Ferric reducing antioxidant potential (FRAP) of antioxidants using reaction flow chromatography.

High performance liquid chromatography coupled with post column derivatisation (HPLC-PCD) may be used to profile the antioxidant content of a sample. There are, however, drawbacks in the use of HPLC-PCD setups; namely the high volume reaction coils that are typically used lowering the observed separation efficiency. Reaction flow chromatography has the ability to overcome these inefficiencies by using a more efficient mixing technique inside the outlet fitting itself, post column reaction loops can be removed with resulting improvement in signal to noise response, plus the separation efficiency is maintained. We assessed two methods of HPLC-PCD antioxidant analysis based on the ferric reducing antioxidant power (FRAP) assay in both conventional and reaction flow HPLC-PCD modes. It was found that the reaction flow technique demonstrated significant advantages over the conventional technique in terms of signal to noise, linear range, precision and observed separation efficiency.

Structural and functional relationship between the receptor recognition and neuraminidase activities of the Newcastle disease virus hemagglutinin-neuraminidase protein: receptor recognition is dependent on neuraminidase activity.

The terminal globular domain of the paramyxovirus hemagglutinin-neuraminidase (HN) glycoprotein spike has a number of conserved residues that are predicted to form its neuraminidase (NA) active site, by analogy to the influenza virus neuraminidase protein. We have performed a site-directed mutational analysis of the role of these residues in the functional activity of the Newcastle disease virus (NDV) HN protein. Substitutions for several of these residues result in a protein lacking both detectable NA and receptor recognition activity. Contribution of NA activity, either exogenously or by coexpression with another HN protein, partially rescues the receptor recognition activity of these proteins, indicating that the receptor recognition deficiencies of the mutated HN proteins result from their lack of detectable NA activity. In addition to providing support for the homology-based predictions for the structure of HN, these findings argue that (i) the HN residues that mediate its NA activity are not critical to its attachment function and (ii) NA activity is required for the protein to mediate binding to receptors.

Structural, spectroscopic, and electrochemical studies of binuclear manganese(II) complexes of bis(pentadentate) ligands derived from bis(1,4,7-triazacyclononane) macrocycles.

Structural, electrochemical, ESR, and H2O2 reactivity studies are reported for [Mn(dmptacn)Cl]ClO4 (1, dmptacn = 1,4-bis(2-pyridylmethyl)-1,4,7-triazacyclononane) and binuclear complexes of bis(pentadentate) ligands, generated by attaching 2-pyridylmethyl arms to each secondary nitrogen in bis(1,4,7-triazacyclononane) macrocycles and linked by ethyl (tmpdtne, [Mn2(tmpdtne)Cl2](ClO4)2.2DMF, 2), propyl (tmpdtnp, [Mn2(tmpdtnp)Cl2](ClO4)2.3H2O, 3), butyl (tmpdtnb, [Mn2(tmpdtnb)Cl2](ClO4)2.DMF.2H2O, 4), m-xylyl (tmpdtn-m-X, [Mn2(tmpdtn-m-X)-Cl2](ClO4)2, 5) and 2-propanol (tmpdtnp-OH, [Mn2(tmpdtnp-OH)Cl2](ClO4)2, 6) groups. 1 crystallizes in the orthorhombic space group P2(1)2(1)2(1) (No. 19) with a = 7.959(7) A, b = 12.30(1) A, and c = 21.72(2) A; 2, in the monoclinic space group P2(1)/c (No. 14) with a = 11.455(4) A, b = 15.037(6) A, c = 15.887(4) A, and beta = 96.48(2) degrees; 3, in the monoclinic space group P2(1)/c (No. 14) with a = 13.334(2) A, b = 19.926(2) A, c = 18.799(1) A, and beta = 104.328(8) degrees; and [Mn2(tmpdtnb)Cl2](ClO4)2.4DMF.3H2O (4'), in the monoclinic space group P2(1)/n (No. 14) with a = 13.361(3) A, b = 16.807(5) A, c = 14.339(4) A, and beta = 111.14(2) degrees. Significant distortion of the Mn(II) geometry is evident from the angle subtended by the five-membered chelate (ca. 75 degrees) and the angles spanned by trans donor atoms (< 160 degrees). The Mn geometry is intermediate between octahedral and trigonal prismatic, and for complexes 2-4, there is a systematic increase in M...M distance with the length of the alkyl chain. Cyclic and square-wave voltammetric studies indicate that 1 undergoes a 1e- oxidation from Mn(II) to Mn(III) followed by a further oxidation to MnIV at a significantly more positive potential. The binuclear Mn(II) complexes 2-5 are oxidized to the Mn(III) state in two unresolved 1e- processes [MnII2-->MnIIMnIII-->MnIII2] and then to the MnIV state [MnIII2-->MnIIIMnIV-->MnIV2]. For 2, the second oxidation process was partially resolved into two 1e- oxidation processes under the conditions of square-wave voltammetry. In the case of 6, initial oxidation to the MnIII2 state occurs in two overlapping 1e- processes as was found for 2-5, but this complex then undergoes two further clearly separated 1e- oxidation processes to the MnIIIMnIV state at +0.89 V and the MnIV2 state at +1.33 V (vs Fc/Fc+). This behavior is attributed to formation of an alkoxo-bridged complex. Complexes 1-6 were found to catalyze the disproportionation of H2O2. Addition of H2O2 to 2 generated an oxo-bridged mixed-valent MnIIIMnIV intermediate with a characteristic 16-line ESR signal.

