A molecular mechanism for autoinhibition of the tandem SH3 domains of p47phox, the regulatory subunit of the phagocyte NADPH oxidase

The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the production of reactive oxygen species. p47(phox) is a cytosolic component of the NADPH oxidase and plays an important role in the assembly of the activated complex. The structural determination of the tandem SH3 domains of p47(phox) is crucial for elucidation of the molecular mechanism of the activation of p47(phox). We determined the X-ray crystal structure of the tandem SH3 domains with the polybasic/autoinhibitory region (PBR/AIR) of p47(phox). The GAPPR sequence involved in PBR/AIR forms a left-handed polyproline type-II helix (PPII) and interacts with the conserved SH3 binding surfaces of the SH3 domains simultaneously. These SH3 domains are related by a 2-fold pseudosymmetry axis at the centre of the binding groove and interact with the single PPII helix formed by the GAPPR sequence with opposite orientation. In addition, a number of intra-molecular interactions among the SH3 domains, PBR/AIR and the linker tightly hold the architecture of the tandem SH3 domains into the compact structure and stabilize the autoinhibited form synergistically. Phosphorylation of the serine residues in PBR/AIR could destabilize and successively release the intra-molecular interactions. Thus, the overall structure could be rearranged from the autoinhibitory conformation to the active conformation and the PPII ligand binding surfaces on the SH3 domains are now unmasked, which enables their interaction with the target sequence in p22(phox).     

Effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in rats in vitro

The phthalate esters have been used as plasticizers for various plastic products, and their testicular toxicity has been reported. In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of the phthalate esters, on prepubertal rat testes in vitro were examined. The testes of 20-day-old Sprague Dawley (SD) rats were cut into smaller pieces and seeded in medium, and then the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, TUNEL-positive spermatogenic cells were identified, and they gradually increased in number in time- and dose-dependent manners. Ultrastructurally, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still-functioning cell organelles, and packed cell contents in membrane-bounded bodies), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), apoptotic Sertoli cells (highly condensed nuclei and nuclear membrane lysis) and necrotic Sertoli cells (marginated chromatins along the nuclear membrane, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, based on transmission electron microscopic observations, MEHP treatment may affect spermatogenic cells, and lead them to necrosis. Thus, testicular tissue cultures and cell cultures are of advantageous for screening testicular toxicity of chemicals.     

Expression of alpha-tryptase and beta-tryptase by human basophils

BACKGROUND: Alpha and beta-tryptase levels in serum are clinical tools for the evaluation of systemic anaphylaxis and systemic mastocytosis. Basophils and mast cells are known to produce these proteins. OBJECTIVE: The current study examines the effect of the alpha,beta-tryptase genotype on basophil tryptase levels and the type of tryptase stored in these cells. METHODS: Tryptase extracted from purified peripheral blood basophils from 20 subjects was examined by using ELISAs measuring mature and total tryptase and by using an enzymatic assay with tosyl-Gly-Pro-Lys-p-nitroanilide. Tryptase genotypes (4:0, 3:1, and 2:2 beta/alpha ratios) were assessed by using a hot-stop PCR technique with alpha,beta-tryptase-specific primers. Total alpha,beta-tryptase mRNA was measured by means of competitive RT-PCR, and ratios of alpha to beta-tryptase mRNA were measured by means of hot-stop RT-PCR. RESULTS: Tryptase in all but one of the basophil preparations was mature and enzymatically active. Tryptase quantities in basophils were less than 1% of those in tissue mast cells. Tryptase genotypes (beta/alpha) among the 20 donors were 4:0 in 7, 3:1 in 7, and 2:2 in 6. Tryptase protein and mRNA levels per basophil were not affected by the tryptase genotype. CONCLUSION: Basophils from healthy subjects contain modest amounts of mature and enzymatically active tryptase unaffected by the tryptase genotype.     

Reduced expression of aquaporin 4 in human muscles with amyotrophic lateral sclerosis and other neurogenic atrophies

Aquaporin 4 (AQP4) is a water channel protein that is widely distributed in human tissues. However, the precise functional role of AQP4 in skeletal muscle tissue has not yet been determined. Expression of AQP4 was reported to be reduced in muscle tissue from Duchenne muscular dystrophy patients. In the regenerating phase of skeletal muscle, AQP4 expression was reduced when nerve supply was not present. However, in diseased human muscles with neurogenic atrophy including amyotrophic lateral sclerosis, there has been no data on the changes in AQP4 expression. In the present study, we investigated the expression of AQP4 at mRNA and protein levels in human muscles with neurogenic atrophy. The mean level of AQP4 mRNA was significantly lower in muscles with neurogenic atrophy than that in muscles from normal controls. The myofiber surface immunostaining with anti-AQP4 antibody in muscles with neurogenic atrophy was reduced on the surface of scattered myofibers, small angulated myofibers, and myofibers in small- and large-group atrophy despite the presence of dystrophin. Based on the present findings, we conclude that the expression of AQP4 is affected by nerve supply and is down-regulated in human muscles with neurogenic atrophy.     

