Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcγ receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcγ receptor-mediated phagocytosis to enhance the innate immune response.Receptors that interact with the constant region of IgG (FcγR)⁴ mediate the recognition and elimination of soluble immune complexes and particles coated (opsonized) with immunoglobulins. Clustering of FcγR on the surface of leukocytes upon attachment to multivalent ligands induces their activation and subsequent internalization. Soluble immune complexes are internalized by endocytosis, a clathrin- and ubiquitylation-dependent process (1). In contrast, large, particulate complexes like IgG-coated pathogens are ingested by phagocytosis, a process that is contingent on extensive actin polymerization that drives the extension of pseudopods (2). In parallel with the internalization of the opsonized targets, cross-linking of phagocytic receptors triggers a variety of other responses that are essential components of the innate immune response. These include degranulation, activation of the respiratory burst, and the synthesis and release of multiple inflammatory agents (3, 4).Like T and B cell receptors, FcγR possesses an immunoreceptor tyrosine-based activation motif that is critical for signal transduction (3, 4). Upon receptor clustering, tyrosyl residues of the immunoreceptor tyrosine-based activation motif are phosphorylated by Src family kinases, thereby generating a docking site for Syk, a tyrosine kinase of the ZAP70 family (3, 4). The recruitment and activation of Syk in turn initiates a cascade of events that include activation of Tec family kinases, Rho- and ARF-family GTPases, phosphatidylinositol 3-kinase, phospholipase Cγ (PLCγ), and a multitude of additional effectors that together remodel the underlying cytoskeleton, culminating in internalization of the bound particle (5, 6).Phosphoinositide metabolism is thought to be critical for FcγR-induced phagocytosis (7, 8). Highly localized and very dynamic phosphoinositide changes have been observed at sites of phagocytosis: phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) undergoes a transient accumulation at the phagocytic cup, which is rapidly superseded by its complete elimination from the nascent phagosome (7). The secondary disappearance of PtdIns(4,5)P₂ is attributable in part to the localized generation of phosphatidylinositol 3,4,5-trisphosphate, which has been reported to accumulate at sites of phagocytosis (9). Activation of PLCγ is also believed to contribute to the acute disappearance of PtdIns(4,5)P₂ in nascent phagosomes. Indeed, the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate has been detected by chemical means during FcγR-evoked particle ingestion (10, 11). Moreover, imaging experiments revealed that DAG appears at the time and at the precise site where PtdIns(4,5)P₂ is consumed (7).Two lines of evidence suggest that the DAG generated upon engagement of phagocytic receptors modulates particle engulfment. First, antagonists of PLC severely impair phagocytosis by macrophages (7, 12). This inhibition is not mimicked by preventing the associated [Ca²⁺] transient, suggesting that DAG, and not inositol 1,4,5-trisphosphate, is the crucial product of the PLC (13). Second, the addition of exogenous DAG or phorbol esters, which mimic the actions of endogenous DAG, augment phagocytosis (14, 15).Selective recognition of DAG by cellular ligands is generally mediated by specific regions of its target proteins, called C1 domains (16). Proteins bearing C1 domains include, most notably, members of the classical and novel families of protein kinase C (PKC), making them suitable candidates to account for the DAG dependence of phagocytosis. Indeed, PKCα, a classical isoform, and PKCϵ and PKCδ, both novel isoforms, are recruited to phagosomes (12, 15, 17, 18). Although the role of the various PKC isoforms in particle engulfment has been equivocal over the years, Cheeseman et al. (12) convincingly demonstrated that PKCϵ contributes to particle uptake in a PLC- and DAG-dependent manner.PKCs are not the sole proteins bearing DAG-binding C1 domains. Similar domains are also found in several other proteins, including members of the RasGRP family, chimaerins, and Munc-13 (19–21). One or more of these could contribute to the complex set of responses elicited by FcγR-induced DAG production. The RasGRP proteins are a class of exchange factors for the Ras/Rap family of GTPases (22). There are four RasGRP proteins (RasGRP1 to -4), and emerging evidence has implicated RasGRP1 and RasGRP3 in T and B cell receptor signaling (23–27).The possible role of DAG-mediated signaling pathways other than PKC in phagocytosis and the subsequent inflammatory response has not been explored. Here, we provide evidence that DAG stimulates Ras and Rap1 at sites of phagocytosis, probably through RasGRPs. Last, the functional consequences of Ras and Rap1 activation were analyzed.     

