Serological Cross-Reactions Between the Hemagglutinin Subunits of H0N1 and H1N1 Influenza Viruses Detected with “Monospecific” Antisera

"Monospecific" antisera to the "fragile" hemaglutinnis of H0N1 (PR8) and H1N1 (FM1) influenza viruses detected an asymmetrical cross-reaction between these two strains that could not be explained by a common neuraminidase.     

Pharmacological characterization of [³H]CHIBA-3007 binding to glycine transporter 1 in the rat brain

Glycine transporter-1 (GlyT-1) in glial cells regulates extracellular levels of glycine, which acts as an obligatory co-agonist at the N-methyl-D-aspartate (NMDA) receptors in the brain. In the present study, we developed a novel radioligand, [³H]3-chloro-N-((S)-((R)-1-methylpiperidin-2-yl)(thiophen- 3-yl)methyl)-4- (trifluoromethyl)picolinamide ([³H]CHIBA-3007), for studying GlyT-1 in the brain. The presence of a single saturable high-affinity binding component for [³H]CHIBA-3007 binding to the rat brain membranes was detected. Scatchard analysis revealed an apparent equilibrium dissociation constant (K_d) of 1.61±0.16 nM and a maximal number of binding sites (B_max) of 692.8±22.8 fmol/mg protein (mean ± SEM, n = 3). The specific binding of [³H]CHIBA-3007 was inhibited by a number of GlyT-1 inhibitors, such as CHIBA-3007, desmethyl-CHIBA-3007, CHIBA-3008, SSR504734, NFPS/ALX5407, LY2365109 and {"type":"entrez-protein","attrs":{"text":"Org24598","term_id":"1179171570","term_text":"ORG24598"}}Org24598, consistent with the pharmacological profiles of GlyT-1 inhibitors. Interestingly, the potency of eight GlyT-1 inhibitors (CHIBA-3007, desmethyl-CHIBA-3007, NFPS/ALX5407, LY2365109, {"type":"entrez-protein","attrs":{"text":"Org24598","term_id":"1179171570","term_text":"ORG24598"}}Org24598, SSR504734, sarcosine, and glycine) for blocking in vitro specific binding of [³H]CHIBA-3007 was significantly correlated with the potency of these inhibitors for inhibiting [¹⁴C]glycine uptake in the rat brain membranes. In contrast, the GlyT-2 inhibitor ALX1393 exhibited very weak for [³H]CHIBA-3007 binding. Furthermore, the regional distribution of [³H]CHIBA-3007 binding in the rat brain was similar to the previously reported distribution of GlyT-1. The present findings suggest that [³H]CHIBA-3007 would be a useful new radioligand for studying GlyT-1 in the brain.     

Effects of point mutations in pVHL on the binding of HIF-1α

The von Hippel-Lindau tumor suppressor protein (pVHL) has an essential role in the regulation of the hypoxia response pathway in animal cells. Under normoxic conditions, the hypoxia-inducible factor (HIF) undergoes trans-4-prolyl hydroxylation and is subsequently recognised by the β-domain of pVHL, leading to the ubiquitination and degradation of HIF. Mutations of pVHL alter the binding of HIF. A subset of relevant clinically observed mutations to pVHL are thought to cause weaker binding of HIF-1α and are associated with cancer and cardiovascular diseases. Here, we present computational studies analyzing the interaction of HIF with mutant forms of pVHL, describing at atomic detail the local structural reorganization caused by substitution of certain residues of pVHL. The results reveal that the canonical configuration in the wild-type system is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the h-bond network in the binding site disrupts HIF binding. Although the experimentally observed ordering of binding energies for mutants of Tyr98 is reproduced, our examination of a broader range of mutations does not support the hypothesis of a correlation between the degree of disruption of the pVHL/HIF-1α interaction caused by a mutation and the phenotype with which the mutation is associated. We suggest that disruption of the binding interaction is one of many factors behind the manifestation of VHL disease.     

