Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease

Tyrosinase is a key enzyme in the synthesis of melanin in skin and hair and has also been proposed to contribute to the formation of neuromelanin (NM). The presence of NM, which is biochemically similar to melanin in peripheral tissues, identifies groups of neurons susceptible in Parkinson's disease (PD). Whether tyrosinase is beneficial or detrimental to neurons is unclear; whilst the enzyme activity of tyrosinase generates dopamine-quinones and other oxidizing compounds, NM may form a sink for such radical species. In the present study, we demonstrated that tyrosinase is expressed at low levels in the human brain. We found that mRNA, protein and enzyme activity are all present but at barely detectable levels. In cell culture systems, expression of tyrosinase increases neuronal susceptibility to oxidizing conditions, including dopamine itself. We related these in vitro observations to the human disease by assessing whether there was any genetic association between the gene encoding tyrosinase and idiopathic PD. We found neither genotypic or haplotypic association with three polymorphic markers of the gene. This argues against a strong genetic association between tyrosinase and PD, although the observed contribution to cellular toxicity suggests that a biochemical association is likely.     

The evolution of stability in a competitive system

The characteristics governing the dynamics of populations can evolve and this evolution can either be towards stability or chaos. Yet it is not obvious how or why such population characteristics can evolve through selection on individuals. In this paper we construct a mathematical model, inspired by experimental results, illustrating the dynamics of a population of competing Drosophila. We demonstrate how selection of life history characteristics and stability influence one another as a population interacts with its environment. We generalize this result and show that population stability can evolve as a consequence of selection on individuals.     

Spatial models of virus-immune dynamics

To date, the majority of theoretical models describing the dynamics of infectious diseases in vivo are based on the assumption of well-mixed virus and cell populations. Because many infections take place in solid tissues, spatially structured models represent an important step forward in understanding what happens when the assumption of well-mixed populations is relaxed. Here, we explore models of virus and virus-immune dynamics where dispersal of virus and immune effector cells was constrained to occur locally. The stability properties of our spatial virus-immune dynamics models remained robust under almost all biologically plausible dispersal schemes, regardless of their complexity. The various spatial dynamics were compared to the basic non-spatial dynamics and important differences were identified: When space was assumed to be homogeneous, the dynamics generated by non-spatial and spatially structured models differed substantially at the peak of the infection. Thus, non-spatial models may lead to systematic errors in the estimates of parameters underlying acute infection dynamics. When space was assumed to be heterogeneous, spatial coupling not only changed the equilibrium properties of the uncoupled populations but also equalized the dynamics and thereby reduced the likelihood of dynamic elimination of the infection. In line with experimental and clinical observations, long-lasting oscillation periods were virtually absent. When source-sink dynamics were considered, the long-term outcome of the infection depended critically on the degree of spatial coupling. The infection collapsed when emigration from source sites became too large. Finally, we discuss the implications of spatially structured models on medical treatment of infectious diseases, and note that a huge gap exists in data accurately describing infection dynamics in solid tissues.     

Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding protein 2, and La protein

Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5'-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.     

Internal point mutations of the capsid modify the serotype of Rice yellow mottle virus

Rice yellow mottle virus is classified in five major serotypes; the molecular diversity of the coat protein (CP) is well established, but the amino acids involved in the recognition by discriminant monoclonal antibodies (MAbs) remain unknown. Reconstruction of a phylogenetic tree and sequence alignment of the CP gene of a sample representative of the continental-large diversity were used to identify 10 serospecific amino acids (i.e., conserved in all isolates belonging to the same serotype and distinct in other serotypes). Positions occupied by serospecific residues were localized on the crystal structure of the CP monomer and on modeled capsomers. Structural, molecular, and serological properties of each serotype were analyzed, and subsequently, hypotheses on the potential role of amino acids in discriminating reactions with antibodies were formulated. The residues 114 and 115 (serospecific of Sr1) and 190 (serospecific of Sr2) were localized on the outer surface of the capsid and might be directly involved in the immunoreactivity with MAb D and MAb A, respectively. In contrast, residues 180 (Sr3) and 178 (Sr5) lay within the inner surface of the capsid. To understand the role of these internal positions in the recognition with the antibodies, two substitutions (T180K and G178D) were introduced in the CP of an infectious clone. These mutations modified the antigenicity with MAb G and MAb E discriminating Sr3 and Sr5, respectively, while the reaction with MAb D remained unaffected. This result suggests an indirect effect of these two internal mutations on local immunostructure while the global structure was maintained.     

