Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1

We recently described the isolation of 2'-fluoropyrimidine-substituted RNA aptamers that bind specifically to the surface glycoprotein (gp 120) of the R5 strain, HIV-1(Ba-L), as presented in a previous study. These aptamers potently neutralize HIV-1 infectivity in human peripheral blood mononuclear cells of both tissue culture lab-adapted strains and diverse R5 clinical isolates from multiple clades. Here, we report a detailed structural characterization of one such neutralizing aptamer, B40, using enzymatic and chemical probing methods. We identify the minimal region of the aptamer essential for binding gp120 and accordingly design a 77-nucleotide truncated aptamer, B40t77. We then quantitatively analyze the binding affinity and neutralization potency of the parental and truncated (minimal) aptamer, and show them to be comparable. Furthermore, using results from secondary structure analysis, RNA mutagenesis and BIAcore surface plasmon resonance (SPR) binding assays, we hypothesize that a folded RNA structure is required to present specific nucleotide sequences to allow gp120-recognition and binding. The information gained from this study may provide leads for development of novel anti-HIV-1 therapies and can be used to extend our understanding of the molecular interactions between the virus and its host cell.     

Bayesian models for the analysis of genetic structure when populations are correlated

MOTIVATION: Population allele frequencies are correlated when populations have a shared history or when they exchange genes. Unfortunately, most models for allele frequency and inference about population structure ignore this correlation. Recent analytical results show that among populations, correlations can be very high, which could affect estimates of population genetic structure. In this study, we propose a mixture beta model to characterize the allele frequency distribution among populations. This formulation incorporates the correlation among populations as well as extending the model to data with different clusters of populations. RESULTS: Using simulated data, we show that in general, the mixture model provides a good approximation of the among-population allele frequency distribution and a good estimate of correlation among populations. Results from fitting the mixture model to a dataset of genotypes at 377 autosomal microsatellite loci from human populations indicate high correlation among populations, which may not be appropriate to neglect. Traditional measures of population structure tend to overestimate the amount of genetic differentiation when correlation is neglected. Inference is performed in a Bayesian framework. CONTACT: fur@ohsu.edu.     

Crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase: extreme asymmetry in a tetramer of identical subunits

Phosphoglycerate dehydrogenases exist in at least three different structural motifs. The first D-3-phosphoglycerate dehydrogenase structure to be determined was from Escherichia coli and is a tetramer composed of identical subunits that contain three discernable structural domains. The crystal structure of D-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 A. This enzyme represents a second structural motif of the D-3-phosphoglycerate dehydrogenase family, one that contains an extended C-terminal region. This structure is also a tetramer of identical subunits, and the extended motif of 135 amino acids exists as a fourth structural domain. This intervening domain exerts quite a surprising characteristic to the structure by introducing significant asymmetry in the tetramer. The asymmetric unit is composed of two identical subunits that exist in two different conformations characterized by rotation of approximately 180 degrees around a hinge connecting two of the four domains. This asymmetric arrangement results in the formation of two different and distinct domain interfaces between identical domains in the asymmetric unit. As a result, the surface of the intervening domain that is exposed to solvent in one subunit is turned inward in the other subunit toward the center of the structure where it makes contact with other structural elements. Significant asymmetry is also seen at the subunit level where different conformations exist at the NAD-binding site and the putative serine-binding site in the two unique subunits.     

Altered expression, polymerisation and cellular distribution of alpha-/beta-tubulins and apoptosis-like cell death in arsenite resistant Leishmania donovani promastigotes

Studies in mammalian systems have shown specific affinity of arsenite for tubulin proteins. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which is a 13-fold increase in resistance compared to the wild type. Data presented in this study shows decreased expression of alpha- and beta-tubulin in wild type L. donovani promastigotes on exposure to NaAsO2 from 0.0016 to 5.0 microM (IC50 in the wild type strain) in a dose-dependent manner. alpha- and beta-tubulins in the resistant strain show decreased expression levels only at 65.0 microM NaAsO2 (IC50 in the resistant strain). Treatment with respective IC50 concentrations of NaAsO2 caused alterations in tubulin polymerisation dynamics and deregulated the cellular distribution of the microtubules in wild type and resistant strains. The NaAsO2-induced cell death exhibited characteristics of apoptosis-like DNA laddering and fragmentation in both the affected wild type and resistant cells. However, poly(ADP-ribose)polymerase cleavage was evident in the wild type strain but not in the resistant strain.     

Phospholipase A2 as a target protein for nonsteroidal anti-inflammatory drugs (NSAIDS): crystal structure of the complex formed between phospholipase A2 and oxyphenbutazone at 1.6 A resolution

Phospholipase A(2) (PLA(2); EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA(2) occurs through a well-formed hydrophobic channel. To determine the viability of PLA(2) as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA(2) with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 A resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA(2) and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA(2) with a dissociation constant of 6.4 x 10(-8) M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA(2). It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr(52) and Lys(69) with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu(3), Phe(5), Met(8), Ile(9), and Ala(18) are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA(2) is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA(2) as well.     