Dynamical behaviour of the human β1-adrenoceptor under agonist binding

The human β₁-adrenoceptor (hβ₁AR) is a transmembrane (TM) protein responsible for the signal transduction pathway via agonist interaction. Despite its importance, hβ₁AR activation mechanism is still unclear. The most studied and widely accepted mechanism is the disruption of a salt bridge between TM3 arginine and TM6 glutamic acid, called ionic lock. In this work, we constructed a functional hβ₁AR-model equilibrated in a membrane environment to study the influence of agonist binding on the dynamical behaviour of hβ₁AR and on the opening of the ionic lock. The results indicate that the agonist (R-noradrenaline) disturbs the hβ₁AR, causing a TM helices rotation, disrupting the ionic lock. This rotational motion occurs in opposite directions in the intercellular and extracellular domains of hβ₁AR, opening the ionic lock.     

Assignment of the 1H, 13C and 15N resonances of the calponin homology-2 domain of α-actinin-4

We report the assignment of the 110 amino acid second calponin homology domain of human α-actinin-4. The two calponin homology domains of α-actinin combine to regulate F-actin binding.     

《柳叶刀》：H7N9死亡风险低于H5N1高于H1N1

中国科学家6月24日在线发表于《柳叶刀》上的研究论文称,H7N9禽流感患者死亡风险远低于人感染H5N1禽流感。但高于季节性流感和甲型H1N1流感。     

Identification of binding partners interacting with the α1-N-propeptide of type V collagen

The predominant form of type V collagen is the [α1(V)]?α2(V) heterotrimer. Mutations in COL5A1 or COL5A2, encoding respectively the α1(V)- and α2(V)-collagen chain, cause classic EDS (Ehlers-Danlos syndrome), a heritable connective tissue disorder, characterized by fragile hyperextensible skin and joint hypermobility. Approximately half of the classic EDS cases remain unexplained. Type V collagen controls collagen fibrillogenesis through its conserved α1(V)-N-propeptide domain. To gain an insight into the role of this domain, a yeast two-hybrid screen among proteins expressed in human dermal fibroblasts was performed utilizing the N-propeptide as a bait. We identified 12 interacting proteins, including extracellular matrix proteins and proteins involved in collagen biosynthesis. Eleven interactions were confirmed by surface plasmon resonance and/or co-immunoprecipitation: α1(I)- and α2(I)-collagen chains, α1(VI)-, α2(VI)- and α3(VI)-collagen chains, tenascin-C, fibronectin, PCPE-1 (procollagen C-proteinase enhancer-1), TIMP-1 (tissue inhibitor of metalloproteinases-1), MMP-2 (matrix metalloproteinase 2) and TGF-β1 (transforming growth factor β1). Solid-phase binding assays confirmed the involvement of the α1(V)-N-propeptide in the interaction between native type V collagen and type VI collagen, suggesting a bridging function of this protein complex in the cell-matrix environment. Enzymatic studies showed that processing of the α1(V)-N-propeptide by BMP-1 (bone morphogenetic protein 1)/procollagen C-proteinase is enhanced by PCPE-1. These interactions are likely to be involved in extracellular matrix homoeostasis and their disruption could explain the pathogenetic mechanism in unresolved classic EDS cases.     

Crystal structure of the N-terminal region of human Topoisomerase IIβ binding protein 1

Human DNA Topoisomerase IIβ binding protein 1 (TopBP1) is a modulating protein that plays an essential role in the response to DNA damage. The N-terminal region of TopBP1, which contains predicted BRCA1-carboxy terminal (BRCT) domains 1 and 2, binds to Rad9, a component of the cell cycle checkpoint clamp Rad9-Hus1-Rad1 complex. Here, we report the crystal structure of the TopBP1 N-terminal region (residues 1-290) at 2.4 Å resolution. Interestingly, in addition to the predicted tandem BRCT1-2 repeats (residues 103-284), residues 7-98 form a previously unreported BRCT domain (here, BRCT0). In contrast to both BRCT1 and BRCT2, which possess the conventional phosphopeptide binding residues within a surface pocket, the corresponding pocket in BRCT0 is largely hydrophobic. Structural comparisons together with peptide binding studies indicate that the tandem BRCT1-2 domains are the binding region for phosphorylated Ser387 in Rad9.     

Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the α-chain of human hemoglobin

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the -chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues 121–135. The present study describes a precise delineation of this Hp-binding site on the -chain. Two overlapping peptides (111–125 and 121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (119–135, 119–133, 119–131, 119–129, and 119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of¹²⁵I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide 119–127 retained a Hp-binding activity equivalent to that of peptide 121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (122–127, 121–127, 120–127, 119–127, 118–127, 117–127, 116–127, and 115–127). The binding of¹²⁵I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the -chain of human Hb comprises residues 121–127.     

Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7–Pat1 complex

Decapping is a critical step in the conserved 5′-to-3′ mRNA decay pathway of eukaryotes. The hetero-octameric Lsm1-7–Pat1 complex is required for normal rates of decapping in this pathway. This complex also protects the mRNA 3′-ends from trimming in vivo. To elucidate the mechanism of decapping, we analyzed multiple lsm1 mutants, lsm1-6, lsm1-8, lsm1-9, and lsm1-14, all of which are defective in decapping and 3′-end protection but unaffected in Lsm1-7–Pat1 complex integrity. The RNA binding ability of the mutant complex was found to be almost completely lost in the lsm1-8 mutant but only partially impaired in the other mutants. Importantly, overproduction of the Lsm1-9p- or Lsm1-14p-containing (but not Lsm1-8p-containing) mutant complexes in wild-type cells led to a dominant inhibition of mRNA decay. Further, the mRNA 3′-end protection defect of lsm1-9 and lsm1-14 cells, but not the lsm1-8 cells, could be partly suppressed by overproduction of the corresponding mutant complexes in those cells. These results suggest the following: (1) Decapping requires both binding of the Lsm1-7–Pat1 complex to the mRNA and facilitation of the post-binding events, while binding per se is sufficient for 3′-end protection. (2) A major block exists at the post-binding steps in the lsm1-9 and lsm1-14 mutants and at the binding step in the lsm1-8 mutant. Consistent with these ideas, the lsm1-9, 14 allele generated by combining the mutations of lsm1-9 and lsm1-14 alleles had almost fully lost the RNA binding activity of the complex and behaved like the lsm1-8 mutant.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

Is the C-terminal region of bradykinin the binding site of polyphenols?

Bradykinin is a bioactive hormone involved in a variety of physiological processes. In various solvents, this peptide adopts beta-turn structures. The C-terminal turn is a structural feature for the receptor affinity of agonists and antagonists while the N-terminal turn might be important for antagonistic activities. Polyphenols like dimeric proanthocyanidin B3 interact with the peptide. Thus to investigate the effects of polyphenols on bradykinin activity and structure, we studied the interaction in the structuring solvent DMSO which can be a close mimic of aqueous physiological environments like receptor-binding sites. Bradykinin alone presented a folded structure with two turns. B3 interacted with the peptide C-terminus and involved the loss of the bend structure of this region, while the N-terminus turn was maintained. Numerous studies have shown that polyphenolic molecules can act upon various biological targets, and the formation of this type of complex might be one of the possible modes of action.     

Supply of Neuraminidase Inhibitors Related to Reduced Influenza A (H1N1) Mortality during the 2009–2010 H1N1 Pandemic: An Ecological Study

Background

The influenza A (H1N1) pandemic swept across the globe from April 2009 to August 2010 affecting millions. Many WHO Member States relied on antiviral drugs, specifically neuraminidase inhibitors (NAIs) oseltamivir and zanamivir, to treat influenza patients in critical condition. Such drugs have been found to be effective in reducing severity and duration of influenza illness, and likely reduced morbidity during the pandemic. However, it is less clear whether NAIs used during the pandemic reduced H1N1 mortality.

Methods

Country-level data on supply of oseltamivir and zanamivir were used to predict H1N1 mortality (per 100,000 people) from July 2009 to August 2010 in forty-two WHO Member States. Poisson regression was used to model the association between NAI supply and H1N1 mortality, with adjustment for economic, demographic, and health-related confounders.

Results

After adjustment for potential confounders, each 10% increase in kilograms of oseltamivir, per 100,000 people, was associated with a 1.6% reduction in H1N1 mortality over the pandemic period (relative rate (RR) = 0.84 per log increase in oseltamivir supply). While the supply of zanamivir was considerably less than that of oseltamivir in each Member State, each 10% increase in kilogram of active zanamivir, per 100,000, was associated with a 0.3% reduction in H1N1 mortality (RR = 0.97 per log increase).

