In situ assessment of mitochondrial calcium transport in tobacco pollen tubes.

Pollen tubes require functional mitochondria in order to achieve fast and sustained growth. In addition, cell wall expansion requires a calcium gradient in the tube apex formed by a dedicated array of calcium pumps and channels. Most studies have traditionally focused on the molecular aspects of calcium interactions and transport across the pollen tube plasmalemma. However, calcium transients across mitochondrial membranes from pollen tubes are beginning to be studied. Here, we report the presence of a ruthenium red-sensitive mitochondrial calcium uniporter-like activity in tobacco pollen tubes with functional oxidative phosphorylation. The present study provides a framework to measure in situ specifics of mitochondrial transport and respiration in pollen tubes from different plants. The relevance of a mitochondrial calcium uniporter for pollen tube growth is discussed.

Transcriptional Regulation of Energy Metabolism in Cancer Cells.

Cancer development, growth, and metastasis are highly regulated by several transcription regulators (TRs), namely transcription factors, oncogenes, tumor-suppressor genes, and protein kinases. Although TR roles in these events have been well characterized, their functions in regulating other important cancer cell processes, such as metabolism, have not been systematically examined. In this review, we describe, analyze, and strive to reconstruct the regulatory networks of several TRs acting in the energy metabolism pathways, glycolysis (and its main branching reactions), and oxidative phosphorylation of nonmetastatic and metastatic cancer cells. Moreover, we propose which possible gene targets might allow these TRs to facilitate the modulation of each energy metabolism pathway, depending on the tumor microenvironment.

Activated protein C resistance and factor V Leiden in Mexico.

A common cause of hereditary thrombophilia is activated protein C resistance (APCR), and most cases result from factor V Leiden mutation. An APCR phenotype without association with factor V Leiden has been described. This transversal, observational, nonrandomized study evaluated these 2 phenomena in healthy indigenous and mestizo Mexican subjects (n = 4345), including 600 Mexican natives. No indigenous subjects had APCR, but 82 mestizo subjects did. After retesting, 50 subjects had a negative test. The remaining 32 subjects had factor V Leiden, giving a 0.85% prevalence of factor V Leiden in the mestizo Mexican population. Only 31% of APCR carriers had factor V Leiden. These results show a very low prevalence of APCR and factor V Leiden in Mexico. Except for factor V Leiden, there are no other mutations in the factor V gene responsible for the APCR phenotype. Acquired APCR is nearly twice as prevalent as the inherited variant.

Dengue Virus Induces the Release of sCD40L and Changes in Levels of Membranal CD42b and CD40L Molecules in Human Platelets.

Platelets are considered as significant players in innate and adaptive immune responses. The adhesion molecules they express, including P-selectin, CD40L, and CD42b, facilitate interactions with many cellular effectors. Upon interacting with a pathogen, platelets rapidly express and enhance their adhesion molecules, and secrete cytokines and chemokines. A similar phenomenon occurs after exposure of platelets to thrombin, an agonist extensively used for in vitro activation of these cells. It was recently reported that the dengue virus not only interacts with platelets but possibly infects them, which triggers an increased expression of adhesion molecule P-selectin as well as secretion of IL-1ß. In the present study, surface molecules of platelets like CD40L, CD42b, CD62P, and MHC class I were evaluated at 4 h of interaction with dengue virus serotype 2 (DENV-2), finding that DENV-2 induced a sharp rise in the membrane expression of all these molecules. At 2 and 4 h of DENV-2 stimulation of platelets, a significantly greater secretion of soluble CD40L (sCD40L) was found (versus basal levels) as well as cytokines such as GM-CSF, IL-6, IL-8, IL-10, and TNF-α. Compared to basal, DENV-2 elicited more than two-fold increase in these cytokines. Compared to the thrombin-induced response, the level generated by DENV-2 was much higher for GM-CSF, IL-6, and TNF-α. All these events induced by DENV end up in conspicuous morphological changes observed in platelets by confocal microscopy and transmission electron microscopy, very different from those elicited by thrombin in a more physiological scenery.

Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets.

Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3-38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation.

Venous thrombosis among patients with AIDS.

