caudal analgesia. Postoperative pain scores, rescue analgesic consumption, and side-effects were evaluated 48 h after operation. RESULTS: Postoperative pain scores at 6 and 24 h post-surgery were significantly lower in Group D than in Group C. Furthermore, the number of subjects who remained pain free up to 48 h after operation was significantly greater in Group D [19 of 38 (50%)] than in Group C [four of 37 (10.8%); P<0.001]. The number of subjects who received oral analgesic was significantly lower in Group D [11 of 38 (28.9%)] than in Group C [20 of 37 (54.1%); P=0.027]. Time to first oral analgesic administration after surgery was also significantly longer in Group D than in Group C (P=0.014). Adverse events after surgery including vomiting, fever, wound infection, and wound dehiscence were comparable between the two groups. CONCLUSIONS: The addition of dexamethasone 0.1 mg kg(-1) to ropivacaine for caudal block can significantly improve analgesic efficacy in children undergoing orchiopexy. Clinical trial registration NCT01604915.

The effects of transanal irrigation as a stepwise bowel management program on the quality of life of children with spina bifida and their caregivers.

STUDY DESIGN: Experimental, prospective study. OBJECTIVES: Fecal incontinence and constipation affect the quality of life (QOL) of children with spina bifida and their caregivers. We evaluated the clinical efficacy of a stepwise bowel management program on QOL for children with spina bifida and their caregivers. SETTING: Republic of Korea. METHODS: Between December 2010 and April 2011, 53 children with constipation, fecal incontinence or both underwent a stepwise bowel management program at our spina bifida clinic. The children and their caregivers were evaluated before and after this program using a self-administered questionnaire. RESULTS: Among the children, 11.3% received only oral laxatives and controlled well, 88.7% received transanal irrigation. After this program, the mean number of episodes of fecal incontinence per week, number of diaper changes and total time for bowel care decreased from 6.9 to 0.5 (P=0.004), from 1.6 to 0.2 (P=0.001) and from 27 to 15.9 min (P=0.003), respectively. Caregivers and children were able to leave their houses more often (P=0.006), and caregivers' bothersomeness, anxiety and depression due to bowel care decreased (P<0.001). Factors related to family relationships (P=0.265) and financial impact (P=0.071) improved, but not significantly. CONCLUSIONS: We observed significant improvement in defecation symptoms and QOL scores of spina bifida patients who underwent this program. We recommend that this simple therapeutic method be considered as a safe and valid choice for the treatment of chronic constipation and fecal incontinence.

Crystallization and preliminary X-ray studies of Trp138Phe/Val185Thr xylose isomerases from Thermotoga neapolitana and Thermoanaerobacterium thermosulfurigenes.

Xylose isomerases from Thermotoga neapolitana (TNXI) and Thermoanaerobacterium thermosulfurigenes (TTXI) share 70.4% sequence identity and are thermostable. The double mutants Trp138Phe/Val185Thr of TNXI and TTXI have higher catalytic efficiencies than TNXI and TTXI, respectively. The Trp138Phe/Val185Thr TNXI and TTXI mutants were overexpressed in Escherichia coli strain BL21(DE3) and purified. Crystals of the two proteins were grown with polyethylene glycol 8000 as the major precipitant by the hanging-drop vapour-diffusion method. Crystals of the TNXI mutant were obtained in the absence of substrate, in complex with glucose and in complex with fructose. Crystals of the TTXI mutant were obtained complexed with glucose. Diffraction data were collected at 1.9, 2.1 and 2.1 A resolution for the fructose-TNXI mutant, glucose-TNXI mutant and substrate-unbound TNXI mutant, respectively. The diffraction data for the glucose-TTXI mutant were collected at 2.0 A resolution. The crystals belong to the orthorhombic space groups C222(1) (TNXI mutant) and P2(1)2(1)2(1) (TTXI mutant). The TNXI and TTXI mutant crystals contain two and four monomers in the asymmetric unit, respectively.

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.

BACKGROUND: The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS: We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS: Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

Crystallization and preliminary X-ray crystallographic analysis of the surE protein from Thermotoga maritima.

The surE protein from Thermotoga maritima is a 247-residue protein of unknown function. Its homologues are well conserved among both the eubacteria and the archaea. It has been overexpressed in soluble form in Escherichia coli. The protein has been crystallized at 296 K using 2-propanol as a precipitant. X-ray diffraction data have been collected to 1.9 A resolution using synchrotron radiation. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 115.96, c = 78.60 A, alpha = beta = 90, gamma = 120 degrees. The asymmetric unit contains two monomers of the surE protein, with a corresponding V(M) of 2.72 A(3) Da(-1) and a solvent content of 54.7%.

Crystallization and preliminary X-ray diffraction studies of the guanylate kinase-like domain of PSD-95 protein from rat.

The PSD-95 (postsynaptic density-95) protein, one of the members of the MAGUK (membrane-associated guanylate kinase) family, is composed of three PDZ domains, one SH3 domain and one guanylate kinase-like (GK) domain. The GK domain mediates the scaffolding function of PSD-95 by protein--protein interaction. Here, the GK domain was subcloned, expressed as an intein fusion protein, purified without the intein and then crystallized at room temperature by the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. The complete native data set was collected to a resolution of 2.35 A using flash-cooling. The crystals belong to the primitive tetragonal space group P4(3) (or P4(1)), with unit-cell parameters a = b = 70.03 (4), c = 37.64 (1) A.

Crystallization and preliminary X-ray crystallographic studies of response regulator for cyanobacterial phytochrome, Rcp1.

The key response-regulator gene of light regulation, rcp1, from Synechocystis sp. has been overexpressed, purified and subsequently crystallized using ammonium sulfate as a precipitant in forms suitable for X-ray crystallographic studies. A native data set was collected to a resolution of 2.5 A at cryogenic temperature. The crystals belong to the hexagonal space group P6(3), with unit-cell parameters a = b = 89.04 (5), c = 60.29 (3) A. The Matthews parameter suggests that Rcp1 crystallizes with two molecules per asymmetric unit.

Sensitivity of plasma membrane H+-ATPase of cucumber root system in response to low root temperature.

Cucumber plants (Cucumis sativus L.) were grown at different root temperatures and the H+-ATPase activities in the root plasma membranes were investigated. Electrolyte leakages and the amount of water transported from root to shoot indicated the sensitivity of cucumber root systems to low root temperature. The increase in H+-ATPase activity after 1 day of low root temperature treatment was reversed and gradually diminished as root temperatures of 10 °C continued for another 6 days. The H+-transport rate of the plasma membranes and the transcriptional activity of the corresponding gene expression closely followed the H+-ATPase activity. The role of H+-ATPase in root plasma membranes in terms of the root acclimation process at low root temperature is discussed.