Interaction of DNA with lysine-rich polypeptides and proteins. The influence of polypeptide composition and secondary structure

Using X-ray diffraction we have studied fibres obtained from complexes of DNA with lysine-rich polypeptides and with proteins that have different conformations, to ascertain whether the conformations of the polypeptides and the DNA are maintained upon interaction. Substances investigated include N-acetyl-Lys-Ala-Tyr-Ala-Lys-ethylamide, random poly(Leu50, Lys50), sequential poly(Leu-Lys), poly(Val-Lys), poly(Ala-Lys), poly(Lys-Ala-Ala-Lys), poly(Lys-Ala-Ala), poly(Lys-Leu-Ala), poly(Lys-Ala-Gly), protein phi 0 from sea cucumber spermatozoa, histone H1 and two fragments of this protein obtained by chemical cleavage. In general, the B form of DNA with ten base-pairs per helical turn is maintained upon interaction at high levels of humidity. The A form is never observed; it appears to be forbidden in a protein environment. No evidence for transition into any novel DNA conformation has been observed, although the B form is altered in some cases, in particular upon dehydration. Such alteration occurs always in the sense of tightening the double helix, so that the number of base-pairs per helical turn diminishes. The polypeptides may interact with DNA in both the alpha and beta conformations. We have found different types of complexes in which either a monolayer or a double layer of beta-pleated sheets is intercalated between layers of DNA molecules. Alternatively, the polypeptide chain may be wrapped around the DNA, following one of the grooves. The polypeptide conformation may be either maintained or changed upon interaction. The charge density of the polypeptide is an important parameter of the interaction. When it matches the charge density of the DNA, the polypeptide conformation is maintained in most cases; otherwise it is modified. The globular part of histone H1 gives a unique X-ray pattern upon interaction, indicative of a loss of order of DNA in the complex. On the other hand, the C-terminal part of histone H1 gives a very well-ordered complex, similar to a nucleoprotamine, in spite of its lower charge density.     

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50 μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3 h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50 nM, 30 min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.     

Contribution of habitat patches to network connectivity: Redundancy and uniqueness of topological indices

In order to increase the efficiency of monitoring and conservation efforts, it is of key importance to develop sound quantitative methods that are able to indicate which key areas and landscape elements play prominent and crucial role in the functioning of habitat mosaics. In particular, network models are being widely used to evaluate the contribution of landscape elements to uphold connectivity and related ecological fluxes. However, monitoring programs and conservation practitioners are overwhelmed by a myriad of network indices without being fully aware of their differences for characterizing the importance of individual habitat patches in fragmented landscapes. We analysed a set of thirteen commonly used graph indices and the forest habitat network of goshawks living in NE Spain in order to (a) evaluate how the patch rank orders derived from these indices differ from each other and (b) identify which indices tend to quantify the same network characteristics and which others are quite unique in addressing topological characteristics that are not considered by the rest. We found that most of the variability in patch rankings can be captured by only three network indices. The largest group of redundant indices corresponded to those that intend to measure the amount of flux received by a given habitat patch. The connector fraction of the integral index of connectivity (IIC) and probability of connectivity (PC) indices and betweenness centrality (BC) stood out as quite unique by focusing on the way habitat patches act as connecting elements between other habitat areas. We discuss which indices can be most beneficial by clearly indicating and differentiating the value of the top patches compared to the others, so that conservation priorities can be established with lower uncertainties. We believe that our results can provide valuable guidelines by facilitating the selection of a few non-redundant and complementary indicators that quantify the important and distinctive roles of habitat patches in maintaining the connectivity of habitat networks.     

Bcl-x L blocks mitochondrial multiple conductance channel activation and inhibits 6-OHDA-induced death in SH-SY5Y cells

Apoptosis is an active process that is regulated by different signalling pathways. One of the more important organelles involved in apoptosis regulation is the mitochondrion. Electron chain transport disruption increases free radical production leading to multiple conductance channel opening, release of cytochrome c and caspase activation. This death pathway can be blocked by anti-apoptotic members of the Bcl-2 protein family that might shift redox potential to a more reduced state, preventing free radical-mediated damage. 6-Hydroxydopamine (6-OHDA) has been widely used to generate Parkinson's disease-like models. It is able to generate free radicals and to induce catecholaminergic cell death. In this paper we have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-x(L) as a model to gain insights into the mechanisms through which Bcl-x(L) blocks 6-OHDA-induced cell death and to identify the molecular targets for this action. Herein, we present evidence supporting that the Bcl-x(L)-anti-apoptotic signal pathway seems to prevent mitochondrial multiple conductance channel opening, cytochrome c release and caspase-3 like activity following 6-OHDA treatment in the human neuroblastoma cell line SH-SY5Y.     