Thrombosis has been considered an uncommon complication in patients with AIDS. In a 42-month period, 28 adult male homosexuals with AIDS experienced 34 thrombotic events. All but three received HAART regimen, two a successful round of double nucleoside analog therapy, and one patient received no treatment. Median age of group was 38.5 years (range, 24 to 56 years). Median time from HIV infection to thrombosis was 40.5 months (range, 3 to 108 months). No patient had previous thrombosis, family history of thrombosis, or prothrombotic conditions. There were 31 deep vein thromboses, two pulmonary thromboembolisms, and one renal vein thrombosis. Six patients had two thrombotic events. The rate of thrombosis during the 42-month study period was 1.52% (cumulative incidence = 0.30%/year), while the rate of thrombosis in 600 patients before the era of protease inhibitor therapy was 0.33% (cumulative incidence approximately 0.055%/year) (p < 0.001). Due to high incidence of thrombotic recurrences and hemorrhagic complications while using oral anticoagulants, acetylsalicylic acid was initiated; no thrombotic episodes were recorded while using this drug. Protein C and protein S deficiency were found in nine and two patients, respectively. Two patients had lupus anticoagulant and two activated protein C resistance (APCR) without FV Leiden mutation (APCR test was negative after initial screening). Fifteen patients had no thrombophilic abnormalities. These data suggest that protease inhibitors could be a risk factor for venous thrombosis not due to thrombophilic abnormalities but likely related to abnormalities in platelets or endothelium.

[National evaluation of the diagnosis of activated protein C resistance]. / Evaluación nacional del diagnóstico de la resistencia a la proteína C activada.

Thrombophilia or prothrombotic state appears when activation of blood hemostatic mechanisms overcomes the physiological anticoagulant capacity allowing a thrombotic event. Thrombosis is the leading worldwide mortality cause and due to its high associated morbidity and mortality, it should be insisted in the opportune identification of a thrombophilic state. The study of thrombophilia identifies individuals at high risk for thrombosis. This meeting was conceived first to analyze the current status of the diagnosis of thrombophilia in Mexico and second to create the base for a national consensus for thrombophilia screening and for the establishment of a national center for laboratory reference and quality control for thrombophilia. Since searching of activated protein C resistance (APCR) and FV Leiden seem to have priority either in the clinical setting and in public health services, it was decided to start with these two abnormalities as a model to analyze the current status of thrombophilia diagnosis in the clinical laboratory. At this time, several thrombophilic abnormalities have been described however, APCR remains the most important cause of thrombophilia, accounting for as much as 20% to 60% of all venous thrombosis. APCR is a consequence of the resistance of activated FV to be inactivated by activated protein C. Procoagulant activity of activated FV increases the risk of thrombosis. Hereditary APCR is almost always due to a point mutation at the nucleotide 1691 of the FV gen inducing an Arg506Glu substitution in FV molecule. This mutation is better known as FV Leiden. Heterocygous carriers of FV Leiden have a thrombotic risk 5 to 10 times higher than general population while the risk for the homocygote state is increased 50 to 100-fold. When activated PC is added to plasma from patients with FV Leiden, this last resists the anticoagulant effect of activated PC. Therefore, thrombin production is not inhibited. This phenomenon is called APCR. The functional test evaluates the partially activated thromboplastin time (aPTT) in a plasma sample before and after adding activated PC. The result is reported as a standardized sensibility index: aPTT post-activated PC/aPTT pre-activated PC. The conclusions of this national reunion pretend to optimize the available resources in our country in order to allow a wide and less-expensive diagnosis of patients with thrombosis.

Mitochondrial Unselective Channels throughout the eukaryotic domain.

Mitochondria from diverse species can undergo a massive permeability increase known as the permeability transition, a process first thought to be an artifact. It is currently accepted that in the inner mitochondrial membrane there is a Mitochondrial Unselective Channel (MUC), also known as the permeability transition pore. Regardless of the species, MUC opening leads to uncoupling of oxidative phosphorylation. In each species, MUC regulation appears to be different, probably as a result of the adaptation of each organism to its specific environment. To date, the components and the putative physiological role of MUCs are still a matter of debate. Current hypothesis suggests that proteins normally participating in diverse metabolic functions constitute MUCs. Among these proteins, the Adenine Nucleotide Translocase and the phosphate carrier have been proposed as putative MUC components in mammalian and yeast mitochondria. In this review, the characteristics of MUCs from different species and strains are discussed. The data from the literature reinforce the current notion that these channels are preserved through evolution albeit with different control factors. We emphasize the knowledge available of Mitochondrial Unselective Channels from different yeast species.

