Interaction of polyriboinosinic acid. Polyribocytidylic acid with human lymphoblastoid cells

The double-stranded RNA, poly(I).poly(C), failed to induce interferon in Namalva lymphoblastoid cells even when tested under varying conditions. Striking differences were observed between lymphoblastoid cells and human diploid fibroblasts in the binding, release and degradation of radiolabelled poly(I).poly(C). The cells were able to take up radiolabelled poly(I).poly(C) for only a short time. Cell-associated radioactivity was immediately released into the supernatant fluid. Although the released material was still TCA-precipitable, partial or complete degradation could not be excluded. Pretreatment of the cells with DEAE-dextran enabled the cells to take up a much larger amount of radiolabelled poly(I).poly(C) and this material was not being released. However, this procedure did not lead to any detectable interferon production.     

1H NMR studies of lac-operator DNA fragments.

The hydrogen-bonded imino protons of a 14 base pair double-stranded DNA fragment comprising one half of the lac operator of E. coli were investigated by 360 MHz H NMR. From combined melting studies of this synthetic 14 b.p. fragment and its two constituent 7 b.p. fragments a nearly complete assignment for the low-field proton resonances was obtained. The experimental spectra are compared with calculated spectra and with the spectrum of a 51 b.p. DNA restriction fragment from E. coli containing the complete lac operator. Structural information on these oligonucleotides is presented. This study is a prerequisite for future 1H NMR investigations of the interaction of the lac operator with the lac repressor.     

1H NMR studies of lambda cro repressor. 2. Sequential resonance assignments of the 1H NMR spectrum

The cro repressor protein from bacteriophage lambda has been studied in solution by two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Following the approach of Wüthrich and co-workers [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311-319], individual spin systems were identified by J-correlated spectroscopy (COSY) supplemented, where necessary, by relayed coherence transfer spectroscopy (RELAY). Nuclear Overhauser effect spectroscopy (NOESY) was used to obtain sequence-specific assignments. From the two-dimensional spectra, the peptide backbone resonances (NH and C alpha H) for 65 of the 66 amino acids were assigned, as well as most of the side chain resonances. The chemical shifts for the assigned protons are reported at 35 degrees C in 10 mM potassium phosphate, pH 6.8, and in 10 mM potassium phosphate, pH 4.6, 0.2 M KCl, and 0.1 mM EDTA. Small shifts were observed for some resonances upon addition of salt, but no major changes in the spectrum were seen, indicating that no global structural change occurs between these ionic strengths. NOE patterns characteristic of alpha-helices, beta-strands, and turns are seen in various regions of the primary sequence. From the location of these regions the secondary structure of cro in solution appears to be virtually identical with the crystal structure [Anderson, W. F., Ohlendorf, D. H., Takeda, Y., & Matthews, B. W. (1981) Nature (London) 290, 754-758]. Missing assignments include the Pro-59 resonances and the peripheral protons of the eight lysine, the three arginine, and three of the five isoleucine residues.     

Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments

We report the complete sequence-specific assignment of the backbone resonances and most of the side-chain resonances in the 1H NMR spectrum of alpha-bungarotoxin by two-dimensional NMR. Problems with resonance overlap were resolved with the assistance of the HRNOESY experiment described in an accompanying paper [Basus, V.J., & Scheek, R.M. (1988) Biochemistry (second paper of three in this issue)]. Significant differences exist between the solution structure described here and the crystal structure of alpha-bungarotoxin, on the basis of the proton to proton distances obtained by nuclear Overhauser enhancement spectroscopy (NOESY) and the corresponding distances from the X-ray crystal structure [Love, R.A., & Stroud, R.M. (1986) Protein Eng. 1, 37]. These differences include a larger beta-sheet in solution and a different orientation of the invariant tryptophan, Trp-28, making the solution structure more consistent with the crystal structure of the homologous neurotoxin alpha-cobratoxin. Four errors in the order of the amino acids in the primary sequence were indicated by the NMR data. These errors were confirmed by chemical means, as described in an accompanying paper [Kosen, P.A., Finer-Moore, J., McCarthy, M.P., & Basus, V.J. (1988) Biochemistry (third paper of three in this issue)].     

Two-dimensional 1H NMR studies of cytochrome c

Two-dimensional nuclear magnetic resonance techniques were used to assign the NH, C alpha H, and C beta H protons of over 60 of the 104 amino acid residues in the 1H NMR spectrum of horse ferrocytochrome c. The majority of these amino acids were completely assigned. Assignments were based on the analysis of two-dimensional J-correlated (COSY), nuclear Overhauser effect (NOESY), and relayed COSY spectra and on comparisons of the J-correlated spectra of various cytochrome c species. Spin diffusion is not a problem with monomeric proteins the size of cytochrome c. Here these advances are illustrated with data that lead to the assignment of the heme-associated residues cysteine-14 and tryptophan-59, the axial ligands methionine-80 and histidine-18, the entire N-terminal helix, and several other amino acid spin systems. With these approaches, structure, structure change, the internal dynamics of cytochrome c, and the interaction of these with function are being studied, especially by observation of the hydrogen exchange behavior of essentially all the H-bonded amides and some side chain protons in both the reduced and oxidized proteins.     

