From dimictic to monomictic: Empirical evidence of thermal regime transitions in three deep alpine lakes in Austria induced by climate change

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Potential risk of biologic pollution associated to the introduction of <i>Pinus radiata</i> in grassland areas

Afforestation is a recommended practice to mitigate global warming. However, their implementation may generate undesirable impacts, mostly if exotic species are used. Plantations of Pinus radiata D Don in Ventania (Bs. As., Argentina) soils showed notorious increments of extractable P (Pe), which could affect the dynamic of this element as well as the degree of phosphorus saturation (GSP_Bray). The objectives of this study were: i) to quantify the GSP_Bray in Mollisols afforested with P. radiata comparing the results with those coming from adjacent, natural grassland areas (base line); ii) to evaluate the potential environmental risk induced by afforestation through the identification of a change point (PC) in the GSP_Bray indicative of a phosphate leaching increment. Treatments included mature stands of P. radiata (TB) and adjacent areas with natural grassland vegetation (TP). Samples were taken at 0-15; 15-30 and 30-45 cm soil depth, and texture, pH, total organic carbon (COT), Pe, soluble reactive phosphorus (PSR), phosphorus sorption index (ISP) and GSP_Bray were determined. The results showed a significant acidification in TB and an increase in the COT stock, indicating an additional atmospheric CO₂ sequestration by the trees. The Pe and PSR values were notoriously higher in TB, and they were reflected in a significant increment in the GSP_Bray with respect to TP. The detection of a significant PC in the GSP_Bray-PSR regression indicates higher chances of phosphate leaching in the forest stands, which could reach water courses, lakes and artificial reservoirs promoting their eutrophication. Because of the potential environmental pollution risk of biologic origin derived from the afforestation with P. radiata in Mollisols areas, their inclusion in clean development practices must be reconsidered.     

Properties of murine and human epidermal cell-derived thymocyte-activating factor

Keratinocytes, the predominant cell within the epidermis, perform some macrophage-like functions, such as endocytosis and phagocytosis. We therefore investigated whether keratinocytes may also exert some nonspecific immunoregulatory functions through the secretion of mediators. Tissue cultures of freshly isolated murine and human keratinocytes as well as keratinocyte cell lines secrete a cytokine, epidermal cell-derived thymocyte-activating factor (ETAF), which augments in vitro lymphoproliferative responses. Keratinocyte cell line cells produce increased levels of ETAF activity after exposure to a variety of cell-damaging agents such as silica, endotoxin, phorbol myristate acetate, hydroxyurea, and mechanical disruption. Biochemical studies showed that murine and human ETAF, like interleukin 1 (IL 1), had a molecular weight between 12,000 and 20,000, interacted with hydrophobic phenyl-Sepharose, and was eluted from anion but not cation exchangers. Like IL 1, murine ETAF had a single isoelectric point whereas human ETAF eluted as three peaks of activity (pI 7.2, 5.8, and 5.0). Partially purified ETAF of either species also had the same biological properties of IL 1. That is, ETAF enhanced IL 2 production by T cells in culture, was chemotactic for polymorphonuclear leukocytes, and was directly mitogenic for fibroblasts. When injected into C3H/HeJ mice ETAF induced hepatocyte production of serum amyloid A, an acute phase protein. Furthermore, ETAF, like IL 1, may act as an endogenous pyrogen and induce fever when injected into rabbits. These findings indicate that production of IL 1-like molecules is not confined to cells of the immune system and that ETAF production by keratinocytes may have important implications in would healing, as well in the pathogenesis of inflammatory and neoplastic diseases.     

Autonomous folding and coenzyme binding of the excised pyridoxal 5'-phosphate binding domain of aspartate aminotransferase from Escherichia coli

The coenzyme (PLP) binding domain (residues 47-329) of the dimeric aspartate aminotransferase from Escherichia coli was produced separately by recombinant DNA methods. It folded autonomously both in vivo and in vitro, that is, independently of the native N- and C-terminal extensions that combine to form the small domain of eAAT. The PLP-domain had one binding site for PLP of relatively high affinity involving a covalent bond to the protein. It was monomeric, although the major subunit-subunit interface at the 2-fold symmetry axis remained unchanged. This effect appears to be due mainly to the absence of the N-terminal extension that contains hydrophobic residues, which interact with the PLP-domain of the second subunit in the wild-type dimer. Judged by circular dichroism, fluorescence, and HPLC gel filtration at increasing concentrations of guanidinium chloride, the PLP-domain underwent a three-state unfolding transition (M' in equilibrium M'* in equilibrium U') involving a compact intermediate M'*. This behavior parallels the unfolding of the dissociated native monomer of cAAT.     

