Determining high value areas for steppe birds in Spain: hot spots,complementarity and the efficiency of protected areas

We have examined the distribution of 26 species of steppe birds in the Iberian Peninsula and the Balearic Islands within a grid of 5,070 10 × 10 km grid cells. The most valuable areas for steppe birds have been identified by selecting the upper 5% of the 10 × 10 km grid cells after a simple ranking based on the following criteria: species-richness, richness of rare species, rarity index, species vulnerability at Spanish, European and Global levels, and using an index combining the previous criteria. We have also used a heuristic algorithm to select those areas which offered most complementarity. The results have been analysed on a national scale and have been compared with those obtained by species assessments in previous status summaries. Finally, we have performed an analysis of the coverage afforded to locations which are valuable for steppe species by Natural Protected Areas (NPAs) and Special Protected Areas (SPAs), and an evaluation of the potential effects of temporal changes in species’ threat-status. The combined index was the most reliable criterion for defining hotspots, encompassing 15% of the species’ distributions within 5% of the total area considered. This index showed a high level of geographical concordance with the other criteria (nearly 70% of the selected grid cells coincided). Analysis of complementarity delivered poorer results than simple rank-scoring. The analysis of the efficiency of NPA network showed a very low coverage (less than 2%) of the hotspots selected according to the Combined Index. Coverage of the SPA network was higher (nearly 45%), although it diminished (to <35%) when only steppe-defined SPAs were included. The geographical concordance between high-value areas selected using current status summaries and those chosen using earlier ones was low for European threat status (53.4%), intermediate (63.2%) for Spanish threat status and high for SPEC status (78.1%). The Combined Index showed a high level of geographical concordance between the old and new data (76.6%). We conclude that automatic scoring methods (identifying hotspots) are useful for selecting valuable areas and for analysing the efficiency of the network of protected natural spaces, as well as for examining the potential effects of status changes on hotspot definition. Furthermore, the Spanish SPA network does not cover the most important areas for steppe birds adequately.     

Homocysteine and inflammation as main determinants of oxidative stress in the elderly

Oxidative stress is commonly observed in the elderly and could be involved in age-related diseases. However, the determinants of superoxide anion overproduction are not clearly understood. Superoxide anion production was evaluated using a lucigenin-based chemiluminescence method in 478 elderly subjects (304 women, 174 men; 79.5 ± 7.1 years). Homocysteine (HCy) metabolism (homocysteinemia, vitamin B12, plasma, and erythrocyte folates), inflammation (CRP, fibrinogen, α-1 acid glycoprotein), lipid parameters (total cholesterol, triglycerides, HDL and LDL cholesterol), and nutritional parameters (albumin, transthyretin) were determined. The results show that HCy levels (p < 0.001) and superoxide anion production (p = 0.04) increase with aging, but CRP does not. Highest HCy (> 20 μM) (OR 1.83 (1.09–3.07), p = 0.02) and CRP over 5 mg/L (adjusted OR 2.01 (1.15–3.51), p = 0.01) are the main determinants in superoxide anion production in the elderly. These clinical data are confirmed in an in vitro study using THP-1 monocyte-like cells. Incubation with HCy thiolactone (HTL) (0–200 μM) and LPS (0–20 ng/ml) dramatically enhances NADPH oxidase expression and activation. Moreover, a synergic action was evidenced for low concentrations of HTL (20 μM) and LPS (5 ng). Taken together, the clinical data and in vitro experiments support the hypothesis that moderate homocysteinemia and low-grade inflammation synergically enhance NADPH oxidase activity in the elderly.     

Characterization of the in vitro antimycotic activity of a novel killer protein from Williopsis saturnus DBVPG 4561 against emerging pathogenic yeasts

A novel killer toxin, labelled as KT4561, secreted by Williopsis saturnus DBVPG 4561, was found to possess a wide antimycotic activity against strains of Candida glabrata, Issatchenkia orientalis and Pichia guillermondii. KT4561 was precipitated by ethanol and purified by ion-exchange chromatography. The active protein migrated as a single band in SDS-PAGE and was characterized by a molecular weight of approximately 62 kDa. Purified KT4561 was active across wide ranges of temperature (5-45 degrees C) and pH (4.5-8.0) and displayed a rapid decrease in viability of yeast cells after 4-8 h. The in vitro activity of KT4561 against 102 yeast isolates (79% of clinical origin) was determined: MIC(50) and MIC(90) of strains were 0.08 and 0.15 microg/ml for C. glabrata, 0.03 and 0.23 microg/ml for I. orientalis and 1.50 and 2.25 microg/ml for P. guilliermondii. Comparative susceptibility tests showed that a high number of strains used in the present study were insensitive to selected azole and polyene antibiotics. The present study demonstrated the potential of KT4561 to be applied as novel control agent against pathogenic yeasts.     

Evidence for a trans-acting activator function regulating the expression of the human CD5 antigen

Interspecies somatic cell hybrids were generated by fusing the mouse T-lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T-cell clone were used as human partners, respectively, in three independent fusions. Irrespective of the human cell partner used for fusion, a certain number of hybrids lost CD5 surface expression over a period of time in culture. Analysis at the phenotype and genetic level showed that lack of CD5 expression was due neither to segregation of human autosome 11, on which the CD5 gene has been mapped, nor to deletion of the CD5 structural gene. Furthermore, loss of CD5 surface expression correlated with the absence of specific mRNA. Since these hybrids preferentially segregate human chromosomes, these results indicate the existence of a non-syntenic trans-active locus, or loci, positively controlling the expression of the human CD5 gene.     

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

Summary Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.     