Conclusion

While there are limitations to the ecologic nature of these data, this analysis offers evidence of a protective relationship between antiviral drug supply and influenza mortality and supports a role for influenza antiviral use in future pandemics.     

The relation of protein binding to function: what is the significance of munc18 and synaptotagmin binding to syntaxin 1, and where are the corresponding binding sites?

The Q-SNARE syntaxin 1 is a central component of the synaptic membrane fusion machinery. Syntaxin probably interacts with multiple proteins during synaptic vesicle exocytosis. In vitro, the tightest binding partners for syntaxin 1 are other SNAREs (synaptobrevin/VAMP and SNAP-25) and munc18-1 (also known as rbsec1/nsec1). Recent studies on Drosophila syntaxin led to the surprising finding that a syntaxin mutant which does not bind the munc18-homolog Rop nevertheless functionally substitutes for wild-type syntaxin in membrane fusion (Wu et al., Neuron 23, 593-605, 1999). This observation suggested that syntaxin 1 binding to munc18-1 is not essential for fusion, a puzzling conclusion in view of the tight binding of munc18 and syntaxin homologs in all organisms. To address this issue, we have now reinvestigated the interaction of syntaxin with munc18 and Rop. We find that the syntaxin sequence that was mutated in the Drosophila studies is not essential for munc18/Rop binding, and that the mutant is in fact able to bind to munc18/Rop. Thus the fact that the mutant syntaxin rescues release cannot be used as an argument that munc18 binding is not essential. In addition to munc18 and SNAREs, several other proteins have been suggested to interact with various domains of syntaxin 1, notably the calcium-sensor synaptotagmin and the vesicle protein CSP. Our results confirm that the SNARE motif in syntaxin binds to synaptotagmin, but this interaction does not require the very C-terminus of the motif. Interestingly, binding of synaptotagmin appears to be decreased in the closed conformation of syntaxin. In contrast, no interaction of CSP with syntaxin was detected even under low-stringency conditions. Our data suggest 1., that assays measuring protein/protein interactions that involve syntaxin may be more difficult to evaluate than is often assumed because of the sticky nature of the proteins involved, and 2., that it is currently not possible to draw conclusions about the importance of the various interactions with the available data from Drosophila or vertebrates.     

The Decision to Vaccinate or Not during the H1N1 Pandemic: Selecting the Lesser of Two Evils?

Background

With the release of the H1N1 vaccine, there was much controversy surrounding its use despite strong encouragements to be vaccinated in the media. Though studies have examined factors influencing people''s decision to be vaccinated, few have focused on how general beliefs about the world or where an individual gathers information might influence that decision.

Methodology/Principal Findings

A cross-sectional web-based survey (N = 817) was conducted during the H1N1 outbreak after the vaccine was available. Variables examined included sociodemographic information, health related behaviours, specific beliefs concerning the H1N1 virus and its vaccine, as well as general beliefs, such as fear of contamination, intolerance of uncertainty, emotional states, coping behaviour, and the source of information concerning the virus. Three converging statistical methods were used to examine the associations – analysis of variance, logistic regression, and recursive partition modelling. The most consistent and strongest association was that negative beliefs about the H1N1 vaccine (e.g. fear of its side effects) was related to the decision not to be vaccinated, whereas beliefs about the dangers of the H1N1 virus was related to the decision to be vaccinated. Most notably, having very strong negative beliefs about the vaccine was a more powerful predictor than even strong beliefs about the dangers of the H1N1 virus. Furthermore, obtaining information from the Internet, as compared to more traditional sources of information (e.g., TV, newspapers) was related to the decision not to be vaccinated.

Conclusions/Significance

These results are consistent with the Health Belief Model. Importantly they suggest that during future pandemics public health officials should not only discuss the dangers of the pandemic but also (i) take additional steps to reassure the public about the safety of vaccines and (ii) monitor the information disseminated over the Internet rather than strictly relying on the more traditional mass media.