A neural network method for identification of RNA-interacting residues in protein

Identification of the most putative RNA-interacting residues in protein is an important and challenging problem in a field of molecular recognition. Structural analysis of protein-RNA complexes reveals a strong correlation between interaction residues and their structure. Building on this viewpoint, we have developed a neural network predictor to correctly identify residues involved in protein-RNA interactions from protein sequence and its structural information. The system has been exhaustedly cross-validated with various strategies differing in input encoding, amount of input information, and network architectures. In addition, we have evaluated performance among functional subsets of complexes. Finally, to reflect the properties of protein-RNA complexes in our dataset, two kinds of post-processing method are adopted. The experimental result shows that our system yields not-trivial performance although the residues in interaction sites are too scarce.     

High‐Temperature Propagation and Termination Kinetics of Styrene to High Conversion Investigated by Electron Paramagnetic Resonance Spectroscopy

Summary: The free radical bulk polymerization of styrene at 120 °C has been investigated over almost the entire conversion range using electron paramagnetic resonance spectroscopy, Fourier‐transform near‐infrared spectroscopy and gel permeation chromatography. The free radical concentration went through a sharp maximum that coincided with the peak in the rate of polymerization during the gel effect, and shifted to higher conversion with increasing initiator concentration. The termination rate coefficient (k_t), decoupled from the initiator efficiency (f) by use of the instantaneous degree of polymerization, remained close to constant up to as high as approximately 80% conversion, at which a dramatic decrease occurred. Both the propagation rate coefficient (k_p) and f fell orders of magnitude near 80% conversion in spite of the temperature being above the glass transition temperature of the system. The value of k_p increased with the initiator concentration at a given conversion in the highest (diffusion‐controlled) conversion range.

Termination rate coefficient (k_t) versus conversion for bulk free radical polymerization of St initiated by TBP at 120 °C. [TBP] = 0.15 (○), 0.10 (?) and 0.05 M ().     

The management of possible fishbone ingestion

OBJECTIVE: We assessed the usefulness of computed tomography (CT) in cases of suspected impaction of fish bones in the esophagus. The findings of this study were also compared with those of studies in which surgery was used to remove or confirm the presence of fish bones. We accordingly propose a management protocol to ensure optimum outcome for patients with a history of fish bone ingestion. METHODS: X-ray and CT imaging were performed in 76 patients in whom esophageal impaction of fish bones was suspected. RESULTS: Plain X-ray revealed impacted fish bones in 17 patients (22%), soft-tissue swelling but no evidence of foreign body in 5 (7%), and no abnormal findings in 54 (71%). These findings were apparent on CT scans in 31 (41%), 8 (10%), and 37 (49%), respectively. Of the 31 patients in whom CT revealed a fishbone, 17 (55%) also exhibited X-ray evidence of foreign body. Of the remainder, X-ray revealed only soft-tissue swelling in 3 (10%), and was unremarkable in 11 (35%). Among the 5 patients in whom X-ray demonstrated only soft-tissue swelling, CT was positive for foreign body in 3 (60%). Of the 54 patients in whom X-ray appearances were normal, CT revealed foreign bodies in 11 (20%) and other abnormalities in 6 (11%). CONCLUSION: In the present study, sensitivity and specificity of plain X-ray was 54.8% (17 of 31) and 100% (45 of 45), respectively. However, for CT, both sensitivity and specificity were 100%. CT was determined to be very useful in the diagnosis of impacted fish bones in the esophagus.     

Inflammation controls B lymphopoiesis by regulating chemokine CXCL12 expression

Inflammation removes developing and mature lymphocytes from the bone marrow (BM) and induces the appearance of developing B cells in the spleen. BM granulocyte numbers increase after lymphocyte reductions to support a reactive granulocytosis. Here, we demonstrate that inflammation, acting primarily through tumor necrosis factor alpha (TNFalpha), mobilizes BM lymphocytes. Mobilization reflects a reduced CXCL12 message and protein in BM and changes to the BM environment that prevents homing by cells from naive donors. The effects of TNFalpha are potentiated by interleukin 1 beta (IL-1beta), which acts primarily to expand the BM granulocyte compartment. Our observations indicate that inflammation induces lymphocyte mobilization by suppressing CXCL12 retention signals in BM, which, in turn, increases the ability of IL-1beta to expand the BM granulocyte compartment. Consistent with this idea, lymphocyte mobilization and a modest expansion of BM granulocyte numbers follow injections of pertussis toxin. We propose that TNFalpha and IL-1beta transiently specialize the BM to support acute granulocytic responses and consequently promote extramedullary lymphopoiesis.     

The significance of lifestyle as a risk factor for the metabolic syndrome

The metabolic syndrome is featured by obesity, dyslipidemia and insulin resistance, additionally associated with inflammatory and fibrinolytic dysfunctions, all of which are risk factors of cardiovascular diseases and type 2 diabetes. The lifestyle factors that could cause and aggravate the metabolic syndrome are physical inactivity, calorie-dense diet, habitual alcohol drinking and smoking, and psychosocial stress. Energy excess along with a lot of saturated fat, i.e. more fast food and sugar-containing drinks, and sedentary lifestyle like television viewing are contributing to the epidemic of obesity. The significance of lifestyle from birth to adulthood will be reviewed in the context of the metabolic syndrome, as well as the background and environment in which the convenient lifestyle spread far and wide among us.     

Diagnostic criteria for acute pancreatitis and evaluation of the severity

A research group supported by grants from the Ministry of Health and Labor has recently proposed new diagnostic criteria of acute pancreatitis and scoring system to predict the severity of the disease. Comparisons with the present criteria and scoring system were described and their potential problems were briefly discussed.