Cooperativity in nucleosomes assembly on supercoiled pBR322 DNA.

Many studies have shown that in reconstituted chromatin model systems, containing only purified DNA and histone octamer, nucleosomes can adopt well defined locations with respect to DNA nucleotide sequence. Recently, nucleosome-nucleosome interactions were suggested as one of the factors underlying preferential nucleosomes positioning. In the present paper this aspect has been studied by topological analysis and electron microscopy visualization of minichromosomes reconstituted at different histone/DNA ratios. Both methods suggest that cooperativity plays a role in nucleosomes formation. A linear cooperative model in which nucleosomes are formed on discrete sites with cooperative interactions occurring only between nearest neighbours allows to calculate the cooperative constant. The reported results show that basic interactions, which are of relevance in the process of chromatin folding, are present also in very simple model system.     

The photochemical trapping rate from red spectral states in PSI-LHCI is determined by thermal activation of energy transfer to bulk chlorophylls

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)-light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the "bulk" acceptor chlorophyll (chl) states in the framework of the Arrhenius-Eyring theory. It is shown that the wavelength-dependent "activation energies" come out in the range between 1.35 and 2.7 kcal mol(-1), increasing with the emission wavelength within the interval 710-770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130-280 K for PSI-LHCI. We conclude that the variable trapping time in PSI-LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.     

Tissue-enhanced plasma proteomic analysis for disease stratification in amyotrophic lateral sclerosis

Background

It is unclear to what extent pre-clinical studies in genetically homogeneous animal models of amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disorder, can be informative of human pathology. The disease modifying effects in animal models of most therapeutic compounds have not been reproduced in patients. To advance therapeutics in ALS, we need easily accessible disease biomarkers which can discriminate across the phenotypic variants observed in ALS patients and can bridge animal and human pathology. Peripheral blood mononuclear cells alterations reflect the rate of progression of the disease representing an ideal biological substrate for biomarkers discovery.

Methods

We have applied TMTcalibrator?, a novel tissue-enhanced bio fluid mass spectrometry technique, to study the plasma proteome in ALS, using peripheral blood mononuclear cells as tissue calibrator. We have tested slow and fast progressing SOD1G93A mouse models of ALS at a pre-symptomatic and symptomatic stage in parallel with fast and slow progressing ALS patients at an early and late stage of the disease. Immunoassays were used to retest the expression of relevant protein candidates.

Results

The biological features differentiating fast from slow progressing mouse model plasma proteomes were different from those identified in human pathology, with only processes encompassing membrane trafficking with translocation of GLUT4, innate immunity, acute phase response and cytoskeleton organization showing enrichment in both species. Biological processes associated with senescence, RNA processing, cell stress and metabolism, major histocompatibility complex-II linked immune-reactivity and apoptosis (early stage) were enriched specifically in fast progressing ALS patients. Immunodetection confirmed regulation of the immunosenescence markers Galectin-3, Integrin beta 3 and Transforming growth factor beta-1 in plasma from pre-symptomatic and symptomatic transgenic animals while Apolipoprotein E differential plasma expression provided a good separation between fast and slow progressing ALS patients.

Conclusions

These findings implicate immunosenescence and metabolism as novel targets for biomarkers and therapeutic discovery and suggest immunomodulation as an early intervention. The variance observed in the plasma proteomes may depend on different biological patterns of disease progression in human and animal model.

     

A novel P2RX2 mutation in an Italian family affected by autosomal dominant nonsyndromic hearing loss

Hereditary hearing loss (HHL) is a common disorder accounting for at least 60% of prelingual deafness. It is characterized by a large genetic heterogeneity, and despite the presence of a major gene, still there is a need to search for new causative mutations/genes. Very recently, a mutation within ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) gene (P2RX2) at DNFA41 locus has been reported leading to a bilateral and symmetrical sensorineural non-syndromic autosomal dominant HHL in two Chinese families. We performed a linkage analysis in a large Italian family with a dominant pattern of inheritance showing a significant 3.31 LOD score in a 2 Mb region overlapping with the DNFA41 locus. Molecular analyses of P2RX2 identified a novel missense mutation (p.Gly353Arg) affecting a residue highly conserved across species. Visual inspection of the protein structure as obtained from comparative modeling suggests that substitution of the small glycine residue with a charged bulky residue such as an arginine that is close to the ‘neck’ of the region responsible for ion channel gating should have a high energetic cost and should lead to a severely destabilization of the fold. The identification of a second most likely causative mutation in P2RX2 gene further supports the possible role of this gene in causing autosomal dominant HHL.     

