Pro-oxidative properties of flavonoids in human lymphocytes

The pro-oxidative properties of the four flavonoids, quercetin, morin, naringenin and hesperetin, in human lymphocyte system were investigated. Naringenin and hesperetin accelerated the oxidation of deoxyribose induced by Fe(3+)/H(2)O(2) in a concentration range of 0-200 microM, but quercetin and morin decreased it when the concentration was greater than 100 microM. The generation of hydrogen peroxide and the superoxide anion and the production of TBARS in lymphocytes were increased with increasing concentration of a flavonoid. Cell membrane protein thiols of the lymphocytes decreased when treated with the four flavonoids. Quercetin and hesperetin had no significant effect (p>0.05) on the activity of glutathione reductase, but morin and naringenin could inhibit the activity of the enzyme at a concentration of 200 microM, when compared to the control group. The glutathione S-transferase activity was slightly decreased by treatment with each of the four flavonoids only at a concentration of 200 microM. Therefore, the DNA damage in lymphocytes induced by the flavonoids in the model system might have been due to their stimulation of oxidative stress in the lymphocytes, which resulted in the decrease of cell membrane protein thiols, increase of lipid peroxidation in cell membrane and in the influence of the antioxidative enzyme activities.     

Prediction of soil depth using a soil-landscape regression model: a case study on forest soils in southern Taiwan

Techniques for conventional forest soil surveys in Taiwan need to be further developed in order to save time and money. Although some soil-landscape regression models have been developed to describe and predict soil properties and depths, they have seldom been studied in Taiwan. This study establishes linear soil-landscape regression models related to soil depths and landscape factors found in the forest soils of southern Taiwan. These models were evaluated by validating the models according to their mean errors and root mean square errors. The study was carried out at the 60,000 ha Chishan Forest Working Circle. About 310 soil pedons were collected. The landscape factors included elevation, slope, aspect, and surface stone contents. Sixty percent of the total field samples were used to establish the soil-landscape regression models, and forty % were used for validation. The sampling strategy indicated that each representative pedon covers an area of about 147 ha. The number of samples was appropriate considering the available time and budget. The single variate and/or multivariate linear regression soil-landscape models were successfully established. Those models revealed significant inter-relations among the soil depths of the B and B+BC horizons, solum thickness, and landscape factors, including slope and surface stone contents (p < 0.003). The mean errors in the validation of the soil-landscape model were low and acceptable for this case study. In addition, the slope data derived from the DEM (digital elevation model) database in this case study were used to predict the soil depths of the B, B+BC horizons and the solum thickness without carrying out a field survey. Surface stone should be collected in a field soil survey to increase the precision of soil depth prediction of the B and B+BC horizons, and the solum thickness.     

Euphol from Euphorbia tirucalli Negatively Modulates TGF-β Responsiveness via TGF-β Receptor Segregation inside Membrane Rafts

Transforming growth factor-β (TGF-β) responsiveness in cultured cells can be modulated by TGF-β partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. Lipid rafts are plasma membrane microdomains with an important role in cell survival signaling, and cholesterol is necessary for the lipid rafts’ structure and function. Euphol is a euphane-type triterpene alcohol that is structurally similar to cholesterol and has a wide range of pharmacological properties, including anti-inflammatory and anti-cancer effects. In the present study, euphol suppressed TGF-β signaling by inducing TGF-β receptor movement into lipid-raft microdomains and degrading TGF-β receptors.     

Multiple cysteine residues are implicated in Janus kinase 2-mediated catalysis

The redox regulation of Janus kinase 2 (JAK2) is poorly understood, and there are contradictory reports as to whether the enzyme's activity is inhibited or stimulated by oxidizing conditions in the cell. Here we demonstrate that multiple cysteine residues within the JAK2 catalytic domain may be crucial for enzymatic activity. The enzyme is catalytically inactive when oxidized; activity can be restored via reduction to the thiol state. A series of recombinant variants of JAK2 were overproduced using the baculoviral expression vector system. A truncated variant of JAK2, GST/(NDelta661)rJAK2, provided evidence that the amino-terminal autoinhibitory domain was not essential for direct redox regulation and that only nine cysteine residues were potentially involved. The effect of individually and combinatorially altering these nine cysteines was examined via cysteine-to-serine mutagenesis. This identified four cysteine residues in the catalytic domain (Cys866, Cys917, Cys1094, and Cys1105) that cooperatively maintain JAK2's catalytic competency. Our data are consistent with a direct mechanism for redox regulation of JAK2 via oxidation and reduction of critical cysteine residues.     

Molecular detection of Babesia canis in Dermacentor reticulatus ticks collected in Slovakia

Dermacentor reticulatus ticks are recognized as the most important vectors of Babesia canis, the aetiological agent of canine babesiosis occurring throughout Europe. Vector competence of D. reticulatus for B. canis is well described and experimentally determined; however, by using molecular analysis it was proven so by one recent study in Russia. Herein, the additional molecular evidence of B. canis infection in D. reticulatus ticks collected in Slovakia is provided. Using PCR followed by sequencing of distinctive amplicons we determined the presence of Babesia canis canis in one of 100 tested adult ticks. Two zoonotic pathogens, Francisella tularensis and Coxiella burnetii, were previously isolated from D. reticulatus ticks in Slovakia. In our samples, we detected only the presence of F. tularensis.     

