Cytochrome P450 2E1-derived reactive oxygen species mediate paracrine stimulation of collagen I protein synthesis by hepatic stellate cells.

To evaluate possible fibrogenic effects of CYP2E1-dependent generation of reactive oxygen species, a model was developed using co-cultures of HepG2 cells, which do (E47 cells) or do not (C34 cells) express cytochrome P450 2E1 (CYP2E1) with stellate cells. There was an increase in intra- and extracellular H(2)O(2), lipid peroxidation, and collagen type I protein in stellate cells co-cultured with E47 cells compared with stellate cells alone or co-cultured with C34 cells. The increase in collagen was prevented by antioxidants and a CYP2E1 inhibitor. CYP3A4 did not mimic the stimulatory effects found with CYP2E1. Collagen mRNA levels remained unchanged, and pulse-chase analysis indicated similar half-lives of collagen I protein between both co-cultures. However, collagen protein synthesis was increased in E47 co-culture. Hepatocytes from pyrazole-treated rats (with high levels of CYP2E1) induced collagen protein in primary stellate cells, and antioxidants and CYP2E1 inhibitors blocked this effect. These results suggest that increased translation of collagen mRNA by CYP2E1-derived reactive oxygen species is responsible for the increase in collagen protein produced by the E47 co-culture. These co-culture models may be useful for understanding the impact of CYP2E1-derived ROS on stellate cell function and activation.     

Isolation of fungi from bats of the Amazon basin

A total of 2,886 bats captured in the Amazon Basin of Brazil were processed for the isolation of fungi. From the livers, spleens, and lungs of 155 bats (5.4%), 186 fungal isolates of the genera Candida (123 isolates), Trichosporon (26 isolates), Torulopsis (25 isolates), Kluyveromyces (11 isolates), and Geotrichum (1 isolate) were recovered. Seven known pathogenic species were present: Candida parapsilosis, C. guilliermondii, C. albicans, C. stellatoidea, C. pseudotropicalis, Trichosporon beigelii, and Torulopsis glabrata. Twenty-three culture-positive bats showed identical fungal colonization in multiple organs or mixed colonization in a single organ. The fungal isolation rates for individual bat species varied from 1 fungus per 87 bats to 3 fungi per 13 bats, and the mycoflora diversity for members of an individual fungus-bearing bat species varied from 16 fungi per 40 bats to 7 fungi per 6 bats. Of the 38 fungal species isolated, 36 had not been previously described as in vivo bat isolates. Of the 27 culture-positive bat species, 21 had not been previously described as mammalian hosts for medically or nonmedically important fungi.     

Differential apoptotic pathways activated in response to Cu-induced or HOCl-induced LDL oxidation in U937 monocytic cell line

We compared the apoptotic mechanism involved in U937 human monocytic cell line in presence of oxidized low-density lipoproteins (oxLDL) obtained after treatment with hypochlorous acid (HOCl) or copper (Cu).Both types of oxLDL induced U937 apoptotic cell death via the mitochondrial pathway. In contrast to HOCl-oxLDL, Cu-oxLDL induced apoptosis via a caspase-independent mechanism, with no activation of pro-caspase-3, but via the release of apoptosis inducing factor (AIF) from mitochondria.The apoptotic program of the monocyte differs depending on the mode of LDL oxidation, based on differences in the oxidatively modified components of the two oxLDL types.     

A predictive model for freshwater bioassessment (Mondego River,Portugal)

We sampled macroinvertebrates at 75 locations in the Mondego river catchment, Central Portugal, and developed a predictive model for water quality assessment of this basin, based on the Reference Condition Approach. Sampling was done from June to September 2001. Fifty-five sites were identified as “Reference sites” and 20 sites were used as “Test sites” to test the model. At each site we also measured 40 habitat variables to characterize water physics and chemistry, habitat type, land use, stream hydrology and geographic location. Macroinvertebrates were generally identified to species or genus level; a total of 207 taxa were found. By Unweighted Pair Group Method with Arithmetic mean (UPGMA) clustering and analysis of species contribution to similarities percentage (SIMPER), two groups of reference sites were established. Using Discriminant Analysis (stepwise forward), four variables correctly predicted 78% of the reference sites to the appropriate group: stream order, pool quality, substrate quality and current velocity. Test sites’ environmental quality was established from their relative distance to reference sites, in MDS ordination space, using a series of bands (BEAST methodology). The model performed well at upstream sites, but at downstream sites it was compromised by the lack of reference sites. As with the English RIVPACS predictive model, the Mondego model should be continually improved with the addition of new reference sites. The adaptation of the Mondego model methodology to the Water Framework Directive is possible and would consist mainly of the integration of the WFD typology and increasing the number of ellipses that define quality bands. Handling editor: K. Martens     

