NMR studies of the quaternary structure and heterogeneity of nitrosyl- and methemoglobin.

NMR was used to study the quaternary structure of nitrosyl- and methemoglobin, the kinetics and equilibrium behavior of nitric oxide binding, and the oxidation of hemoglobin. The -9.6 ppm (from H2O) resonance was used as a measure of nitrosylhemoglobin molecules in the T quaternary structure. We found that stripped nitrosylhemoglobin is 70% in the T state below pH 6.4, and is in the R state above. Inositol hexaphosphate (IHP) raises this transition point to pH 7.5. For stripped aquomethemoglobin, the T marker at -10 ppm is absent. In IHP, at pH 6.5 all of the molecules are in the T state. At both higher and lower pH they shift to the R state. The intensity decreases to half of its maximum at pH 5.5 and 7.4. The relative affinity of nitric oxide binding to the alpha and beta subunits was inferred from the intensities of the resonances at -12 and -18 ppm. Under conditions in which nitrosylhemoglobin exists in the T state, NO binds to the alpha subunit 10 times more strongly than it does to the beta subunit. The kinetic experiments reveal that it binds to the two subunits at the same rate and that it dissociates at 5 x 10(-3) s-1 from the beta subunit and at 5 x 10(-4) s-1 from alpha subunit. At high pH, the two subunits are ligated at the same rate. Potassium ferricyanide oxidation, at pH 6.0 in the absence of IHP, is about 3 times more favorable for the alpha than the beta subunit. Addition of IHP raises this preferential oxidation slightly. At pH 8.44, both alpha and beta subunits were oxidized at the same rate.     

In memoriam: Professor Ray WU

<正>During the Chinese New Year holidays in 2008, Professor Zhang Nai-Heng, former chair of the Department of Biochemistry, Beijing Medical College, informed me of the sad news that Professor Ray Wu had passed away in Ithaca, New York on     

Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1^f/f), and the other with a homozygous disruption (Fbh1^?/?). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1^?/? cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1^f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1^f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1^f/f and Fbh1^?/? cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.     

Sterically stabilized liposomes: a hypothesis on the molecular origin of the extended circulation times.

Therapeutic applications of intravenously injected liposomes have been limited by their rapid clearance from the bloodstream and their uptake by the macrophage cells of the liver and spleen (RES). Recently, however, liposomes which substantially evade the rapid uptake by the RES have been introduced. Since these liposomes exhibit dramatically different pharmacokinetics and biodistribution, new therapeutic opportunities have appeared. These include enhanced efficacy of antineoplastic agents against tumors, sites of inflammation, and targeting ligand-coupled liposomes to extravascular targets. Despite extensive experimental work, the mechanism underlying the ability of liposomes to avoid the rapid uptake by the RES is still not fully understood. Our approach is an alternative to seeking the answers in complex differential interactions of liposomes with various components of blood. We believe that the effect can be easily explained, at least in qualitative terms, by the fundamental principles of colloid stability. In this communication, we propose that steric stabilization of liposomes is responsible for their prolonged circulation times. We propose that stabilization results from local surface concentration of highly hydrated groups that sterically inhibit both electrostatic and hydrophobic interactions of a variety of blood components at the liposome surface.     

Rat liver cytosolic azoreductase. Purification and characterization.

An azoreductase has been purified to apparent homogeneity from the hepatic 105,000 x g supernatant fraction of 3-methylcholanthrene-treated rats. In the presence of sodium dodecyl sulfate, the purified enzyme preparation electrophoreses on polyacrylamide gels as a single protein band with a molecular weight of 30,000. In the absence of detergent, chromatography of the azoreductase on Sephadex G-100 gives a molecular weight of about 52,000 suggesting that the native enzyme may exist as a dimer. The purified azoreductase has a typical flavoprotein absorption spectrum and contains 2 mol of FAD/mol of enzyme. The enzyme catalyzes the reductive fission of methyl red (2'-carboxy-4-N,N-dimethylaminoazobenzene) and a structure-activity study indicates that the 2'-carboxyl group of methyl red is essential for catalysis since other structurally related analogs are totally inactive.     

A Role for Toll-like Receptor 3 Variants in Host Susceptibility to Enteroviral Myocarditis and Dilated Cardiomyopathy

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3β (MAP1LC3β). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3β fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.     

Screening and Identification of Marburg Virus Entry Inhibitors Using Approved Drugs

正Dear Editor,Marburg virus(MARV) belongs to the Filoviridae family,along with the related Ebola virus(EBOV). Although MARV is less renowned than EBOV, it causes an equally devastating disease, with clinical symptoms similar to EBOV infection and a remarkably high mortality rate. To