1H NMR relaxation studies of protein-polysaccharide mixtures

NMR water proton relaxation was used to characterize the structure of plant proteins and plant protein-polysaccharide mixtures in aqueous solutions. The method is based on the mobility determination of the water molecules in the biopolymer environment in solutions through relaxation time measurements. Differences of conformation between pea globulin and alpha gliadin seem to control the water molecules mobility in their environment. As deduced from the study of complexes, the electrostatic interactions may also play a major role in the water molecule motions. The phase separation induced under specific conditions seems to promote the translational diffusion of structured water molecules whereas the rotational motion was more restricted.     

Solid-state NMR studies of the prion protein H1 fragment.

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.     

1H NMR studies on lanthanides substituted transferrins

The binding of lanthanide(III) ions to human serum apotransferrin has been investigated through 1H NMR spectroscopy. Several well resolved isotropically shifted signals have been observed between 100/-100 ppm for the Tm, Tb, Yb and Dy derivatives. Significant spectroscopic inequivalence of the two metal binding sites has been revealed. Differences in the behavior of signals assigned to the C- and to the N-terminal site have been observed upon titration with sodium perchlorate.     

Interaction of silver nanoparticles with HIV-1

The interaction of nanoparticles with biomolecules and microorganisms is an expanding field of research. Within this field, an area that has been largely unexplored is the interaction of metal nanoparticles with viruses. In this work, we demonstrate that silver nanoparticles undergo a size-dependent interaction with HIV-1, with nanoparticles exclusively in the range of 1–10 nm attached to the virus. The regular spatial arrangement of the attached nanoparticles, the center-to-center distance between nanoparticles, and the fact that the exposed sulfur-bearing residues of the glycoprotein knobs would be attractive sites for nanoparticle interaction suggest that silver nanoparticles interact with the HIV-1 virus via preferential binding to the gp120 glycoprotein knobs. Due to this interaction, silver nanoparticles inhibit the virus from binding to host cells, as demonstrated in vitro.     

1H NMR studies of mastoparan binding by calmodulin

Association with the cytoactive tetradecapeptide mastoparan perturbs the downfield 1H NMR spectrum of the calmodulin-Ca42+ complex. Changes occur in the resonances assigned to His-107 and Tyr-138. Composite peaks assigned to Phe-16 and Phe-89 and to Phe-68 and Tyr-99 are also affected. Both the upfield and downfield 1H NMR spectra contain evidence for spectroscopically distinct intermediates in Ca2+ binding by the calmodulin-mastoparan complex.     

1H NMR studies of Chromatium vinosum cytochrome c'

The cytochrome c' from Chromatium vinosum has been studied through 1H NMR in the pH range 4-11 in both the oxidized and the reduced forms. The 1H NMR spectra are similar to those of the other cytochrome c' systems. Three pKa values of 5.1, 7.0, and 9.2 have been observed for the oxidized species and tentatively assigned to the two carboxylate propionic residues of the heme moiety and to the iron-coordinated histidine 125, respectively. The spectra are consistent with an essentially S = 5/2 state in all the pH ranges investigated. Some evidence is provided for conformational flexibilities. Among the oxidized cytochromes c' the present one is capable of binding cyanide, giving rise to a low spin state. The reduced species is a typical high spin iron(II) system.     

2D 1H NMR studies of monomeric insulin

In order to establish the conditions required for the observation of monomeric insulin in solution, a series of proton nuclear magnetic resonance studies of insulin in a variety of solvents was undertaken. Optimal spectra were recorded in trifluoroethanol- water mixtures in a 1:2 ratio. Using the sequential assignment approach the proton nuclear magnetic resonance spectrum of insulin was then assigned. Aspects of the structure of monomeric insulin in solution have been determined using the observed NOE cross peaks and slow exchange protons.     

1H NMR studies on the oxidized ferredoxin from Clostridium pasteurianum.

The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR. Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented. Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time.     

Interactions of HIV-1 with antigen-presenting cells.

There is currently much interest in the numerical and functional loss of antigen-presenting cells (APC) in HIV-1 disease and the contribution that this may make to HIV-1 pathology. The HIV-1 virus can interfere with the normal function of APC in a number of ways involving inappropriate signalling. These include changes in cytokine balance, cell-surface molecule expression and intracellular signalling pathways. This review examines how HIV-1 is able to disregulate APC function and discusses possible outcomes for the function of the immune system.     

HSV-2 infection of dendritic cells amplifies a highly susceptible HIV-1 cell target

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α₄β₇ on lymphocytes. α₄β₇ can be engaged by HIV-1 on the cell-surface and CD4⁺ T cells expressing high levels of this integrin (α₄β₇ ^high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α₄β₇ ^high CD4⁺ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c⁺ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α₄β₇ expression on CD4⁺ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α₄β₇ ^highCD4⁺ T cells. These factors may play a role in increasing the susceptibility to HIV-1.     

1H NMR studies: dynamics of water in gelatin

 Proton magnetic resonance was used to characterize the dynamics of water in gelatin. Both sol and gel states were investigated. Transverse relaxation rates (R ₂) were dependent on the proton frequency measurement. (R ₂) measured with the Carr-Purcell-Meiboom-Gill pulse sequence was dependent on pulse spacing. These observations were interpreted in terms of chemical exchanges between water protons and those of the macromolecules in the sol state, whereas in the gel state the contribution of diffusion through microheterogeneities in the sample seems to provide an additional transverse relaxation mechanism. Received: 10 May 1999 / Revised version: 13 December 1999 / Accepted: 25 January 2000