The Peculiar Facets of Nitric Oxide as a Cellular Messenger: From Disease-Associated Signaling to the Regulation of Brain Bioenergetics and Neurovascular Coupling

In this review, we address the regulatory and toxic role of ^·NO along several pathways, from the gut to the brain. Initially, we address the role on ^·NO in the regulation of mitochondrial respiration with emphasis on the possible contribution to Parkinson’s disease via mechanisms that involve its interaction with a major dopamine metabolite, DOPAC. In parallel with initial discoveries of the inhibition of mitochondrial respiration by ^·NO, it became clear the potential for toxic ^·NO-mediated mechanisms involving the production of more reactive species and the post-translational modification of mitochondrial proteins. Accordingly, we have proposed a novel mechanism potentially leading to dopaminergic cell death, providing evidence that NO synergistically interact with DOPAC in promoting cell death via mechanisms that involve GSH depletion. The modulatory role of NO will be then briefly discussed as a master regulator on brain energy metabolism. The energy metabolism in the brain is central to the understanding of brain function and disease. The core role of ^·NO in the regulation of brain metabolism and vascular responses is further substantiated by discussing its role as a mediator of neurovascular coupling, the increase in local microvessels blood flow in response to spatially restricted increase of neuronal activity. The many facets of NO as intracellular and intercellular messenger, conveying information associated with its spatial and temporal concentration dynamics, involve not only the discussion of its reactions and potential targets on a defined biological environment but also the regulation of its synthesis by the family of nitric oxide synthases. More recently, a novel pathway, out of control of NOS, has been the subject of a great deal of controversy, the nitrate:nitrite:NO pathway, adding new perspectives to ^·NO biology. Thus, finally, this novel pathway will be addressed in connection with nitrate consumption in the diet and the beneficial effects of protein nitration by reactive nitrogen species.

     

Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.     

Lysosomal accumulation and recycling of lipopolysaccharide to the cell surface of murine macrophages, an in vitro and in vivo study.

In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate of Brucella abortus LPS in murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses. The intracellular pathway of B. abortus LPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitro after LPS incubation with macrophages. We also followed LPS trafficking after infection of macrophages with B. abortus strain 19. After binding to the cell surface and internalization, Brucella LPS is routed from early endosomes to lysosomes with unusual slow kinetics. It accumulates there for at least 24 h. Later, LPS leaves lysosomes and reaches the macrophage cell surface. This recycling pathway is also observed for LPS released by Brucella S19 following in vitro infection. Indeed, by 72 h postinfection, bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery. From 72 h onward, LPS is gradually detected at the plasma membrane. In each case, the LPS present at the cell surface is found in large clusters with the O-chain facing the extracellular medium. Both the antigenicity and heterogenicity of the O-chain moiety are preserved during the intracellular trafficking. We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposing its immunogenic moiety toward the extracellular medium.     

Biogeographical patterns and Rapoport's rule in southeastern Pacific benthic polychaetes of the Chilean coast

Recently three biogeographical units were identified along the Chilean coast (the Magellanic Province, an Intermediate Area, and the Peruvian Province), however few studies have focused on the factors and dynamic processes that formed these spatial units (e.g. Rapoport's rule and its causal mechanisms). In this study we used benthic polychaetes of the Chilean coast to evaluate patterns of latitudinal distribution and species richness, and the existence of the three main biogeographical provinces described for the Chilean coast. Additionally, we evaluated the latitudinal Rapoport effects and geometric constraint as a null hypothesis explaining the species richness distribution.
We found that benthic polychaete diversity increased towards southern latitudes. The cluster and ordination (non-metric MultiDimensional Scaling, nMDS) analyses of the distribution data, presented only two statistically significant (bootstrapping techniques) biogeographic provinces along the Chilean coast, with a break occurring between 41° and 42°S. While, our results did not support a latitudinal Rapoport effect, they did support the view that latitudinal Rapoport effects are a local phenomenon, occurring only for the Northeastern Pacific marine taxa. The relationship between latitudinal range extent and mean latitude indicated the existence of two hard boundaries at either extreme of the Chilean coast, limiting the geographical ranges of the species. However, geometric constraints tested using a Monte Carlo simulation approach showed a weak level of mid-domain effect on species richness. Finally, we propose that geometric constraint together with the geomorphology and historical characteristics of the Chilean coast explain the biogeographical patterns of benthic polychaete taxa in Chile.