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.     

A Lotus japonicus nodulation system based on heterologous expression of the fucosyl transferase NodZ and the acetyl transferase NoIL in Rhizobium leguminosarum

Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL protein obtained from Mesorhizobium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-alpha-(2-aminoethoxyvinyl) glycine (AVG).     

Description of the male of Tityus kuryi Louren?o, 1997 and notes about males of Tityus stigmurus (Thorell, 1877) and Tityus serrulatus Lutz & Mello, 1922 (Scorpiones,Buthidae)

The male of Tityus kuryi Lourenço, 1997 is described for the first time. Despite being very similar to the female, the male presents more robust metasomal segments. Additionally, the distribution of the sexual populations of another two species of the T. stigmurus complex is reported herein: T. serrulatus Lutz & Mello, 1922 and T. stigmurus (Thorell, 1877). Males of T. serrulatus were, until now, restricted to the Minas Gerais State (Southwestern region of Brazil), and with new records reported here, its known distribution now encompasses the Northeastern region of Brazil. Males of T. stigmurus were previously recorded only for two municipalities in the State of Bahia, and here we present eight new records for Bahia State and one for Pernambuco State. We present a key to related species of the T. stigmurus complex based on morphology and coloration pattern.     

Identification and genomic characterization of a new virus (Tymoviridae family) associated with citrus sudden death disease

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.     

Chromaffin cell death induced by 6-hydroxydopamine is independent of mitochondrial swelling and caspase activation

Our results provide evidence that 6-hydroxydopamine induced, after auto-oxidation, toxic levels of hydrogen peroxide (H2O2) that caused bovine chromaffin cell toxicity and death. 6-Hydroxydopamine (6-OHDA) treatment markedly reduced, in a dose-response fashion, chromaffin cell viability. Cell death was accompanied by cell shrinkage, nuclear condensation and DNA degradation. Under our experimental conditions, 6-OHDA auto-oxidation formed quinones and reactive oxygen species (ROS) that mainly contributed to 6-OHDA-induced cytotoxicity in bovine chromaffin cells. Accordingly, different antioxidants, including catalase, vitamin E, Mn(IIItetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) or ascorbic acid, provided protection against 6-OHDA-induced toxicity. Further evidence that 6-OHDA induces oxidative stress is provided by the fact that this compound decreased total mitochondrial reduced NAD(P)H levels. Our results also suggest that mitochondrial swelling and caspase activation do not play a direct role in 6-OHDA-induced death in bovine chromaffin cells.     

Non-Invasive Imaging Provides Spatiotemporal Information on Disease Progression and Response to Therapy in a Murine Model of Multiple Myeloma

Background

Multiple myeloma (MM) is a B-cell malignancy, where malignant plasma cells clonally expand in the bone marrow of older people, causing significant morbidity and mortality. Typical clinical symptoms include increased serum calcium levels, renal insufficiency, anemia, and bone lesions. With standard therapies, MM remains incurable; therefore, the development of new drugs or immune cell-based therapies is desirable. To advance the goal of finding a more effective treatment for MM, we aimed to develop a reliable preclinical MM mouse model applying sensitive and reproducible methods for monitoring of tumor growth and metastasis in response to therapy.

Material and Methods

A mouse model was created by intravenously injecting bone marrow-homing mouse myeloma cells (MOPC-315.BM) that expressed luciferase into BALB/c wild type mice. The luciferase in the myeloma cells allowed in vivo tracking before and after melphalan treatment with bioluminescence imaging (BLI). Homing of MOPC-315.BM luciferase+ myeloma cells to specific tissues was examined by flow cytometry. Idiotype-specific myeloma protein serum levels were measured by ELISA. In vivo measurements were validated with histopathology.

Results

Strong bone marrow tropism and subsequent dissemination of MOPC-315.BM luciferase⁺ cells in vivo closely mimicked the human disease. In vivo BLI and later histopathological analysis revealed that 12 days of melphalan treatment slowed tumor progression and reduced MM dissemination compared to untreated controls. MOPC-315.BM luciferase⁺ cells expressed CXCR4 and high levels of CD44 and α4β1 in vitro which could explain the strong bone marrow tropism. The results showed that MOPC-315.BM cells dynamically regulated homing receptor expression and depended on interactions with surrounding cells.

Conclusions

This study described a novel MM mouse model that facilitated convenient, reliable, and sensitive tracking of myeloma cells with whole body BLI in living animals. This model is highly suitable for monitoring the effects of different treatment regimens.