HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

Sensitivity analysis of coexistence in ecological communities: theory and application

Sensitivity analysis, the study of how ecological variables of interest respond to changes in external conditions, is a theoretically well‐developed and widely applied approach in population ecology. Though the application of sensitivity analysis to predicting the response of species‐rich communities to disturbances also has a long history, derivation of a mathematical framework for understanding the factors leading to robust coexistence has only been a recent undertaking. Here we suggest that this development opens up a new perspective, providing advances ranging from the applied to the theoretical. First, it yields a framework to be applied in specific cases for assessing the extinction risk of community modules in the face of environmental change. Second, it can be used to determine trait combinations allowing for coexistence that is robust to environmental variation, and limits to diversity in the presence of environmental variation, for specific community types. Third, it offers general insights into the nature of communities that are robust to environmental variation. We apply recent community‐level extensions of mathematical sensitivity analysis to example models for illustration. We discuss the advantages and limitations of the method, and some of the empirical questions the theoretical framework could help answer.     

Photoassimilate partitioning in nodulated soybean I. 11C methodology

An established method using ¹¹C for the in vivo measurement of photoassimilate partitioning within intact plants was applied to the characterization of partitioning of photoassimilate to soybean nodules. The method describes partitioning in terms of the magnitude and stability of partitioning flows, i.e. sink 'activity' and 'priority', and the transit time of tracer to a given sink. Leaflet labelling with ¹¹CO2 was recommended over whole shoot labelling to allow information on transport properties of the shoot to be acquired. The assumptions inherent in the method, that labelled and unlabelled photoassimilate in passage within the stem to the root system were well mixed and that tracer flow is unidirectional between source and sink (nodule), were validated. Tracer was re-exported from root to shoot, but this re-export process did not invalidate the assumption of unidirectional flow because the transit time of the re-export process was long relative to the half-life of the isotope. The transit time of tracer between entry to, and respiration from, the root system was also long (>60 min) relative to the half-life of the isotope. However, a significant fraction of tracer entering the root system was respired (c. 10% within 200 min), mainly by nodules (37% of tracer entering a nodule cluster was respired with 200 min). Therefore root-respired tracer was trapped and attributed to the nodule in partitioning calculations. A case study is presented using the method to assess changes in partitioning to nodules following treatment of the root system with nitrate, highlighting the limitation to this method of ontogenetic changes in the pattern of export from the load leaflet.     

Polyclonal activation of rat B cells. II. Dextran sulfate as a cofactor in mitogen-induced and antigen-induced differentiation of rat B lymphocytes

Salmonella typhimurium mitogen (STM) is a polyclonal activator of rat B lymphocytes, triggering them to proliferate, but not differentiate, to antibody-secreting cells. When lymphokines in the form of a supernatant from Con A-stimulated splenocytes (CAS) are added to B cell cultures activated by STM, only a small number of cells are driven to differentiate. Only with the addition of a third signal provided by the polyanionic polysaccharide dextran sulfate (DXS) is significant rat B cell differentiation observed. In this study, we have shown that this requirement for DXS is not unique to the STM mitogen. LPS, Staphylococcus aureus Cowan I-fixed cells, and anti-Ig antibody all induced rat B cell proliferation with little differentiation, even in the presence of CAS. DXS was necessary to induce differentiation in all cultures costimulated with mitogen and CAS. The requirement for DXS for optimal B cell differentiation is also observed with other lymphokine preparations such as the supernatants from PMA-stimulated EL-4 cells and PHA-stimulated human T cells. Furthermore, this augmentative effect of DXS in rat B cell differentiation was not confined to polyclonal activation systems. Ag-specific IgG secretion was also increased when DXS was added to Ag and CAS costimulated cultures of B cells harvested from the draining lymph nodes of rats immunized with DNP-keyhole limpet hemocyanin. Within the polyclonal activation system, a method of staged additions of STM, DXS, and CAS to B cell cultures was used to investigate the role of DXS during B cell differentiation. Optimal differentiation occurred only when DXS was present in the B cell cultures in conjunction with CAS. The augmentation in differentiation seen with DXS did not appear to be due to the recruitment of an additional CAS-responsive B cell subset, because cycling, low density B cell blasts showed large increases in IgM secretion with subsequent exposure to DXS and CAS. These studies suggest tha DXS acts as a cofactor to various differentiation factors, augmenting polyclonal and Ag-specific rat B cell differentiation. The relevance of DXS to in vivo immune responses is discussed.