Direct probing of RNA structures and RNA-protein interactions in the HIV-1 packaging signal by chemical modification and electrospray ionization fourier transform mass spectrometry

RNA hairpins of the HIV-1 packaging signal and their complexes with the nucleocapsid protein p7 (NC) were probed by solvent-accessibility reagents and electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The combination of dimethylsulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMCT) offers the full range of information on base-pairing and solvent exposure concerning the four more abundant ribonucleotides. ESI-FTMS provides a universal method to achieve a direct and unambiguous characterization of alkylated structures, with no need for the different probe-specific procedures required by established methodologies based on gel electrophoresis. It enables us to streamline the optimization of the conditions for probe administration to minimize the incidence of probe-induced distortion of the structures under investigation. Nucleotides located in the single-stranded loops of hairpins SL2, SL3 and SL4 manifested different levels of protection, which were correlated directly to their conformation and structural surroundings. A common feature noted for all the hairpins was the limited susceptibility observed for the guanine base located at the 5'-end of each tetraloop, which assumes a stacked position upon the last base-pair of the double-stranded stems. The remaining loop bases were found to be clearly accessible by modifying reagents in free RNA, but were effectively protected in the NC-hairpin complexes. While this finding is consistent with the proven participation of SL2 and SL3 loops in interactions with NC, it contrasts with prior suggestions that tetraloop bases in SL4 might not be involved directly in NC binding. Alkylation was detected for stem nucleotides, which are not involved in the normal base-pairing and stacking typical of double-stranded structures, such as adenine 15 of the SL2 triple-base platform. Modification of the blunt ends of the double-stranded stems was found to be absent or extremely limited, due to the annealing stabilization introduced by the presence of G-C pairs at the end of the stems structures. Previously undetected alkylation of guanine 3 and guanine 13 in SL4 provides direct evidence of the destabilizing effects induced by the tandem G.U wobbles on the double-stranded structure of this stem, which is thought to be important for the hairpin's biological function.     

cDNA array technology in melanoma: an overview

Genetic aberrations, mostly resulting in changes in gene expression, are critical events in cancer onset and progression. The advent of the cDNA array technology allows the screening and the efficient measurement of expression of thousands genes simultaneously in a wide spectrum of experimental and clinical models. This genomic scale approach is being currently used to obtain global views of human cancer gene expression and to identify genetic markers that might be important for diagnosis, prognosis, and therapy. This review discusses some recent findings obtained by means of cDNA arrays investigating the human melanoma.     

Direct mass spectrometric determination of the stoichiometry and binding affinity of the complexes between nucleocapsid protein and RNA stem-loop hairpins of the HIV-1 Psi-recognition element

The formation of noncovalent complexes between the HIV-1 nucleocapsid protein p7 (NC) and RNA hairpins SL2-SL4 of the Psi-recognition element was investigated by direct infusion electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The high resolution afforded by this method provided the unambiguous characterization of the stoichiometry and composition of complexes formed by multiple equilibria in solution. For each hairpin, the formation of a 1:1 complex was found to be the primary binding mode in solutions of intermediate salt content (150 mM ammonium acetate). Binding of multiple units of NC was observed with lower affinity and a maximum stoichiometry matching the limit calculated from the number of nucleotides in the construct and the size of the footprint of NC onto single-stranded nucleic acids, thus implying the defolding of the hairpin three-dimensional (3D) structure. Dissociation constants of 62 +/- 22 nM, 178 +/- 64 nM, and 1.3 +/- 0.5 microM were determined for SL2, SL3-2, and SL4, respectively, which are similar to values obtained by spectroscopic and calorimetric methods with the additional confidence offered by a direct, rather than inferred, knowledge of the binding stoichiometry. Competitive binding experiments carried out in solutions of intermediate ionic strength, which has the effect of weakening the electrostatic interactions in solution, provided a direct way of evaluating the stabilizing contributions of H-bonding and hydrophobic interactions that are more sensitive to the sequence and structural context of the different hairpins. The relative scale of binding affinity obtained in this environment reflects the combination of contributions provided by the different structures of both the tetraloop and the double-stranded stem. The importance of the stem 3D structure in modulating the binding activity was tested by a competitive binding experiment that included the SL3-2 RNA construct, a DNA analogue of SL3 (SL3(DNA)), and a DNA analogue in which all four loop bases were replaced with abasic nucleotides (SL3(abasic)). NC was found to bind the A-type double-stranded stem of SL3-2 RNA at least 30 times more tightly than the B-type helical structure of SL3(DNA). Eliminating the stabilization provided by the interactions with the tetraloop bases made the binding of SL3(abasic) approximately 50 times weaker than that of SL3(DNA).     

