Hyper-processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli

BACKGROUND: A strong mutator mutation, dnaE173, leads to a Glu612 --> Lys amino acid change in the alpha subunit of Escherichia coli DNA polymerase III (PolIII) holoenzyme and abolishes the proofreading function of the replicative enzyme without affecting the 3' --> 5' exonuclease activity of the epsilon subunit. The dnaE173 mutator is unique in its ability to induce sequence-substitution mutations, suggesting that an unknown function of the alpha subunit is hampered by the dnaE173 mutation. RESULTS: A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the beta clamp subunit) and the beta subunit showed a strong resistance to replication-pausing on the template DNA and readily promoted strand-displacement DNA synthesis. Unlike wild-type PolIII*, dnaE173 PolIII* was able to catalyse highly processive DNA synthesis without the aid of the beta-clamp subunit. The rate of chain elongation by the dnaE173 holoenzyme was reduced to one-third of that determined for the wild-type enzyme. In contrast, an exonuclease-deficient PolIII holoenzyme was vastly prone to pausing, but had the same rate of chain elongation as the wild-type. CONCLUSIONS: The hyper-processivity and slower DNA chain elongation rate of the dnaE173 holoenzyme are distinct effects caused by the dnaE173 mutation and are likely to be involved in the sequence-substitution mutagenesis. A link between the proofreading and chain elongation processes was suggested.     

Thromboembolic events associated with Guglielmi detachable coil embolization with use of diffusion-weighted MR imaging. Part II. Detection of the microemboli proximal to cerebral aneurysm

BACKGROUND AND PURPOSE: The purpose of this study was to document the incidence and radiologic appearance of thromboembolic events during Guglielmi detachable coil (GDC) embolization for asymptomatic basilar artery (BA) bifurcation and BA-superior cerebellar artery (SCA) aneurysms by using diffusion-weighted (DW) MR imaging, with special emphasis on the evidence of thromboembolic events in vascular territories proximal from the treated aneurysm, which cause cerebellar infarction, and to discuss which step of the procedure (aneurysm or catheter manipulation) may play a role for most thromboembolic events. METHODS: Since 1999, 38 asymptomatic BA bifurcation and BA-SCA aneurysms were treated with GDCs at the National Cardiovascular Center. DW studies were performed for 26 patients between 2 and 5 days after GDC embolizations. All DW images were reviewed by two radiologists for depiction of abnormalities. These findings were retrospectively evaluated with clinical and technical factors of thromboembolic events. RESULTS: DW images showed new hyperintense lesions in 18 patients (69%), with seven (27%) incurring neurologic deteriorations. All symptomatic patients fully recovered by discharge. Fourteen (78%) of 18 patients showed new lesions proximal to the treated aneurysm; that is, in the cerebellar hemispheres. In three cases treated with the balloon-assisted technique, new hyperintense lesions were seen. CONCLUSION: In our experience, most thromboembolic events related to the use of the GDC embolization may be caused by catheter manipulation, especially in the case of the balloon-assisted technique. Caution should be exercised in the handling of catheters. Furthermore, a softer and smaller caliber catheter and simple GDC technique should be considered.     

Case Report: Adjuvant Therapy with a High Dose of Mitotane for Adrenocortical Carcinoma in a 4-year-old Boy

This report concerns control of adrenocortical carcinoma in a 4-yr-old boy by adjuvant mitotane therapy. He presented precocious puberty and was diagnosed with adrenocortical carcinoma. He underwent surgical resection, and adjuvant mitotane therapy was initiated, leading to a final dose of 5.0 g/day. Despite monitoring of the plasma mitotane level, encephalopathy developed 5 mo after initiation. Although he recovered from the encephalopathy, careful follow-up of his growth and development is necessary. On the other hand, he has been free of recurrence and metastases for 3 yr since discontinuation of mitotane. A high dose of mitotane is potentially effective as an adjuvant chemotherapy for adrenocortical carcinoma, although optimal and safe usage needs to be established for children.     

Visualization of newly deposited tau in neurofibrillary tangles and neuropil threads

Neurofibrillary tangles (NFTs) and neuropil threads (NTs), the major hallmark of Alzheimer disease (AD), are composed of the microtubule-associated protein tau that has undergone posttranslational modifications, including deamidation and isomerization on asparaginyl or aspartyl residues. Because such modifications represent protein aging, we generated 2 antibodies, TM4, specific for Asp-387 of tau, and iD387, specific for isoAsp-387 of tau, to investigate the evolution of NFTs and NTs. On Western blots of Sarkosyl-insoluble fractions, TM4 strongly labeled paired helical filament-tau (PHF-tau), whereas iD387 preferentially labeled PHF smear. Thus, it is reasonable to postulate that TM4-labeled tau (unmodified tau species) represents more recent deposition, and iD387-labeled tau (modified tau species) represents earlier deposition. Unexpectedly, TM4 immunostained even highly evolved NFTs, suggesting that deposition of newly produced tau continues until neuronal death. iD387 labeled the whole profile of NFTs up to distal dendritic branches, whereas TM4 staining was localized to particular portions of NFTs in proximal dendrites and neuronal perikarya. In NTs, TM4 preferentially labeled the outer portion, whereas iD387 intensely labeled the core portion. Based on TM4-positive NFT counts and total NFT counts, we speculate that NFTs in the human hippocampus are produced at a constant rate irrespective of the disease stage.     