IFN-beta 2, B cell differentiation factor 2, or hybridoma growth factor (IL-6) is expressed and released by human epidermal cells and epidermoid carcinoma cell lines

IL-6, which is also known as IFN-beta 2, hybridoma growth factor, hepatocyte-stimulating factor, and B cell differentiation factor, mediates acute phase responses including fever, has lymphocyte-stimulating capacities, and antiviral activity. IL-6 is produced by monocytes, fibroblasts, certain lymphocytes, and various tumor cells. The present study demonstrates that this multifunctional cytokine is released also by normal human epidermal cells (EC) and human epidermoid carcinoma cell lines (A431, KB). Accordingly, supernatants derived from freshly isolated EC, long term keratinocyte cultures, A431, or KB cells stimulated the proliferation of a hybridoma growth factor/IL-6-dependent plasmacytoma cell line (B9). IL-6 constitutively was produced in the presence of serum proteins. The addition of IL-1 alpha, IL-1 beta, or the tumor promoter PMA significantly enhanced the synthesis and release of EC-derived IL-6 (EC-IL 6). Like monocyte or fibroblast-derived IL-6, EC-IL-6 exhibited Mr microheterogeneity within 21 and 28 kDa. Similarly in Western blotting experiments an antiserum directed against human rIFN-beta 2/IL-6 detected the different Mr forms of EC-IL-6. Moreover, this antiserum was able to block the B9 cell growth-promoting capacity of EC-IL-6 strongly suggesting that this EC-derived mediator is closely related, if not identical with IL-6. This was further confirmed by Northern blot analysis detecting IL-6 specific mRNA both in long term cultured keratinocytes and A431 cells by hybridization with a cDNA fragment encoding for B cell differentiating factor 2/IL-6. Therefore, in addition to the production of other cytokines as previously reported, EC and in particular keratinocytes also synthesize and release IL-6. This further supports the important regulatory role of the epidermis during the pathogenesis of inflammatory, autoimmune, and neoplastic diseases.     

Human epidermal cells and squamous carcinoma cells synthesize a cytokine that augments natural killer cell activity

Normal as well as transformed epidermal cells (EC) have recently been reported to produce a cytokine--EC-derived thymocyte-activating factor (ETAF), which according to its biologic as well as biochemical properties is indistinguishable from macrophage-derived interleukin 1 (IL 1). In the present study, the effect of supernatants (SN) derived from normal EC and a human squamous carcinoma cell (SCC) line were tested for their effects on natural killer (NK) cell activity. EC- as well as SCC-derived SN were able to augment in vitro NK cell activity of peripheral blood lymphocytes against K 562 cells. In contrast, adherent cell-derived, IL 1-containing SN did not affect NK cell activity. Upon high-pressure liquid chromatography (HPLC) gel filtration, ETAF and the EC-derived NK cell activity-augmenting factor (ENKAF) exhibited a similar m.w. However, by using reverse-phase HPLC, ETAF and ENKAF eluted as distinct peaks of activity, indicating that SCC cell-derived ENKAF is different from ETAF. Furthermore, ENKAF does not contain interleukin 2 (IL 2) or interferon (IFN) activity. The enhancement of NK cell activity was dose dependent and evident after 20 hr of preincubation of effector cells. Pretreatment of target cells with ENKAF did not affect the susceptibility of the target cells. The NK activity of large granular lymphocytes (LGL) purified by discontinuous Percoll gradient centrifugation and further depleted of high-affinity sheep erythrocyte rosetting cells was enhanced by ENKAF. In contrast, no NK cell activity was expressed by LGL-depleted T cell populations before or after treatment with ENKAF. In a single cell cytotoxicity assay in agarose, the number of lymphocyte binding to K 562 was not affected by ENKAF, but the frequency of dead conjugated target cells and presumably of active killer cells was increased by pretreatment with ENKAF. Additional incubation of LGL with ETAF did not further increase ENKAF-mediated augmentation of NK activity. In contrast to ETAF, ENKAF was not chemotactic for polymorphonuclear leukocytes. These results indicate that normal as well as transformed EC release a unique cytokine--ENKAF--which augments NK cell activity of LGL but is distinct from ETAF, IL 2, and IFN.     

Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer

Immunodeficiency is a barrier to successful vaccination in individuals with cancer and chronic infection. We performed a randomized phase 1/2 study in lymphopenic individuals after high-dose chemotherapy and autologous hematopoietic stem cell transplantation for myeloma. Combination immunotherapy consisting of a single early post-transplant infusion of in vivo vaccine-primed and ex vivo costimulated autologous T cells followed by post-transplant booster immunizations improved the severe immunodeficiency associated with high-dose chemotherapy and led to the induction of clinically relevant immunity in adults within a month after transplantation. Immune assays showed accelerated restoration of CD4 T-cell numbers and function. Early T-cell infusions also resulted in significantly improved T-cell proliferation in response to antigens that were not contained in the vaccine, as assessed by responses to staphylococcal enterotoxin B and cytomegalovirus antigens (P < 0.05). In the setting of lymphopenia, combined vaccine therapy and adoptive T-cell transfer fosters the development of enhanced memory T-cell responses.     

A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome

Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the histone octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)(2) tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z.