Rat carotid artery dilation by PTCA balloon catheter induces neointima formation in presence of IEL rupture

The best animal angioplasty model is the porcine model, which is expensive and not available in all laboratories. The aim of this study was to describe a new rat model of angioplasty. An injury was induced with the use of a standard percutaneous transluminal coronary angioplasty (PTCA) 1.5-mm balloon catheter. The neointimal tissue, arterial dimensions, and the injury index were assessed following angioplasty. Ki-67 expression was detected to evaluate cell turnover after balloon angioplasty. In contrast with the standard Clowes model, a significant neointimal formation was detected only in the presence of ruptured internal elastic lamina (IEL). A positive correlation between the percentage of ruptured IEL and the amount of neointimal tissue was also demonstrated. The percentage of IEL fracture correlates with the proliferation index by anti-Ki-67 immunolabeling 7 and 14 days after the angioplasty. Significant arterial negative remodeling was observed following PTCA balloon dilation. In conclusion, our inexpensive animal model of restenosis after angioplasty may have great relevance toward a better understanding of the mechanisms and toward assessment of new therapeutical strategies for this phenomenon.     

A role for monoglyceride lipase in 2-arachidonoylglycerol inactivation

2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain (where its levels are 170-folds higher than those of anandamide), is produced by neurons in an activity- and calcium-dependent manner, and is rapidly eliminated. The mechanism of 2-AG inactivation is not completely understood, but is thought to involve carrier-mediated transport into cells followed by enzymatic hydrolysis. We examined the possible role of the serine hydrolase, monoglyceride lipase (MGL), in brain 2-AG inactivation. We identified by homology screening a cDNA sequence encoding for a 303-amino acid protein, which conferred MGL activity upon transfection to COS-7 cells. Northern blot and in situ hybridization analyses revealed that MGL mRNA is unevenly present in the rat brain, with highest levels in regions where CB1 cannabinoid receptors are also expressed (hippocampus, cortex, anterior thalamus and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased the degradation of endogenously produced 2-AG in these cells, whereas no such effect was observed on anandamide degradation. These results indicate that hydrolysis via MGL may be a primary route of 2-AG inactivation in intact neuronal cells.     

Ischemic preconditioning changes the pattern of coronary reactive hyperemia regardless of mitochondrial ATP-sensitive K(+) channel blockade

Ischemic preconditioning increases the velocity of vasodilatation and reduces the total hyperemic flow (THF) of a subsequent coronary reactive hyperemia (CRH). The increase in the velocity of vasodilatation has been shown to depend on an up-regulation of the endothelial release of nitric oxide, while the reduction of THF is attributed to an adenosine A(1) receptor-mediated mechanism. We investigated whether the changes in CRH induced by preconditioning ischemia (PI) can still be obtained after blockade of mitochondrial ATP-sensitive K(+) channels by sodium 5-hydroxydecanoate (5-HD), and whether the blockade per se affects the pattern of CRH.In anesthetized goats, flow was recorded from the left circumflex coronary artery (LCCA). CRH was obtained with the occlusion of LCCA for 15 s. PI was obtained by 2 cycles of 2.5 min of LCCA occlusion with a 5 min interval of reperfusion between the two occlusions. CRH was studied before and after i.v. administration of 5-HD (20 mg/kg), as well as in the presence of 5-HD after PI. Following 5-HD, the pattern of CRH remained unchanged. After 5-HD and PI, velocity of vasodilatation and total hyperemic flow of CRH showed the same changes as in previous studies after PI alone. It was concluded that the blockade of mitochondrial ATP-sensitive K(+) channels, which is reported to prevent myocardial protection, does not affect CRH and does not prevent PI from increasing the velocity of vasodilatation and reducing THF. These results demonstrate that the changes induced in CRH by preconditioning are independent of the opening of the mitochondrial ATP-sensitive K(+) channels.     

Strains of JC virus in Amerind-speakers of North America (Salish) and South America (Guaraní), Na-Dene-speakers of New Mexico (Navajo), and modern Japanese suggest links through an ancestral Asian population

Previously we showed that strains of human polyoma virus JC among the Navajo in New Mexico, speakers of an Athapaskan language in the Na-Dene language phylum, and among the Salish people in Montana, speakers of a language of the Salishan group in the Amerind family, were mainly of a northeast Asian genotype found in Japan (type 2A). We now report partial VP1-gene, regulatory region, and complete genome sequences of JC virus (JCV) from the Guaraní Indians of Argentina. The Tupí-Guaraní language represents the Equatorial branch of the Amerind language family proposed by Greenberg ([1987] Language in the Americas, Stanford: Stanford University Press). The partial VP1 gene sequences of the Guaraní revealed several variants of strains found in northeast Asia (Japan), as did the Salish. In contrast, the strains in the Navajo largely conformed to the prototype type 2A sequence (MY). Phylogenetic reconstruction with both the neighbor-joining and maximum parsimony methods utilized three complete Guaraní JCV genome sequences, three genomes from the Salish people, and 27 other complete JCV genomes, including three from the Navajo and three from Japan. Both trees showed that all type 2A JCV strains from the North and South Americans are closely related phylogenetically to strains in present-day Japan. However, variant sites in the coding regions, the T-antigen intron, and the regulatory region link the type 2A strains in Amerind groups (Guaraní and Salish), but differentiate them from those in a Na-Dene-speaking (Navajo) population. The data suggest separation from a population ancestral to modern Japanese, followed by a second division within the ancestral group that led to Amerind- and Na-Dene-speaking groups. The data cannot, however, localize the latter split to the Asian mainland (two migrations) or to North America (one migration).     

