Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.     

Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.     

The coronary effects of parathyroid hormone

OBJECTIVE: The aim of the present study was to characterize the role of the ATP-sensitive potassium channels (K(+)(ATP)) in the coronary dilator action of parathyroid hormone (PTH). METHODS: Dose-response curves of intracoronary administrated PTH (0.15-1.33 nmol) were obtained in control phases and during continuous intracoronary administration of the K(+)(ATP) channel-selective antagonist glibenclamide (0.1-1.0 micromol/min) in dogs (n = 13). RESULTS: Increments of integrated coronary conductance (excess coronary conductance) at PTH doses of 0.15 and 1.33 nmol were 1.17 versus 0.03 ml/mm Hg (p < 0.05) and 4.03 versus 0.94 ml/mm Hg (p < 0.05) in the control versus during maximal blockade, respectively. CONCLUSION: The results indicate that the activation of K(+)(ATP) channels significantly contributes to the PTH-induced coronary vasodilation.     

Galactolipase activity and free fatty acid levels in chloroplasts of domestic and wild tomatoes with different chilling tolerance

In order to establish differences in the chilling sensitivity of domestic and wild Lycopersicon species, galactolipase (EC 3.1.1.26) activity, free fatty acid (FFA) level and Hill reaction activity were measured in chloroplasts isolated from control and cold treated leaves of L. esculentum Mill., cv. Norton, L. hirsutum Humb. and Bonpl., L. peruvianum var. glandulosum Mill. Galactolipase activity was higher in chloroplasts from Lycopersicon species with high chilling sensitivity than in chloroplasts of more chilling-resistant ones. A similar relationship was observed for FFA level in chloroplasts from both cold-stored and control leaves. Decrease in Hill reaction activity due to cold stress was greater in chloroplasts of more chilling-sensitive species. The changes are accompanied by a decline of photochemical activity. Considering the changes in the three parameters noted above, an increasing order of chilling tolerance was established: L. esculentum < L. hirsutum (700 m) < L. hirsutum (3100 m) < L. peruvianum (3400 m). It is suggested that measurements of galactolipase activity and FFA may be useful in an evaluation of differences in resistance to chilling injury of closely related species.     

Elemental sulfur and acetate can support life of a novel strictly anaerobic haloarchaeon

Archaea domain is comprised of many versatile taxa that often colonize extreme habitats. Here, we report the discovery of strictly anaerobic extremely halophilic euryarchaeon, capable of obtaining energy by dissimilatory reduction of elemental sulfur using acetate as the only electron donor and forming sulfide and CO₂ as the only products. This type of respiration has never been observed in hypersaline anoxic habitats and is the first example of such metabolic capability in the entire Archaea domain. We isolated and cultivated these unusual organisms, selecting one representative strain, HSR2, for detailed characterization. Our studies including physiological tests, genome sequencing, gene expression, metabolomics and [¹⁴C]-bicarbonate assimilation assays revealed that HSR2 oxidized acetate completely via the tricarboxylic acid cycle. Anabolic assimilation of acetate occurred via activated glyoxylate bypass and anaplerotic carboxylation. HSR2 possessed sulfurtransferase and an array of membrane-bound polysulfide reductase genes, all of which were expressed during the growth. Our findings suggest the biogeochemical contribution of haloarchaea in hypersaline anoxic environments must be reconsidered.     

Oxidative Stress in Mammalian Cells Impinges on the Cysteines Redox State of Human XRCC3 Protein and on Its Cellular Localization

In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR), a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3) in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our in silico prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO) cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein) does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT), which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway.     

Hydrostatic pressure affects membrane and storage lipid compositions of the piezotolerant hydrocarbon‐degrading Marinobacter hydrocarbonoclasticus strain #5

A new piezotolerant alkane‐degrading bacterium (Marinobacter hydrocarbonoclasticus strain #5) was isolated from deep (3475 m) Mediterranean seawater and grown at atmospheric pressure (0.1 MPa) and at 35 MPa with hexadecane as sole source of carbon and energy. Modification of the hydrostatic pressure influenced neither the growth rate nor the amount of degraded hexadecane (≈ 90%) during 13 days of incubation. However, the lipid composition of the cells sharply differed under both pressure conditions. At 0.1 MPa, M. hydrocarbonoclasticus #5 biosynthesized large amounts (≈ 62% of the total cellular lipids) of hexadecane‐derived wax esters (WEs), which accumulated in the cells under the form of individual lipid bodies. Intracellular WEs were also synthesized at 35 MPa, but their proportion was half that at 0.1 MPa. This lower WE content at high pressure was balanced by an increase in the total cellular phospholipid content. The chemical composition of WEs formed under both pressure conditions also strongly differed. Saturated WEs were preferentially formed at 0.1 MPa whereas diunsaturated WEs dominated at 35 MPa. This increase of the unsaturation ratio of WEs resembled the one classically observed for bacterial membrane lipid homeostasis. Remarkably, the unsaturation ratio of membrane fatty acids of M. hydrocarbonoclasticus grown at 35 MPa was only slightly higher than at 0.1 MPa. Overall, the results suggest that intracellular WEs and phospholipids play complementary roles in the physiological adaptation of strain #5 to different hydrostatic pressures.