Acute knockdown of uncoupling protein-2 increases uncoupling via the adenine nucleotide transporter and decreases oxidative stress in diabetic kidneys

Increased O(2) metabolism resulting in chronic hypoxia is common in models of endstage renal disease. Mitochondrial uncoupling increases O(2) consumption but the ensuing reduction in mitochondrial membrane potential may limit excessive oxidative stress. The present study addressed the hypothesis that mitochondrial uncoupling regulates mitochondria function and oxidative stress in the diabetic kidney. Isolated mitochondria from kidney cortex of control and streptozotocin-induced diabetic rats were studied before and after siRNA knockdown of uncoupling protein-2 (UCP-2). Diabetes resulted in increased UCP-2 protein expression and UCP-2-mediated uncoupling, but normal mitochondria membrane potential. This uncoupling was inhibited by GDP, which also increased the membrane potential. siRNA reduced UCP-2 protein expression in controls and diabetics (-30-50%), but paradoxically further increased uncoupling and markedly reduced the membrane potential. This siRNA mediated uncoupling was unaffected by GDP but was blocked by ADP and carboxyatractylate (CAT). Mitochondria membrane potential after UCP-2 siRNA was unaffected by GDP but increased by CAT. This demonstrated that further increased mitochondria uncoupling after siRNA towards UCP-2 is mediated through the adenine nucleotide transporter (ANT). The increased oxidative stress in the diabetic kidney, manifested as increased thiobarbituric acids, was reduced by knocking down UCP-2 whereas whole-body oxidative stress, manifested as increased circulating malondialdehyde, remained unaffected. All parameters investigated were unaffected by scrambled siRNA. In conclusion, mitochondrial uncoupling via UCP-2 regulates mitochondria membrane potential in diabetes. However, blockade of the diabetes-induced upregulation of UCP- 2 results in excessive uncoupling and reduced oxidative stress in the kidney via activation of ANT.     

Quantitative analysis of venation patterns of Arabidopsis leaves by supervised image analysis

The study of transgenic Arabidopsis lines with altered vascular patterns has revealed key players in the venation process, but details of the vascularization process are still unclear, partly because most lines have only been assessed qualitatively. Therefore, quantitative analyses are required to identify subtle perturbations in the pattern and to test dynamic modeling hypotheses using biological measurements. We developed an online framework, designated Leaf Image Analysis Interface (LIMANI), in which venation patterns are automatically segmented and measured on dark-field images. Image segmentation may be manually corrected through use of an interactive interface, allowing supervision and rectification steps in the automated image analysis pipeline and ensuring high-fidelity analysis. This online approach is advantageous for the user in terms of installation, software updates, computer load and data storage. The framework was used to study vascular differentiation during leaf development and to analyze the venation pattern in transgenic lines with contrasting cellular and leaf size traits. The results show the evolution of vascular traits during leaf development, suggest a self-organizing mechanism for leaf venation patterning, and reveal a tight balance between the number of end-points and branching points within the leaf vascular network that does not depend on the leaf developmental stage and cellular content, but on the leaf position on the rosette. These findings indicate that development of LIMANI improves understanding of the interaction between vascular patterning and leaf growth.     

Fungicidal effects of chemical disinfectants, UV light, desiccation and heat on the amphibian chytrid Batrachochytrium dendrobatidis

The efficacy of a number of disinfection treatments was tested on in vitro cultures of the fungus Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis in amphibians. The aim was to evaluate the fungicidal effects of chemical disinfectants, sterilising ultraviolet (UV) light, heat and desiccation, using methods that were feasible for either disinfection in the field, in amphibian husbandry or in the laboratory. The chemical disinfectants tested were: sodium chloride, household bleach (active ingredient: sodium hypochlorite), potassium permanganate, formaldehyde solution, Path-X agricultural disinfectant (active ingredient: didecyl dimethyl ammonium chloride, DDAC), quaternary ammonium compound 128 (DDAC), Dithane, Virkon, ethanol and benzalkonium chloride. In 2 series of experiments using separate isolates of B. dendrobatidis, the fungicidal effect was evaluated for various time periods and at a range of chemical concentrations. The end point measured was death of 100% of zoospores and zoosporangia. Nearly all chemical disinfectants resulted in 100%, mortality for at least one of the concentrations tested. However, concentration and time of exposure was critical for most chemicals. Exposure to 70% ethanol, 1 mg Virkon ml(-1) or 1 mg benzalkonium chloride ml(-1) resulted in death of all zoosporangia after 20 s. The most effective products for field use were Path-X and the quaternary ammonium compound 128, which can be used at dilutions containing low levels (e.g. 0.012 or 0.008%, respectively) of the active compound didecyl dimethyl ammonium chloride. Bleach, containing the active ingredient sodium hypochlorite, was effective at concentrations of 1% sodium hypochlorite and above. Cultures did not survive complete drying, which occurred after <3 h at room temperature. B. dendrobatidis was sensitive to heating, and within 4 h at 37 degrees C, 30 min at 47 degrees C and 5 min at 60 degrees C, 100% mortality occurred. UV light (at 1000 mW m(-2) with a wavelength of 254 nm) was ineffective at killing B. dendrobatidis in culture.     