Ectomycorrhizae between <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. and <Emphasis Type="Italic">Naucoria escharoides</Emphasis> (Fr.:Fr.) Kummer from Argentina

Field ectomycorrhizae of Naucoria escharoides on Alnus acuminata ("andean alder", "aliso del cerro") are described in detail for the first time. Naturally occurring ectomycorrhizal roots were sampled beneath sporocarps of N. escharoides. The samples were taken from four natural forest plots at two homogeneous A. acuminata sites (Tucumán and Catamarca Provinces, Argentina). The ectomycorrhizae were characterized morphologically and compared by means of PCR/RFLP analysis of the internal transcribed spacer region of the nuclear rDNA. The most important morphological features of the ectomycorrhizae are a white to pale yellow mantle, simple to monopodial branches, hyaline emanating hyphae, abundant hyphal bundles emerging more or less perpendicularly from a plectenchymatous mantle, and an acute or rounded apex with or without a mantle. N. escharoides fruitbodies have white basal mycelium with emanating hyphae similar to those of andean alder ectomycorrhizae. The RFLP profiles of sporocarps and mycorrhizae were the same.     

Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy

HIV-associated nephropathy is a clinicopathologic entity that includes proteinuria, focal segmental glomerulosclerosis often of the collapsing variant, and microcystic tubulointerstitial disease. Increasing evidence supports a role for HIV-1 infection of renal epithelium in the pathogenesis of HIV-associated nephropathy. Using in situ hybridization, we previously demonstrated HIV-1 gag and nef mRNA in renal epithelial cells of patients with HIV-associated nephropathy. Here, to investigate whether renal epithelial cells were productively infected by HIV-1, we examined renal tissue for the presence of HIV-1 DNA and mRNA by in situ hybridization and PCR, and we molecularly characterized the HIV-1 quasispecies in the renal compartment. Infected renal epithelial cells were removed by laser-capture microdissection from biopsies of two patients, DNA was extracted, and HIV-1 V3-loop or gp120-envelope sequences were amplified from individually dissected cells by nested PCR. Phylogenetic analysis of kidney-derived sequences as well as corresponding sequences from peripheral blood mononuclear cells of the same patients revealed evidence of tissue-specific viral evolution. In phylogenetic trees constructed from V3 and gp120 sequences, kidney-derived sequences formed tissue-specific subclusters within the radiation of blood mononuclear cell-derived viral sequences from both patients. These data, along with the detection of HIV-1-specific proviral DNA and mRNA in tubular epithelium cells, argue strongly for localized replication of HIV-1 in the kidney and the existence of a renal viral reservoir.     

Developmental roles of heparan sulfate proteoglycans: a comparative review in Drosophila,mouse and human

In recent years, progress in the fields of development and proteoglycan biology have produced converging evidence of the role of proteoglycans in morphogenesis. Numerous studies have demonstrated that proteoglycans are involved in several distinct morphogenetic pathways upon which they act at different levels. In particular, proteoglycans can determine the generation of morphogen gradients and be required for their signal transduction. The surface of most cells and the extracellular matrix are decorated by heparan sulfates which are the most common glycosaminoglycans, normally present as heparan sulfate proteoglycans. Considerable structural heterogeneity is generated in proteoglycans by the biosynthetic modification of their heparan sulfate chains as well as by the diverse nature of their different core proteins. This heterogeneity provides an impressive potential for protein-protein and protein-carbohydrate interactions, and can partly explain the diversity of proteoglycan involvement in different morphogenetic pathways. In this review, we summarize the current knowledge about mutations affecting heparan sulfate proteoglycans that influence the function of growth factor pathways essential for tissue assembly, differentiation and development. The comparison of data obtained in Drosophila, rodents and humans reveals that mutations affecting the proteoglycan core proteins or one of the biosynthetic enzymes of their heparan sulfate chains have profound effects on growth and morphogenesis. Further research will complete the picture, but current evidence shows that at the very least, heparan sulfate proteoglycans need to be counted as legitimate elements of morphogenetic pathways that have been maintained throughout evolution as determinant mechanisms of pattern formation.