Arginine side-chain dynamics in the HIV-1 rev-RRE complex

The binding of human immunodeficiency virus type 1 (HIV-1) Rev protein to its viral RNA target, stem-loop IIB (SLIIB) within the Rev Response element (RRE), mediates the export of singly-spliced and unspliced viral mRNA from the nucleus to the cytoplasm of infected cells; this Rev-mediated transport of viral RNA is absolutely required for the replication of infectious virus. To identify important features that influence the binding affinity and specificity of this Rev-RRE interaction, we have characterized the arginine side-chain dynamics of the Rev arginine-rich motif (ARM) while bound to a 34 nt RNA oligomer that corresponds to SLIIB. As the specificity of the Rev-RRE interaction varies with salt concentration, arginine side-chain dynamics were characterized at two different salt conditions. Following NMR measurements of (15)N spin relaxation parameters for the arginine (15)N(epsilon) nuclei, the dynamics of the corresponding N(epsilon)-H(epsilon) bond vectors were interpreted in terms of Lipari-Szabo model-free parameters using anisotropic expressions for the spectral density functions. Results from these analyses indicate that a number of arginine side-chains display a surprising degree of conformational freedom when bound to RNA, and that arginine residues having known importance for specific RRE recognition show striking differences in side-chain mobility. The (15)N relaxation measurements at different salt conditions suggest that the previously reported increase in Rev-RRE specificity at elevated salt concentrations is likely due to reduced affinity of non-specific Rev-RNA interactions. The observed dynamical behavior of the arginine side-chains at this protein-RNA interface likely plays an important role in the specificity and affinity of Rev-SLIIB complex formation.     

Functional characterization of the dimer linkage structure RNA of Moloney murine sarcoma virus

Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255-7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.     

A C-glucosylflavone from leaves of Piper lhotzkyanum

The dichloromethane fraction obtained from the methanolic extract of leaves of P. lhotzkyanum afforded a C-glucosylflavone (kaplanin) together with sakuranetin, methyl 4-methoxydihydroferulate and a mixture of methyl ferulate and dihydroferulate derivatives. Spectrometric methods were used to elucidate the structures of these compounds.     

Cyclic-nucleotide- and Ca2+/calmodulin-regulated channels in plants: targets for manipulating heavy-metal tolerance, and possible physiological roles

Recently we discovered a tobacco protein (designated NtCBP4) that modulates heavy-metal tolerance in transgenic plants. Structurally, NtCBP4 is similar to mammalian cyclic-nucleotide-gated non-selective cation channels containing six putative transmembrane domains, a predicted pore region, a conserved cyclic-nucleotide-binding domain, and a high-affinity calmodulin-binding site that coincides with its cyclic-nucleotide-binding domain. Transgenic tobacco expressing the plasma-membrane-localized NtCBP4 exhibit improved tolerance to Ni(2+) and hypersensitivity to Pb(2+), which are associated with a decreased accumulation of Ni(2+) and an enhanced accumulation of Pb(2+) respectively. Transgenic plants expressing a truncated version of NtCBP4, from which regulatory domains had been removed, have a different phenotype. Here we describe our approach to studying the involvement of NtCBP4 in heavy-metal tolerance and to elucidate its physiological role.     

Carbacyclic peptide mimetics as VCAM-VLA-4 antagonists

Substitution of carbon for sulfur in a potent 13-membered cyclic disulfide containing peptide was accomplished via an intramolecular Wittig reaction and resulted in a series of 'carba' analogues. Potency in the VCAM-VLA-4 assay was sensitive to ring size and lower than that of the parent disulfide.     

The design and synthesis of potent cyclic peptide VCAM-VLA-4 antagonists incorporating an achiral Asp-Pro mimetic

The Asp-Pro sequence of the cyclic peptide Ac-HN-Tyr-Cys*-Asp-Pro-Cys*-OH (1) could be replaced with the achiral dipeptide mimetic 1-(2-aminoethyl)cyclpentylcarboxylic acid with retention of potent inhibition of the VCAM-VLA-4 interaction.     

Protists as opportunistic pathogens: public health impact in the 1990s and beyond

Protist organisms (protozoa and fungi) have become increasingly prominent as opportunistic pathogens among persons infected with human immunodeficiency virus (HIV) and among organ transplant recipients--two immunocompromised populations that have increased dramatically in the past two decades. Pneumocystis carinii pneumonia continues to be the most common serious opportunistic infection (OI) among HIV-infected persons in the United States, occurring frequently among persons not previously receiving medical care. Toxoplasmosis, cryptococcosis, cryptosporidiosis, and isosporiasis occur frequently in HIV-infected persons in the developing world. Candidiasis and aspergillosis are common OIs in organ transplant recipients. As these populations of immunosuppressed patients continue to expand worldwide new OIs caused by protist pathogens are likely to emerge.     

Coinfection of fibroblasts with Coxiella burnetti and Toxoplasma gondii: to each their own

Intracellular pathogens have evolved distinct strategies to subvert host cell defenses. At diametrically opposed ends of the spectrum with regard to the host endosomal/lysosomal defenses are the obligate intracellular protozoan Toxoplasma gondii and the bacterium Coxiella burnetti. While the intracellular replication of T. gondii requires complete avoidance of the host endocytic cascade, C. burnetti actively subverts it. This results in these organisms establishing and growing in very different vacuolar compartments. In this study we examined the potential interaction between these distinct compartments following coinfection of mammalian fibroblasts. When present within the same cell, these organisms exhibit minimal interaction with each other. Colocalization of T. gondii and C. burnetti within the same vacuole occurs at a low frequency in doubly infected cells. In such instances only one of the organisms appears to be replication competent, emphasizing the different requirements for survival and/or intracellular growth. The potential basis for both the lack of interaction between these distinct pathogen-containing compartments, and the mechanisms to address their low frequency of colocalization are discussed in the context of our understanding of the biology of the organisms and membrane traffic in eukaryotic cells.     

Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix that holds the bacteria together in a mass and firmly attaches the bacterial mass to the underlying surface. A major component of the extracellular polysaccharide matrix in several phylogenetically diverse bacteria is PGA, a linear polymer of N-acetylglucosamine residues in beta(1,6)-linkage. PGA is produced by the Gram-negative periodontopathogen Actinobacillus actinomycetemcomitans as well as by the Gram-positive device-associated pathogen Staphylococcus epidermidis. We recently reported that A.actinomycetemcomitans produces a soluble glycoside hydrolase named dispersin B, which degrades PGA. Here, we present the crystal structure of dispersin B at 2.0A in complex with a glycerol and an acetate ion at the active site. The enzyme crystallizes in the orthorhombic space group C222(1) with cell dimensions a=41.02A, b=86.13A, c=185.77A. The core of the enzyme consists a (beta/alpha)(8) barrel topology similar to other beta-hexosaminidases but significant differences exist in the arrangement of loops hovering in the vicinity of the active site. The location and interactions of the glycerol and acetate moieties in conjunction with the sequence analysis suggest that dispersin B cleaves beta(1,6)-linked N-acetylglucosamine polymer using a catalytic machinery similar to other family 20 hexosaminidases which cleave beta(1,4)-linked N-acetylglucosamine residues.