Water deficits affect caffeate O-methyltransferase, lignification, and related enzymes in maize leaves. A proteomic investigation

Drought is a major abiotic stress affecting all levels of plant organization and, in particular, leaf elongation. Several experiments were designed to study the effect of water deficits on maize (Zea mays) leaves at the protein level by taking into account the reduction of leaf elongation. Proteomic analyses of growing maize leaves allowed us to show that two isoforms of caffeic acid/5-hydroxyferulic 3-O-methyltransferase (COMT) accumulated mostly at 10 to 20 cm from the leaf point of insertion and that drought resulted in a shift of this region of maximal accumulation toward basal regions. We showed that this shift was due to the combined effect of reductions in growth and in total amounts of COMT. Several other enzymes involved in lignin and/or flavonoid synthesis (caffeoyl-CoA 3-O-methyltransferase, phenylalanine ammonia lyase, methylenetetrahydrofolate reductase, and several isoforms of S-adenosyl-l-methionine synthase and methionine synthase) were highly correlated with COMT, reinforcing the hypothesis that the zone of maximal accumulation corresponds to a zone of lignification. According to the accumulation profiles of the enzymes, lignification increases in leaves of control plants when their growth decreases before reaching their final size. Lignin levels analyzed by thioacidolysis confirmed that lignin is synthesized in the region where we observed the maximal accumulation of these enzymes. Consistent with the levels of these enzymes, we found that the lignin level was lower in leaves of plants subjected to water deficit than in those of well-watered plants.     

Production and validation of CR-39-based dishes for alpha-particle radiobiological experiments

The study of radiobiological effects induced in vitro by low fluences of alpha particles would be significantly enhanced if the precise localization of each particle track in the cell monolayer was known. From this perspective, we developed a new method based on tailor-made UV-radiation-cured CR-39, the production of which is described. Its validation both as a petri dish and as solid-state nuclear track detectors is demonstrated. With respect to the demands on solid-state nuclear track detectors in such experiments, these biologically compatible detectors have a controlled micrometric thickness that allows them to be crossed by the alpha particles. In this study, we present a method for obtaining 10-mum-thick CR-39, its chemical characterization, and its properties as a solid-state nuclear track detector under the environmental conditions of radiobiological experiments. The experimental studies performed with 3.5 MeV alpha particles show that their transmitted energy is sufficient enough to cross the entire cellular volume. Under optimal conditions, etched tracks are clearly defined 2 h after etching. Moreover, the UV-radiation-cured CR-39 represents an essentially zero background that is due to the short time between the production and use of the polymer. Under a confocal microscope, this thin solid-state nuclear track detector allows the precise localization of the impact parameter at the subcellular level.     

Effect of CD44 deficiency on in vitro and in vivo osteoclast formation

In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.     

Effects of ambient air particles on the endothelin system in human pulmonary epithelial cells (A549)

Inhalation of urban particles results in higher circulating levels of the vasoconstrictor peptide endothelin-1 (ET-1), which may account for the adverse cardiovascular impacts associated with air pollution. The objective of this study was to examine the direct effects of urban particles on the production of ET-1 by human epithelial cells (A549). A549 cells were exposed to TiO₂, SiO₂, Ottawa urban particulate matter EHC-93, and fractions of the urban particles. The levels of ET-1, interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) in the culture medium were detected by ELISA. The mRNA levels of preproET-1, endothelin converting enzyme (ECE-1), ETa receptor and ETb receptor, matrix metalloproteinase (MMP-2), tissue inhibitor of MMP (TIMP-2), and heat shock protein (HSP-70) were determined by quantitative real-time RT-PCR. Cluster analysis of the variables identified similarities in the patterns of effects. Cluster I comprised variables that were primarily inhibited by particles: ET-1 and MMP-2 mRNAs, ET-1 and bigET-1 peptides, and cell viability. Clusters II and III comprised variables that were either inhibited or induced, depending on the test material: HSP-70, ETaR and ECE mRNAs, and IL-8 and VEGF proteins. Cluster IV comprised variables that were mainly induced by particle preparations: ETbR and TIMP-2 mRNAs. The decreased expression of preproET-1 in A549 cells suggests that epithelial cells may not be the source of higher pulmonary ET-1 spillover in the circulation measured in vivo in response to inhaled urban particles. However, higher ECE-1 in A549 cells after exposure to particles suggests an increased ability to process bigET-1 into the mature ET-1 peptide, while increased receptor expression implies higher responsiveness. The increased release of IL-8 and VEGF by epithelial cells in response to particles could possibly upregulate ET-1 production in the adjacent pulmonary capillary endothelial cells, with concomitant increased ET-1 spillover in the systemic circulation.     

Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion

Fibroblasts often constitute the majority of the stromal cells within a breast carcinoma, yet the functional contributions of these cells to tumorigenesis are poorly understood. Using a coimplantation tumor xenograft model, we demonstrate that carcinoma-associated fibroblasts (CAFs) extracted from human breast carcinomas promote the growth of admixed breast carcinoma cells significantly more than do normal mammary fibroblasts derived from the same patients. The CAFs, which exhibit the traits of myofibroblasts, play a central role in promoting the growth of tumor cells through their ability to secrete stromal cell-derived factor 1 (SDF-1); CAFs promote angiogenesis by recruiting endothelial progenitor cells (EPCs) into carcinomas, an effect mediated in part by SDF-1. CAF-secreted SDF-1 also stimulates tumor growth directly, acting through the cognate receptor, CXCR4, which is expressed by carcinoma cells. Our findings indicate that fibroblasts within invasive breast carcinomas contribute to tumor promotion in large part through the secretion of SDF-1.