Identification of a redox-sensitive switch within the JAK2 catalytic domain

Four cysteine residues (Cys866, Cys917, Cys1094, and Cys1105) have direct roles in cooperatively regulating Janus kinase 2 (JAK2) catalytic activity. Additional site-directed mutagenesis experiments now provide evidence that two of these residues (Cys866 and Cys917) act together as a redox-sensitive switch, allowing JAK2's catalytic activity to be directly regulated by the redox state of the cell. We created several variants of the truncated JAK2 (GST/(NΔ661)rJAK2), which incorporated cysteine-to-serine or cysteine-to-alanine mutations. The catalytic activities of these mutant enzymes were evaluated by in vitro autokinase assays and by in situ autophosphorylation and transphosphorylation assays. Cysteine-to-alanine mutagenesis revealed that the mechanistic role of Cys866 and Cys917 is functionally distinct from that of Cys1094 and Cys1105. Most notable is the observation that the robust activity of the CC866,917AA mutant is unaltered by pretreatment with dithiothreitol or o-iodosobenzoate, unlike all other JAK2 variants previously examined. This work provides the first direct evidence for a cysteine-based redox-sensitive switch that regulates JAK2 catalytic activity. The presence of this redox-sensitive switch predicts that reactive oxygen species can impair the cell's response to JAK-coupled cytokines under conditions of oxidative stress, which we confirm in a murine pancreatic β-islet cell line.     

Neural transmembrane protease and endothelial Gs protein activation in cell contact-dependent signaling between neural stem/progenitor cells and brain endothelial cells

Vasculature is an important component of the neural stem cell niche in brain. It regulates neural stem/progenitor (NS/P) cell self-renewal, differentiation, and migration. In the neurogenic niches of adult brain, NS/P cells lie close to blood vessels, and proliferating NS/P cells frequently contact the vasculature. In the present study we showed that NS/P cells in co-culture with brain endothelial (bEND) cells activated endothelial G proteins and p38 mitogen-activated protein kinase (p38 MAPK) and stimulated cytokine/chemokine expression. These NS/P cell-induced endothelial responses took place during NS/P cell and bEND cell direct contact and were critically dependent on the expression of the type II transmembrane serine protease matriptase (MTP) by NS/P cells, because knocking down of MTP in NS/P cells impaired and re-expression of MTP restored their ability to induce endothelial cytokine/chemokine expression, p38 MAPK, or G protein activation. Cholera toxin blocked NS/P cell-induced endothelial responses, suggesting that the endothelial G protein activated by NS/P MTP is in the G(s) subfamily. The addition of p38 MAPK inhibitor impaired NS/P cell-induced endothelial cytokine/chemokine expression. The known G protein-coupled receptor substrate of MTP, protease-activated receptor 2, was not involved in this system. These results revealed a novel signaling pathway in neural stem cell vascular niches that is mediated by neural MTP and endothelial G(s) protein signaling at the cell-cell interface. This is the first report of direct cell-cell signaling between NS/P and bEND cells.     

Human Cytomegalovirus UL76 Elicits Novel Aggresome Formation via Interaction with S5a of the Ubiquitin Proteasome System

HCMV UL76 is a member of a conserved Herpesviridae protein family (Herpes_UL24) that is involved in viral production, latency, and reactivation. UL76 presents as globular aggresomes in the nuclei of transiently transfected cells. Bioinformatic analyses predict that UL76 has a propensity for aggregation and targets cellular proteins implicated in protein folding and ubiquitin-proteasome systems (UPS). Furthermore, fluorescence recovery after photobleaching experiments suggests that UL76 reduces protein mobility in the aggresome, which indicates that UL76 elicits the aggregation of misfolded proteins. Moreover, in the absence of other viral proteins, UL76 interacts with S5a, which is a major receptor of polyubiquitinated proteins for UPS proteolysis via its conserved region and the von Willebrand factor type A (VWA) domain of S5a. We demonstrate that UL76 sequesters polyubiquitinated proteins and S5a to nuclear aggresomes in biological proximity. After knockdown of endogenous S5a by RNA interference techniques, the UL76 level was only minimally affected in transiently expressing cells. However, a significant reduction in the number of cells containing UL76 nuclear aggresomes was observed, which suggests that S5a may play a key role in aggresome formation. Moreover, we show that UL76 interacts with S5a in the late phase of viral infection and that knockdown of S5a hinders the development of both the replication compartment and the aggresome. In this study, we demonstrate that UL76 induces a novel nuclear aggresome, likely by subverting S5a of the UPS. Given that UL76 belongs to a conserved family, this underlying mechanism may be shared by all members of the Herpesviridae.     

Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.