Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

A statistical approach for detecting genomic aberrations in heterogeneous tumor samples from single nucleotide polymorphism genotyping data

We describe a statistical method for the characterization of genomic aberrations in single nucleotide polymorphism microarray data acquired from cancer genomes. Our approach allows us to model the joint effect of polyploidy, normal DNA contamination and intra-tumour heterogeneity within a single unified Bayesian framework. We demonstrate the efficacy of our method on numerous datasets including laboratory generated mixtures of normal-cancer cell lines and real primary tumours.     

Implication of the visual system in the regulation of activity cycles in the absence of solar light: 2-[125I]iodomelatonin binding sites and melatonin receptor gene expression in the brains of demersal deep-sea gadiform fish

Relative eye size, gross brain morphology and central localization of 2-[¹²⁵I]iodomelatonin binding sites and melatonin receptor gene expression were compared in six gadiform fish living at different depths in the north-east Atlantic Ocean: Phycis blennoides (capture depth range 265 to 1260 m), Nezumia aequalis (445 to 1512 m), Coryphaenoides rupestris (706 to 1932 m), Trachyrincus murrayi (1010 to 1884 m), Coryphaenoides guentheri (1030 m) and Coryphaenoides (Nematonurus) armatus (2172 to 4787 m). Amongst these, the eye size range was 0.15 to 0.35 of head length with a value of 0.19 for C. (N.) armatus, the deepest species. Brain morphology reflected behavioural differences with well-developed olfactory regions in P. blennoides, T. murrayi and C. (N.) armatus and evidence of olfactory deficit in N. aequalis, C. rupestris and C. guentheri. All species had a clearly defined optic tectum with 2-[¹²⁵I]iodomelatonin binding and melatonin receptor gene expression localized to specific brain regions in a similar pattern to that found in shallow-water fish. Melatonin receptors were found throughout the visual structures of the brains of all species. Despite living beyond the depth of penetration of solar light these fish have retained central features associated with the coupling of cycles of growth, behaviour and reproduction to the diel light–dark cycle. How this functions in the deep sea remains enigmatic.     

Activity banding of human chromosomes as shown by histone acetylation

The expression of genes in mammalian cells depends on many factors including position in the cell cycle, stage of differentiation, age, and environmental influences. As different groups of genes are expressed, their packaging within chromatin changes and may be detected at the chromsomal level. The organization of DNA within a chromosome is determined to a large extent by the positively charged, highly conserved histones. Histone subtypes and the reversible chemical modifications of histones have been associated with gene activity. Active or potentially active genes have been associated with hyperacetylated histones and inactive genes with nonacetylated histones. Sodium butyrate increases the acetylation levels of histones in cell cultures and acts as both an inducer of gene activity and as a cell-cycle block. We describe a method to label the interphase distribution of DNA associated with various histone acetylation stages on chromosomes. Nucleosomes from untreated and butyrate-treated HeLa cells were fractionated by their acetylation level and the associated DNA labeled, and hybridized to normal human chromosomes. In the sodium butyrate-treated cells the resulting banding patterns of the high- and low-acetylated fractions were strikingly different. DNA from low-acetylated chromatin labeled several pericentric regions, whereas hybridization with DNA from highly acetylated chromatin resulted in a pattern similar to inverse G-bands on many chromsomes. The results from noninduced cells at both high and low acetylation levels were noticeably different from their induced counterparts. The capture and hybridization of DNA from interphase chromatin at different acetylation states provides a “snap-shot” of the distribution of gene activity on chromosomes at the time of cell harvest. Edited by: P.B. Moens     

Enzyme-catalyzed hydrolysis of the supported phospholipid bilayers studied by atomic force microscopy

Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A₂ (PLA₂) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA₂ toward l-enantiomer phospholipids, five kinds of supported bilayers made of l- and d-dipalmitoylphosphatidylcholines (DPPC), including l-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (or l/l in short), l/d, d/l, d/d, and racemic ld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the l/l bilayer show that the hydrolysis rate for l-DPPC is significantly increased by PLA₂ and most of the hydrolysis products desorb from substrate surface in 40 min. As d-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the l/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA₂ increases in the sequence of l/l, l/d, ld/ld, and d/l (d/d is inert to the enzyme action). d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA₂ preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA₂-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA₂ mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that d-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA₂.     

Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

BackgroundThe overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients.MethodsThis randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay.ResultsOur findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration.ConclusionsThe results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.