Altered proglucagon processing in an alpha-cell line derived from prohormone convertase 2 null mouse islets

The endoproteolytic processing of proproteins in the secretory pathway depends on the expression of selected members of a family of subtilisin-like endoproteases known as the prohormone convertases (PCs). The main PC family members expressed in mammalian neuroendocrine cells are PC2 and PC1/3. The differential processing of proglucagon in pancreatic alpha-cells and intestinal L cells leads to production of distinct hormonal products with opposing physiological effects from the same precursor. Here we describe the establishment and characterization of a novel alpha-cell line (alphaTC-DeltaPC2) derived from PC2 homozygous null animals. The alphaTC-DeltaPC2 cells are shown to be similar to the well characterized alphaTC1-6 cell line in both morphology and overall gene expression. However, the absence of PC2 activity in alphaTC-DeltaPC2 leads to a complete block in the production of mature glucagon. Surprisingly, alphaTC-DeltaPC2 cells are able to efficiently cleave the interdomain site in proglucagon (KR 70-71). Further analysis reveals that alphaTC-DeltaPC2 cells, unlike alphaTC1-6 cells, express low levels of PC1/3 that lead to the generation of glicentin as well as low amounts of oxyntomodulin, GLP-1, truncated GLP-1, and N-terminally extended GLP-2. We conclude that alphaTC-DeltaPC2 cells provide additional evidence for PC2 as the major convertase in alpha-cells leading to mature glucagon production and provide a robust model for further analysis of the mechanisms of proprotein processing by the prohormone convertases.     

Cyclooxygenase-1-dependent prostaglandin synthesis modulates tumor necrosis factor-alpha secretion in lipopolysaccharide-challenged murine resident peritoneal macrophages

Comprehensive studies of prostaglandin (PG) synthesis in murine resident peritoneal macrophages (RPM) responding to bacterial lipopolysaccharide (LPS) revealed that the primary PGs produced by RPM were prostacyclin and PGE(2). Detectable increases in net PG formation occurred within the first hour, and maximal PG formation had occurred by 6-10 h after LPS addition. Free arachidonic acid levels rose and peaked at 1-2 h after LPS addition and then returned to baseline. Cyclooxygenase-2 (COX-2) and microsomal PGE synthase levels markedly increased upon exposure of RPM to LPS, with the most rapid increases in protein expression occurring 2-6 h after addition of the stimulus. RPM constitutively expressed high levels of COX-1. Studies using isoform-selective inhibitors and RPM from mice bearing targeted deletions of ptgs-1 and ptgs-2 demonstrated that COX-1 contributes significantly to PG synthesis in RPM, especially during the initial 1-2 h after LPS addition. Selective inhibition of either COX isoform resulted in increased secretion of tumor necrosis factor-alpha (TNF-alpha); however, this effect was much greater with the COX-1 than with the COX-2 inhibitor. These results demonstrate autocrine regulation of TNF-alpha secretion by endogenous PGs synthesized primarily by COX-1 in RPM and suggest that COX-1 may play a significant role in the regulation of the early response to endotoxemia.     

Indian file pattern of adenocarcinoma cells in effusions

OBJECTIVE: To determine the prevalence of the Indian file pattern of adenocarcinoma cells in malignant serous effusions. STUDY DESIGN: Twenty-five malignant and 10 benign effusion samples were selected. Clinical data, including the sites of the primary neoplasms, were noted. The presence or absence of Indian file arrangement of carcinoma cells and other patterns were recorded. RESULTS: Seven of the 25 positive cases showed Indian file arrangement, whereas none of the negative cases did. Their number did not show any correlation with the type and site of the primary neoplasms. In most of the 7 cases other patterns, such as 3-dimensional balls and papillations, were also present. CONCLUSION: Cells in Indian file in serous effusions are strongly suggestive of metastatic carcinoma. They are not specific to any particular site of the primary adenocarcinoma.     

NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus

The full length, positive-strand genome of the Moloney Murine Leukemia Virus contains a "core encapsidation signal" that is essential for efficient genome packaging during virus assembly. We have determined the structure of a 101-nucleotide RNA that contains this signal (called mPsi) using a novel isotope-edited NMR approach. The method is robust and should be generally applicable to larger RNAs. mPsi folds into three stem loops, two of which (SL-C and SL-D) co-stack to form an extended helix. The third stem loop (SL-B) is connected to SL-C by a flexible, four-nucleotide linker. The structure contains five mismatched base-pairs, an unusual C.CG base-triple platform, and a novel "A-minor K-turn," in which unpaired adenosine bases A340 and A341 of a GGAA bulge pack in the minor groove of a proximal stem, and a bulged distal uridine (U319) forms a hydrogen bond with the phosphodiester of A341. Phylogenetic analyses indicate that these essential structural elements are conserved among the murine C-type retroviruses.     

Nerve growth factor-enhanced airway responsiveness involves substance P in ferret intrinsic airway neurons

Nerve growth factor (NGF), a member of the neurotrophin family, enhances synthesis of neuropeptides in sensory and sympathetic neurons. The aim of this study was to examine the effect of NGF on airway responsiveness and determine whether these effects are mediated through synthesis and release of substance P (SP) from the intrinsic airway neurons. Ferrets were instilled intratracheally with NGF or saline. Tracheal smooth muscle contractility to methacholine and electrical field stimulation (EFS) was assessed in vitro. Contractions of isolated tracheal smooth muscle to EFS at 10 and 30 Hz were significantly increased in the NGF treatment group (10 Hz: 33.57 +/- 2.44%; 30 Hz: 40.12 +/- 2.78%) compared with the control group (10 Hz: 27.24 +/- 2.14%; 30 Hz: 33.33 +/- 2.31%). However, constrictive response to cholinergic agonist was not significantly altered between the NGF treatment group and the control group. The NGF-induced modulation of airway smooth muscle to EFS was maintained in tracheal segments cultured for 24 h, a procedure that causes a significant anatomic and functional loss of SP-containing sensory fibers while maintaining viability of intrinsic airway neurons. The number of SP-containing neurons in longitudinal trunk and superficial muscular plexus and SP nerve fiber density in tracheal smooth muscle all increased significantly in cultured trachea treated with NGF. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the NGF-induced increased contraction to EFS in cultured segments but had no effect in saline controls. These results show that the NGF-enhanced airway smooth muscle contractile responses to EFS are mediated by the actions of SP released from intrinsic airway neurons.