Relevance of disease- and organ-specific endothelial cells for in vitro research

The endothelium is a dynamic, heterogeneous, disseminated organ that possesses vital secretory, synthetic, metabolic and immunological functions. Endothelial dysfunction has been implicated as a key factor in the development of organ-specific vascular diseases. This minireview gives a brief overview on EC (endothelial cell) biomarkers in arterial and venous endothelium and critically discusses the different sources of ECs that are most frequently applied in in vitro assays and research. The relevance of organ- and disease-specific endothelial cell cultures for studying cellular responses as a basis for improving therapeutic interventions is highlighted with particular emphasis on endothelial dysfunction in transplant-associated coronary artery disease, in atherosclerotic lesions and in response to diabetes mellitus.     

The kSORT Assay to Detect Renal Transplant Patients at High Risk for Acute Rejection: Results of the Multicenter AART Study

BackgroundDevelopment of noninvasive molecular assays to improve disease diagnosis and patient monitoring is a critical need. In renal transplantation, acute rejection (AR) increases the risk for chronic graft injury and failure. Noninvasive diagnostic assays to improve current late and nonspecific diagnosis of rejection are needed. We sought to develop a test using a simple blood gene expression assay to detect patients at high risk for AR.ConclusionsThe kSORT blood QPCR assay is a noninvasive tool to detect high risk of AR of renal transplants.Please see later in the article for the Editors'' Summary     

The primary structure of the Fab fragment of protein KAU, a monoclonal immunoglobulin M cold agglutinin

The complete amino acid sequence of the Fab fragment of protein KAU, a human monoclonal cold agglutinin (IgMk) with anti-I activity, was determined. The light chain (L-chain) consists of 215 residues; the variable (V)L region belongs to the Hum/Kv325/kIIIb sub-subgroup that is preferentially selected in human IgM autoimmune response. The joining (J) region is encoded by the Jk4 gene, and the constant region (C)L domain expresses the km3 allotypic marker. The Fd fragment contains 232 amino acids, and 120 of them comprise the variable domain. The VH region corresponds to the VHIV subgroup and is closely related to the VHIV 2.1 gene isolated from genomic DNA expressed in peripheral blood of a healthy Caucasian. The complementary-determining region 1 has a unique amino acid (Asp) at position 31, and the complementary-determining region 3 codified by the diversity segment (D) gene, shows poor homology with other known D sequences. The joining segment with two unusual substitutions at the D-J junction is encoded by the JH4 gene. Thus, cold agglutinin KAU is an IgM, VkIIIb-Jk4-km3; VHIV-JH4-C mu.     

HIV induces lymphocyte apoptosis by a p53-initiated, mitochondrial-mediated mechanism.

HIV-1 induces apoptosis and leads to CD4+ T-lymphocyte depletion in humans. It is still unclear whether HIV-1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV-1-induced apoptosis, we infected human CD4+ T cells with HIV-1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro-caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria was observed in HIV-infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. H IV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up-regulation of surface FasL expression, accompanied by a down-regulation of Fas-associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.     

Role of B lymphocytes in the infarcted mass in patients with acute myocardial infarction

Despite early reperfusion, patients with ST segment elevation myocardial infarction (STEMI) may present large myocardial necrosis and significant impairment of ventricular function. The present study aimed to evaluate the role of subtypes of B lymphocytes and related cytokines in the infarcted mass and left ventricular ejection fraction obtained by cardiac magnetic resonance imaging performed after 30 days of STEMI. This prospective study included 120 subjects with STEMI submitted to pharmacoinvasive strategy. Blood samples were collected in subjects in the first (D1) and 30th (D30) days post STEMI. The amount of CD11b+ B1 lymphocytes (cells/ml) at D1 were related to the infarcted mass (rho = 0.43; P=0.033), measured by cardiac MRI at D30. These B1 cells were associated with CD4+ T lymphocytes at D1 and D30, while B2 classic lymphocytes at day 30 were related to left ventricular ejection fraction (LVEF). Higher titers of circulating IL-4 and IL-10 were observed at D30 versus D1 (P=0.013 and P<0.001, respectively). Titers of IL-6 at D1 were associated with infarcted mass (rho = 0.41, P<0.001) and inversely related to LVEF (rho = −0.38, P<0.001). After multiple linear regression analysis, high-sensitivity troponin T and IL-6 collected at day 1 were independent predictors of infarcted mass and, at day 30, only HDL-C. Regarding LVEF, high-sensitivity troponin T and high-sensitivity C-reactive protein were independent predictors at day 1, and B2 classic lymphocytes, at day 30. In subjects with STEMI, despite early reperfusion, the amount of infarcted mass and ventricular performance were related to inflammatory responses triggered by circulating B lymphocytes.