Conformational Equilibrium of NADPH-Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction.

Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme-HO-1, whereas the reduction in heme-HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme-HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme-HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme-HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme-HO-1 in solution. We conclude that the "open-close" transition of CPR is indispensable for electron transfer from CPR to heme-HO-1.

Autonomic nervous system function in adolescent migraineurs.

BACKGROUND: Migraine is a common and disabling neurological disorder. Studies on the relationship between migraine and the autonomic nervous system (ANS) have been inconclusive. Moreover, pediatric studies are extremely limited. Therefore, the present study investigated interictal ANS function in adolescent migraineurs. METHODS: We studied 21 patients with migraine and 26 healthy controls. Beat-to-beat blood pressure (BP) and heart rate (HR) were non-invasively and continuously measured in the supine and standing positions. ANS function was evaluated on power spectral analysis of HR variability and diastolic BP (DBP) variability. RESULTS: Heart rate and systolic BP were not different between the two groups in either the supine or standing position. DBP did not differ between groups in the standing position, but was significantly higher in migraineurs in the supine position. The vasoconstrictor index was significantly higher in migraineurs. High-frequency (HF) RR interval variability (RR-HF) and the ratio of the low-frequency (LF) to HF component of RR interval variability (RR-LF/HF) were not different between the two groups in both positions. The LF component of DBP variability (DBP-LF) in the supine position was significantly lower in migraineurs, while DBP-LF during standing in migraineurs was significantly higher than in controls. CONCLUSION: Migraineurs have significantly lower sympathetic vasomotor activity in the supine position, while sympathetic vasomotor activity was hyperresponsive during standing.

Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein.

Sorcin, a 22 kDa Ca(2+) binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between -716 and -709 bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR.

Variant cardiovascular regulation in children with postural tachycardia syndrome.

BACKGROUND: Postural tachycardia syndrome (POTS) manifests as marked tachycardia while standing. We noticed two forms of circulatory response to orthostatic stress in POTS. We investigated cardiovascular and autonomic nervous response to orthostatic stress in the two forms. METHODS: We studied 79 patients with POTS and 38 healthy control subjects (Ct). Beat-to-beat blood pressure (BP) and heart rate (HR) were non-invasively and continuously measured in the supine and standing positions. Autonomic nervous function was evaluated on power spectral analysis of HR variability and diastolic BP variability. We divided the subjects into two groups: standing-induced tachycardia (SI group; increase in HR ≥35 beats/min) and supine tachycardia (Su group; standing HR ≥115 beats/min with standing-induced HR increase <35 beats/min). RESULTS: The Su group had higher supine BP and HR compared with the other groups, indicating dominant sympathetic control of the heart in the supine position. While rising, the SI group had a higher increase in HR than the Ct group, indicating excessive withdrawal of vagal tone. The Su group had a smaller increase in HR and a greater decrease of systolic BP and cardiac index by standing compared with the SI group. These results suggest that compensatory mechanisms of sympathetic function during standing failed in the Su group, probably because of exhaustion by the nearly maximum effort to generate sympathetic drive even in the supine position with low central blood volume. CONCLUSION: There is a difference between the two types of POTS, in the balance of resting autonomic function and hemodynamic response to standing.

GNB3 C825T polymorphism is associated with postural tachycardia syndrome in children.

AIM: Postural tachycardia syndrome (POTS) is one of the most frequent forms of chronic orthostatic intolerance in children and adolescents. The aim of the present study was to examine the influence of a genetic background on POTS. METHODS: A total of 96 children and adolescents with orthostatic dysregulation were studied. The polymorphism of the G protein ß3 subunit (GNB3) C825T and G protein α subunit (GNAS1) T131C of genes encoding components of the autonomic nervous system were determined and compared with circulatory responses to active standing. RESULTS: In the GNB3 gene C825T polymorphism, the CT and TT genotype had a significant lower supine heart rate and a larger increase of heart rate by standing than the CC, associated with evaluated power of the high-frequency component of heart rate variability. According to the criteria of the Japanese clinical guidelines, 48 children were diagnosed as POTS and 30 were as normal responder with somatoform disorder (SD). In GNB3 C825T polymorphism, the TT genotype was more frequently found in the POTS group (45.8%) than in the SD group (20.0%; P = 0.036) [corrected]. In the GNAS1 T393C, the genotype frequencies for the T393C polymorphisms of GNA1 did not differ significantly between the groups. CONCLUSION: The gene polymorphisms GNB3 C825T might be a risk factor for POTS through the enhanced vagal withdrawal of the heart in children and adolescents.