Ascidians as a vertebrate-like model organism for physiological studies of Rho GTPase signaling

GTPases of the Rho family are evolutionarily conserved proteins that control cell shape dynamics during physiological processes as diverse as cell migration and polarity, axon outgrowth and guidance, apoptosis and phagocytosis. In mammals, 18 Rho proteins are distributed in 7 subfamilies. Rho, Rac and Cdc42 are the best-characterized ones, benefiting from the use of worm and drosophila, which only express these 3 subfamilies. An additional model would therefore help understand the physiological role of other mammalian subfamilies. We identified in genome databases the complete Rho family of two ascidians, Ciona intestinalis and Ciona savignyi, and showed that these families contain single ancestors of most mammalian Rho subfamilies. In Ciona intestinalis, all Rho genes are expressed and display specific developmental variations of mRNA expression during tadpole formation. Although C. intestinalis expresses five additional Rac compared to the closely related Ciona savignyi, only two appeared fully active in functional assays. Last, we identified in Ciona intestinalis database more than 50 Rho regulators (RhoGEFs and RhoGAPs) and 20 effector targets, whose analysis further supports the notion that Rho signaling components are of comparable complexity in mammals and ascidians. Since the tadpole of ascidians combines vertebrate-like developmental features with reduced cell number, particularly adapted to evolutionary and developmental biology studies, our data advocate this model for physiological studies of Rho signaling pathways.     

Regulation of nitric oxide-dependent vasodilation in coronary arteries of estrogen receptor-alpha-deficient mice

Estrogen has been shown to increase endothelium-dependent vasodilation and expression of endothelial nitric oxide (NO) synthase (eNOS); however, the role of estrogen receptors in mediating estrogen effects on endothelial function remains to be elucidated. The purpose of this study was to test the hypothesis that estrogen modulates NO-dependent vasodilation of coronary arteries through its action on estrogen receptor-alpha (ER-alpha) to increase protein levels of eNOS and Cu/Zn superoxide dismutase (SOD-1). Vasodilation to acetylcholine (ACh) and sodium nitroprusside was assessed in isolated coronary arteries from intact and ovariectomized female wild-type (WT) and ER-alpha knockout (ERalphaKO) mice. Protein levels for eNOS and SOD-1 were also evaluated. Vasodilation to ACh was not significantly altered in ERalphaKO mice compared with WT mice. Ovariectomy reduced responsiveness to ACh in ERalphaKO mice but not WT mice. Responses to sodium nitroprusside were not altered by disruption of ER-alpha or by ovariectomy. Supplementation with estrogen restored ACh-induced vasodilation in ovariectomized ERalphaKO mice. eNOS protein was reduced in ERalphaKO mice compared with WT mice. Ovariectomy caused a further reduction in eNOS protein in ERalphaKO mice, but this reduction was reversed by estrogen treatment. SOD-1 protein levels were increased by disruption of ER-alpha. Ovariectomy reduced SOD-1 protein in ERalphaKO mice, but this reduction was partially reversed by estrogen replacement. These results suggest that estrogen modulation of eNOS protein content is mediated in part through ER-alpha. NO-dependent responses are preserved in ERalphaKO mice, possibly through increased SOD-1 expression and enhanced bioavailability of NO.     

Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus

Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells.     

Rh2, a compound extracted from ginseng, hypersensitizes multidrug-resistant tumor cells to chemotherapy

Rh2 is a ginsenoside extracted from ginseng that has drawn attention in a few laboratories in Asian countries because of its potential tumor-inhibitory effect. In the present study, we tested Rh2 on many tumor-cell lines for its effects on cell proliferation, induction of apoptosis, and potential interaction with conventional chemotherapy agents. Our results showed that Rh2 inhibited cell growth by G1 arrest at low concentrations and induced apoptosis at high concentrations in a variety of tumor-cell lines, possibly through activation of caspases. The growth arrest and apoptosis may be mediated by 2 separate mechanisms. Apoptosis is not dependent on expression of the wild-type p53 nor the caspase 3. In addition, the apoptosis induced by Rh2 was mediated through glucocorticoid receptors. Most interestingly, Rh2 can act either additively or synergistically with chemotherapy drugs on cancer cells. Particularly, it hypersensitized multidrug-resistant breast cancer cells to paclitaxel. These results suggest that Rh2 possesses strong tumor-inhibiting properties, and potentially can be used in treatments for multidrug-resistant cancers, especially when it is used in combination with conventional chemotherapy agents.