Mutations in the Newcastle disease virus hemagglutinin-neuraminidase protein that interfere with its ability to interact with the homologous F protein in the promotion of fusion.

Recent evidence suggests that the attachment (HN) and fusion (F) glycoproteins of Newcastle disease virus interact at the cell surface in a virus-specific manner to promote syncytium formation. Consistent with the existence of such an interaction, we have shown that it is possible to coimmunoprecipitate (co-IP) the two proteins from the surface of transiently expressing cells using a monoclonal antibody to either protein. Further, we show that a point mutation in the globular domain of HN that abolishes its receptor recognition and neuraminidase (NA) and fusion activities also abolishes its ability to interact with F in the co-IP assay. The mechanism by which this mutation might interfere with the interaction between the two proteins is discussed in terms of the postulate that recognition by HN of cellular receptors triggers its interaction with F and the apparently conflicting evidence for an interaction between the two proteins in the endoplasmic reticulum. Also, characterization of a set of chimeric HN proteins, having short overlapping sequences from a heterologous HN protein in the F-specific domain in the protein stalk, reveals that a weakened interaction between HN and F is still sufficient to trigger fusion.

Functional chimeric HN glycoproteins derived from Newcastle disease virus and human parainfluenza virus-3.

Newcastle disease virus (NDV) is primarily a respiratory tract pathogen of birds, particularly chickens, but it occasionally produces infection in man. Human parainfluenza virus type 3 (hPIV3) is a common respiratory pathogen, particularly in young children. These two viruses gain entry to host cells via direct fusion between the viral envelope and the cell membrane, mediated by the two surface glycoproteins: the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. Promotion of fusion by HN and F requires that they are derived from homologous viruses. We have constructed chimeric proteins composed of domains from heterologous HN proteins. Their ability to bind cellular receptors and to complement the F protein of each virus in the promotion of fusion were evaluated in a transient expression system. The fusion specificity was found to segregate with a segment extending from the middle of the transmembrane anchor to the top of the putative stalk region of the ectodomain. All of the chimeras, in which the globular domain is derived from the NDV HN and various lengths of the stalk region are derived from the hPIV3 HN maintain receptor binding activity, but some have markedly reduced neuraminidase (NA) activity. Decrease in the NA activity of the chimeras correlates with alteration in the antigenic structure of the globular domain. This suggests that the stalk region of the HN spike is important for maintenance of the structure and function of the globular domain of the HN protein spike.

Cooperative neuraminidase activity in a paramyxovirus.

Paramyxoviruses possess neuraminidase (NA) activity, i.e., the ability to cleave sialic acid from membrane-bound and soluble glycoconjugates. The activity is associated with a homotetrameric, surface glycoprotein spike, called the hemagglutinin-neuraminidase. This structure also mediates viral attachment to sialic acid-containing receptors and constitutes the major neutralizing antigen for the virus. Cooperativity has been demonstrated for the NA activity of an isolate of one of the paramyxoviruses, Newcastle disease virus. Although all known viral NA proteins are homooligomeric, this is the first demonstration of cooperativity in this family of proteins. A variant virus, selected by escape from neutralization by a monoclonal antibody thought to bind close to the NA active site, has lost cooperativity. Conversion to the noncooperative state correlates with increases in both avidity for cellular receptors and fusogenic activity.