Scientific collaboration of Cuban researchers working in Europe: understanding relations between origin and destination countries.

The global network of scientific collaboration created by researchers opens new opportunities for developing countries to engage in the process of knowledge creation historically lead by institutions in the developed world. The results discussed here explore how Cubans working in European science and technology might contribute to extending the scientific collaboration of the country through their ties with Cuban institutions mainly in the academic sector. A bibliometric method was used to explore the pattern of collaboration of Cuban researchers in Europe using the institutional affiliation of authors and collaborators. The records of scientific publications of the defined sample were obtained from Scopus database for the period between 1995 and 2014. The network of collaboration was generated using the affiliations of Cuban authors in Europe and co-authors with worldwide affiliations shown in the records of publications of each Cuban researcher of the study. The analysis of aggregate values of the output of Cuban researchers in Europe (1995-2014) reveals that their collaboration with Cuba correlates moderately with their performance in Europe. However, when taking into account their time publishing in Europe, the collaboration with Cuba decreases the longer they remain away from home. The network of collaborating Cuban researchers in Europe comprises 991 different affiliations from 58 countries: 698 from Europe, 118 from North America, 96 from Latin America and 79 from the rest of the world. K-core analysis of centrality shows two Cuban universities sharing the central position with another 24 institutions worldwide of which 18 belong to higher education.

Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells.

During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/cyclosome-Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein-ß-transducin repeat-containing protein (SCF-ß-TrCP), which control the transient appearance and metabolic activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S(273)) within the conserved recognition site for SCF-ß-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-Glu-Asn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-ß-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-to-late G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.

Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes.

Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 which controls G1-to-S-phase transition by targeting degradation motifs, notably the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture. We now show that APC/C-Cdh1 controls the proliferative response of human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating T lymphocytes. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression.

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions.

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O(2)) conditions. The system measures the concentrations of O(2) and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O(2) concentration and electron turnover of the enzyme. At a high O(2) concentration (70 microM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O(2). At low O(2) (15 microM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O(2) consumption. At both high and low O(2) concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.

Cytochrome c oxidase regulates endogenous nitric oxide availability in respiring cells: a possible explanation for hypoxic vasodilation.

One of the many routes proposed for the cellular inactivation of endogenous nitric oxide (NO) is by the cytochrome c oxidase of the mitochondrial respiratory chain. We have studied this possibility in human embryonic kidney cells engineered to generate controlled amounts of NO. We have used visible light spectroscopy to monitor continuously the redox state of cytochrome c oxidase in an oxygen-tight chamber, at the same time as which we measure cell respiration and the concentrations of oxygen and NO. Pharmacological manipulation of cytochrome c oxidase indicates that this enzyme, when it is in turnover and in its oxidized state, inactivates physiological amounts of NO, thus regulating its intra- and extracellular concentrations. This inactivation is prevented by blocking the enzyme with inhibitors, including NO. Furthermore, when cells generating low concentrations of NO respire toward hypoxia, the redox state of cytochrome c oxidase changes from oxidized to reduced, leading to a decrease in NO inactivation. The resultant increase in NO concentration could explain hypoxic vasodilation.

Cytochrome c oxidase maintains mitochondrial respiration during partial inhibition by nitric oxide.

Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 microM L-arginine at 70 microM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0+/-0.8 and 24.8+/-0.8 microM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism regulated by the flux of electrons in the mitochondria and by the changing ratio of O2:NO, either during hypoxia or, as a consequence of increases in NO, as a result of cell stress.

Expression and activity patterns of nitric oxide synthases and antioxidant enzymes reveal a substantial heterogeneity between cardiac and vascular aging in the rat.

We investigated the effects of aging and ischemia-reperfusion (I/R) injury on the expression and activity of nitric oxide (*NO) synthases and superoxide dismutase (SOD) isoforms. To this end we perfused excised hearts from young (6 months old) and old (31-34 months old) rats according to the Langendorff technique. The isolated hearts were, after baseline perfusion for 30 min, either subjected to 20 min of global no-flow ischemia followed by 40 min of reperfusion or were control-perfused (60 min normoxic perfusion). Both MnSOD and Cu,ZnSOD expression remained unchanged with increasing age and remained unaltered by I/R. However, SOD activity decreased from 7.55 +/- 0.1 U/mg protein in young hearts to 5.94 +/- 0.44 in old hearts (P<0.05). Furthermore, I/R led to a further decrease in enzyme activity (to 6.35 +/- 0.41 U/mg protein; P<0.05) in myocardium of young, but not in that of old animals. No changes in myocardial protein-bound 3-nitrotyrosine levels could be detected. Endothelial NOS (eNOS) expression and activity remained unchanged in aged left ventricles, irrespective of I/R injury. This was in steep contrast to peripheral (renal and femoral) arteries obtained from the same animals where a marked age-associated increase of eNOS protein expression could be demonstrated. Inducible NOS expression was undetectable either in the peripheral arteries or in the left ventricle, irrespective of age. In particular when associated with an acute pathology, which is furthermore limited to a certain time frame, changes in the aged myocardium with respect to enzymes crucially involved in maintaining the redox homeostasis, seem to be much less pronounced or even absent compared to the vascular aging process. This may point to heterogeneity in the molecular regulation of the cardiovascular aging process.

Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol.

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and proapoptotic activity. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF1alpha) and to induce apoptosis in tumour cells by generating reactive oxygen species (ROS). In this study we report that 2ME2 inhibits mitochondrial respiration in both intact cells and submitochondrial particles, and that this effect is due to inhibition of complex I of the mitochondrial electron transport chain (ETC). The prevention by 2ME2 of hypoxia-induced stabilisation of HIF1alpha in HEK293 cells was found not to be due to an effect on HIF1alpha synthesis but rather to an effect on protein degradation. This is in agreement with our recent observation using other inhibitors of mitochondrial respiration which bring about rapid degradation of HIF1alpha in hypoxia due to increased availability of oxygen and reactivation of prolyl hydroxylases. The concentrations of 2ME2 that inhibited complex I also induced the generation of ROS. 2ME2 did not, however, cause generation of ROS in 143B rho(-) cells, which lack a functional mitochondrial ETC. We conclude that inhibition of mitochondrial respiration explains, at least in part, the effect of 2ME2 on hypoxia-dependent HIF1alpha stabilisation and cellular ROS production. Since these actions of 2ME2 occur at higher concentrations than those known to inhibit cell proliferation, it remains to be established whether they contribute to its therapeutic effect.

Endogenous NO regulates superoxide production at low oxygen concentrations by modifying the redox state of cytochrome c oxidase.

We have investigated in whole cells whether, at low oxygen concentrations ([O(2)]), endogenous nitric oxide (NO) modulates the redox state of the mitochondrial electron transport chain (ETC), and whether such an action has any signaling consequences. Using a polarographic-and-spectroscopic-coupled system, we monitored redox changes in the ETC cytochromes b(H), cc(1), and aa(3) during cellular respiration. The rate of O(2) consumption (VO(2)) remained constant until [O(2)] fell below 15 microM, whereas the onset of reduction of cytochromes aa(3), part of the terminal ETC enzyme cytochrome c oxidase, occurred at approximately 50 microM O(2). Incubation of the cells with an inhibitor of NO synthase lowered significantly (P < 0.05) the [O(2)] at which reduction of the cytochromes occurred. We also measured intracellular superoxide (O(2)(-)) production at different [O(2)] and found there was no increase in O(2)(-) generation in control cells, or those treated with the NO synthase inhibitor, when incubated at 21% O(2). However, after 30-min exposure of control cells to 3% O(2), an increase in O(2)(-) generation was observed, accompanied by translocation to the nucleus of the transcription factor NF-kappa B. Both of these responses were diminished by NO synthase inhibition. Our results suggest that endogenous NO, by enhancing the reduction of ETC cytochromes, contributes to a mechanism by which cells maintain their VO(2) at low [O(2)]. This, in turn, favors the release of O(2)(-), which initiates the transcriptional activation of NF-kappa B as an early signaling stress response.

Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy.

We have developed an optical system based on visible light spectroscopy for the continuous study of changes in the redox states of mitochondrial cytochromes in intact mammalian cells. Cells are suspended in a closed incubation chamber in which oxygen and nitric oxide (NO) concentrations can be monitored during respiration. Simultaneously the cells are illuminated with a broad-band tungsten-halogen light source. Emergent light in the visible region (from 490-650 nm) is detected using a spectrophotometer and charge-coupled device camera system. Intensity spectra are then converted into changes in optical attenuation from a 'steady-state' baseline. The oxidised-minus-reduced absorption spectra of the mitochondrial cytochromes are fitted to the attenuation spectra using a multi-wavelength least-squares algorithm. Thus, the system can measure changes in the redox states of the cytochromes during cellular respiration. Here we describe this novel methodology and demonstrate its validity by monitoring the action of known respiratory chain inhibitors, including the endogenous signalling molecule NO, on cytochrome redox states in human leukocytes.

Macrophage endothelial nitric-oxide synthase autoregulates cellular activation and pro-inflammatory protein expression.

Expression of inducible nitric-oxide (NO) synthase (iNOS) and "high-output" production of NO by macrophages mediates many cytotoxic actions of these immune cells. However, macrophages have also been shown to express a constitutive NOS isoform, the function of which remains obscure. Herein, bone marrow-derived macrophages (BMDMØs) from wild-type and endothelial NOS (eNOS) knock-out (KO) mice have been used to assess the role of this constitutive NOS isoform in the regulation of macrophage activation. BMDMØs from eNOS KO animals exhibited reduced nuclear factor-kappaB activity, iNOS expression, and NO production after exposure to lipopolysaccharide (LPS) as compared with cells derived from wild-type mice. Soluble guanylate cyclase (sGC) was identified in BMDMØs at a mRNA and protein level, and activation of cells with LPS resulted in accumulation of cyclic GMP. Moreover, the novel non-NO-based sGC activator, BAY 41-2272, enhanced BMDMØ activation in response to LPS, and the sGC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one attenuated activation. These observations provide the first demonstration of a pathophysiological role for macrophage eNOS in regulating cellular activation and suggest that NO derived from this constitutive NOS isoform, in part via activation of sGC, is likely to play a pivotal role in the initiation of an inflammatory response.