Free vascularized fibula grafting for reconstruction of the wrist following wide tumor excision

Free vascularized fibula transfer is an established method for reconstruction of the wrist following tumor resection. In cases of resection of the radial articular surface, three reconstructive options are possible: fibular head transfer along with the shaft to replace the radial joint surface, fixation of the fibula to the scaphoid and lunate, or a complete wrist fusion. Three patients with a tumor involving the distal end of the radius were treated with wide resection, and subsequent wrist reconstruction was performed, using the above-mentioned procedures. Although our experience included only a small number of patients, both radio-carpal hemiarthroplasty and fibulo-scapho-lunate fusion similarly provided successful wrist stability and functional range of motion in these cases. Even when the wrist was totally fused with the fibula, its function was still acceptable.     

Oxidative stress and metal content in blood and cerebrospinal fluid of amyotrophic lateral sclerosis patients with and without a Cu, Zn-superoxide dismutase mutation

Hydroxyl radical, ascorbate free radical, superoxide dismutase (SOD) activities, Cu,Zn-SOD protein, Mn-SOD protein, 8-hydroxy-2' -deoxyguanosine (8-OHdG) and metals were compared in red blood cells (RBC), plasma and/or cerebrospinal fluid (CSF) between patients with sporadic amyotrophic lateral sclerosis (SALS), familial ALS (FALS) showing the Leu126Ser mutation in the Cu, Zn-SOD gene and controls. In patients with FALS or SALS, concentrations of hydroxyl radical in blood and ascorbate free radical and 8-OHdG in CSF were higher than control group values, while SOD activities in RBC and CSF were lower. In contrast, Cu, Zn-SOD protein concentrations in RBC were low only in FALS patients. Concentrations of Cu in CSF of SALS patients were higher than in controls. Thus, the pathogenesis of increased oxidative stress differs between SALS patients and FALS patients with a mutant Leu126Ser SOD1 gene.     

HRAS mutants identified in Costello syndrome patients can induce cellular senescence: possible implications for the pathogenesis of Costello syndrome

Costello syndrome (CS) is a congenital disease that is characterized by a distinctive facial appearance, failure to thrive, mental retardation and cardiomyopathy. In 2005, we discovered that heterozygous germline mutations in HRAS caused CS. Several studies have shown that CS-associated HRAS mutations are clustered in codons 12 and 13, and mutations in other codons have also been identified. However, a comprehensive comparison of the substitutions identified in patients with CS has not been conducted. In the current study, we identified four mutations (p.G12S, p.G12A, p.G12C and p.G12D) in 21 patients and analyzed the associated clinical manifestations of CS in these individuals. To examine functional differences among the identified mutations, we characterized a total of nine HRAS mutants, including seven distinct substitutions in codons 12 and 13, p.K117R and p.A146T. The p.A146T mutant demonstrated the weakest Raf-binding activity, and the p.K117R and p.A146T mutants had weaker effects on downstream c-Jun N-terminal kinase signaling than did codon 12 or 13 mutants. We demonstrated that these mutant HRAS proteins induced senescence when overexpressed in human fibroblasts. Oncogene-induced senescence is a cellular reaction that controls cell proliferation in response to oncogenic mutation and it has been considered one of the tumor suppression mechanisms in vivo. Our findings suggest that the HRAS mutations identified in CS are sufficient to cause oncogene-induced senescence and that cellular senescence might therefore contribute to the pathogenesis of CS.     

Successful living related liver transplantation in a case with biliary atresia associated with corrected transposition of the great arteries

Little is known about the safety of LRLTx in children with end stage liver disease associated with congenital cardiac anomalies. We report the successful LRLTx in a case with extrahepatic biliary atresia associated with cTGA, VSD, and PS. Preoperative cardiac function was evaluated by cardiac echogram and cardiac catheterization. The recipient's cardiac function was preserved (EF; 54%); however, because of the left to right shunt disease, oxygen saturation was 91%. At operation, carbon dioxide insufflation into the abdominal cavity was attempted to prevent sudden air embolism. Hemodynamic variables were stabilized during partial clamping of the inferior vena cava, and at reperfusion of the portal vein. However, a sudden decrease in blood oxygenation was observed during hepatico-jejunostomy, which was easily normalized by graft mobilization. Post-operatively, neither heart failure nor cerebral infarction because of air embolism was observed. In conclusion, together with preserved cardiac function and carbon dioxide insufflation, LRLTx was successful. Further studies are required to establish the algorithm for the strategy of treating both congenital cardiac anomalies and liver failure.     

Discovery of Novel Benzo[a]phenoxazine SSJ-183 as a Drug Candidate for Malaria

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