Japanese clinical guidelines for chronic pain in children and adolescents.

Chronic pain is a common problem in pediatric practice. The prevalence of chronic pain in children is >30%. Because pain indicates emotional expression as well as the physiological reaction toward infection, injury, and inflammation, both physiological and psychological assessments are essential to determine primary interventions for chronic pain. The Japanese Society of Psychosomatic Pediatrics Task Force of clinical practice guidelines for chronic pain in children and adolescents compiled clinical evidence and opinions of specialists associated with the primary care of pediatric chronic pain in the Japanese 'clinical guidelines for chronic pain in children and adolescents' in 2009, which are presented herein. The guidelines consist of three domains: general introduction to chronic pain; chronic abdominal pain; and chronic headache. Each section contains information on the physiological mechanism, psychological aspects, assessment methods, and primary interventions for pediatric chronic pain. These guidelines are expected to help disseminate knowledge on primary interventions for chronic pain in children and adolescents.

TGF-beta superfamily regulates a switch that mediates differentiation either into adipocytes or myocytes in left atrium derived pluripotent cells (LA-PCS).

Many stem cell studies have focused on the subject of cell fate and the signal molecules that modulate the regulatory switches for a given differentiation pathway. Genome-wide screens for cell fate determination signals require a cell source that differentiates purely into a single cell type. From adult rat left atrium, we established LA-PCs that differentiates into cardiac/skeletal myocytes or adipocytes with almost 100% purity. In this study, we compared gene expression profiles of undifferentiated LA-PCs with those of differentiated cells [adipocytes (Adi) or cardiac/skeletal myocytes (Myo)] to identify the signals that set the regulatory switch for adipocyte or myocyte differentiation. Microarray analysis verified the feasibility of genome-wide screening by this method. Using a pathway analysis screen, we found that members of the TGF-beta superfamily signal transduction pathways modulate the adipocyte/myocyte differentiation switch. Further analysis determined that recombinant TGF-beta inhibits adipogenesis and induces myogenesis simultaneously in a dose-dependent manner. Moreover, noggin induces differentiation into fully developed beating cardiac myocytes in vitro. These results provided new insight into the molecules that modulate the differentiation switch and validated a screening method for their identification.

Myogenic differentiation in atrium-derived adult cardiac pluripotent cells and the transcriptional regulation of GATA4 and myogenin on ANP promoter.

We established cardiac pluripotent stem-like cells from the left atrium (LA-PCs) of adult rat hearts. These cells could differentiate not only into beating myocytes but also into cells of other lineages, including adipocytes and endothelial cells in the methylcellulose-based medium containing interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF). In particular, IL-3 and SCF contributed to the differentiation into cardiac troponin I-positive cells. Notably, small population of LA-PCs coexpressed GATA4 and myogenin, which are markers specific to cardiomyocytes and skeletal myocytes, respectively, and could differentiate into both cardiac and skeletal myocytes. Therefore, we investigated the involvement of these two tissue-specific transcription factors in the cardiac transcriptional activity. Coexpression of GATA4 and myogenin synergistically activated GATA4-specific promoter of the atrial natriuretic peptide gene. This combinatorial function was shown to be dependant on the GATA site, but independent of the E-box. The results of chromatin immunoprecipitation and electrophoretic mobility shift assays suggested that myogenin bound to GATA4 on the GATA elements and the C-terminal Zn-finger domain of GATA4 and the N-terminal region of myogenin were required for this synergistic activation of transcription. Taken together, these two transcription factors could be involved in the myogenesis of LA-PCs.