Effect of combined administration of clopidogrel and lysine acetylsalicylate versus clopidogrel and aspirin on platelet aggregation and activated GPIIb/IIIa expression in healthy volunteers.

Platelet activation contributes to thrombotic events in cardiovascular disease. Acetylsalicylic acid (ASA) is used in combination with clopidogrel to reduce cardiovascular events. Lysine acetylsalicylate (L-ASA), also inhibits platelet activation with fewer gastrointestinal side effects than ASA. Dual therapy with L-ASA and clopidogrel may result in an antiplatelet effect with fewer side effects. We compared the antiplatelet effect of combined ASA/clopidogrel versus L-ASA/clopidogrel in healthy subjects. Fourteen volunteers (seven men and seven women, aged 25-45 years) received antiplatelet therapy during 14-day periods in the following sequence: 75 mg ASA; 160 mg L-ASA; 75 mg clopidogrel; 160 mg L-ASA plus 75 mg clopidogrel, and 75 mg ASA plus 75 mg clopidogrel. We evaluated platelet aggregation and glycoprotein IIb/IIIa activation. Our results show that administration of L-ASA/clopidogrel is as effective as ASA/clopidogrel combination.

Polyamines inhibit both platelet aggregation and glycoprotein IIb/IIIa activation.

Platelet aggregation is inhibited by the polyamines putrescine, spermidine, and spermine. To date, the mechanism of action has not been clearly identified. Evidence suggests that polyamines may interact with the fibrinogen receptor (GP IIb/IIIa), interfering with platelet-platelet attachment. The effect of polyamines on human platelet aggregation and GP IIb/IIIa activation was evaluated. For the aggregation experiments, platelets were obtained from heparin- or citrate-collected blood. Our results indicate that the polyamines putrescine, spermidine, and spermine cause a dose-dependent inhibition of ADP- or collagen-mediated platelet aggregation with an order of potency spermine>spermidine>putrescine. In addition, spermine arrests or inhibits thrombin-, epinephrine-, arachidonate-, or ristocetin-induced platelet aggregation. Expression of platelet membrane glycoproteins IIb, IIIa, and IX is not reduced by polyamines. However, spermine inhibits the ADP- or thrombin-induced activation of GP IIb/IIIa. It is concluded that the final step in aggregation, common to all agonists, ie, fibrinogen binding to GP IIb/IIIa, is inhibited by spermine through inhibition of the agonist-induced activation of GP IIb/IIIa that precedes fibrinogen-ligand binding.

Aterosclerosis experimental: cambios bioquímicos y vasculares / Experimental atherosclerosis: biochemical and vascular changes

Se estudió el desarrollo de placas ateros-clerósicas en conejos Nueva Zelanda alimentados con una dieta rica en colesterol. Para ello se comparó el contenido sérico de lípidos y glucosa en conejos sanos y conejos alimentados con 1 y 10 por ciento de colesterol por 10 semanas. Además, se hicieron estudios histológicos de las aortas de dichos animales para evaluar las lesiones ateromatosas. En los animales que recibían una dieta con 10 por ciento de colesterol, los niveles séricos de éste aumentaron significativamente de 26.3 ñ 8.1 mg/dL a 1485 ñ 26.8 mg/dL (p < 0.05). El colesterol asociado con LDL también se incrementó, de 15.9 ñ 5.9 a 1383.8 ñ 58.9 (p < 0.5); y los triglicéridos de 88.3 ñ 35.6 a 411 ñ 154.5. Se encontraron lesiones ateros-clerósicas solamente en los conejos alimentados con 10 por ciento de colesterol. Este modelo es reproducible y puede ser útil en el estudio de la aterosclerosis per se y de la aterogénesis asociada con enfermedades como la diabetes mellitus