Loss of cardiolipin in palmitate-treated GL15 glioblastoma cells favors cytochrome c release from mitochondria leading to apoptosis

Unlike oleate and linoleate, palmitate induced mitochondrial apoptosis in GL15 glioblastoma cells. Decrease in membrane potential in a subpopulation of mitochondria of palmitate-treated cells was revealed using the 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide probe. The diminished ability to reduce a tetrazolium salt indicated an impairment of mitochondrial function. Up to 50% cytochrome c (cyt c ) was detached from the inner mitochondrial membrane and released outside mitochondria in palmitate-treated cells, whereas no release was detected after oleate and linoleate treatments. Cyt c release into the cytosol was followed by caspase 3 activation. Released cyt c and caspase 3 activity were not affected by neutral and acid sphingomyelinase inhibitors and by the inhibitor of serine palmitoyltransferase cycloserine, indicating that apoptosis was independent of the ceramide pathway, nor the mitochondrial pro-apoptotic AIF or Bcl-2/Bax factors appeared to be involved in the effect. Utilization of palmitate by GL15 cells altered phospholipid composition. Cardiolipin (CL), the lipid involved in cyt c interaction with the inner mitochondrial membrane, was decreased and highly saturated. This produced an imbalance in hydrophilic/hydrophobic interactions underlying the anchorage of cyt c , by weakening the hydrophobic component and facilitating detachment of the protein and activation of downstream processes. The primary role of CL was explored by supplying GL15 with exogenous CL through a fusion process of CL liposomes with cell plasma membrane. Fused CL moved to mitochondria, as detected by nonylacridine orange probe. Enrichment of mitochondrial membranes with CL prior to palmitate treatment of cells caused decreased cyt c release and caspase 3 activity.     

Osteoclast receptors and signaling

Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Besides the well known Receptor Activator of Nuclear factor-κB (RANK), RANK ligand and osteoprotegerin axis, a variety of factors tightly regulate osteoclast formation, adhesion, polarization, motility, resorbing activity and life span, maintaining bone resorption within physiological ranges. Receptor-mediated osteoclast regulation is rather complex. Nuclear receptors, cell surface receptors, integrin receptors and cell death receptors work together to control osteoclast activity and prevent both reduced or increased bone resorption. Here we will discuss the signal transduction pathways activated by the main osteoclast receptors, integrating their function and mechanisms of action.     

IRES-driven translation is stimulated separately by the FMDV 3'-NCR and poly(A) sequences

The 3′ end region of foot-and-mouth disease virus (FMDV) consists of two distinct elements, a 90 nt untranslated region (3′-NCR) and a poly(A) tract. Removal of either the poly(A) tract or both the 3′-NCR and the poly(A) tract abrogated infectivity in susceptible cells in the context of a full-length cDNA clone. We have addressed the question of whether the impairment of RNA infectivity is related to defects at the translation level using a double approach. First, compared to the full-length viral RNA, removal of the 3′ sequences reduced the efficiency of translation in vitro. Secondly, a stimulatory effect of the 3′ end sequences on IRES-dependent translation was found in vivo using bicistronic constructs. RNAs carrying the FMDV 3′ end sequences linked to the second cistron showed a significant stimulation of IRES-dependent translation, whereas cap-dependent translation was not affected. Remarkably, IRES-dependent stimulation exerted by the poly(A) tract or the 3′-NCR seems to be the result of two separate events, as the 3′-NCR alone enhanced IRES activity on its own. Under conditions of FMDV Lb protease-induced translation shut-off, the stimulation of IRES activity reached values above 6-fold in living cells. A northern blot analysis indicated that IRES stimulation was not the consequence of a change in the stability of the bicistronic RNA produced in transfected cells. Analysis of the RNA-binding proteins interacting with a mixture of 3′ end and IRES probes showed an additive pattern. Altogether, our results strongly suggest that individual signals in the viral 3′ end ensure stimulation of FMDV translation.     

Subpopulations of mononuclear leukocytes associated with inhibition of Ehrlich ascites tumor growth by treatment with Bothrops jararaca venom

Snake venoms have been used as antineoplastic substances in several experimental models. We demonstrated in previous studies that Bothrops jararaca venom (BjV) induces inhibition of Ehrlich ascites tumor (EAT) growth accompanied by an increase of mononuclear (MN) leukocytes in all groups inoculated with EAT and/or venom. The objective of the present study was to characterize the subpopulations of MN leukocytes involved in the inhibition of EAT growth by treatment with BjV. Swiss mice were inoculated with 1.0x10(3) EAT cells by the intraperitoneal route and treated with 0.4 mg/kg of BjV by the same route (Group TV). Treatment was started 24 h after tumor cell inoculation and consisted of five intraperitoneal injections performed at 72 h intervals. After 2, 8 and 14 days, groups of animals were sacrificed and the number of B, TCD4 and TCD8 lymphocytes, macrophages and natural killer cells present in the peritoneal cavity was determined by flow cytometry. The control group consisted of animals inoculated with EAT and treated with 0.1 ml of saline under the same conditions as the experimental group (Group T). Two additional control groups consisted of animals not inoculated with EAT and treated with saline or venom. Data were analyzed statistically by the Kruskal-Wallis non-parametric test for independent samples. On the 2nd and 8th day we observed a difference between groups T and TV (group T > group TV) for all cell types, except natural killer cells, that only differed on the 2nd day. However, on the 14th day there was no difference in MN cells among groups. These data suggest that the inhibition of EAT is related to the toxic action of BjV on tumor cells and/or to the proteolytic effect of the venom on the mediators produced by the cells for growth modulation.