Complex subcellular distributions of enzymatic markers in intestinal epithelial cells

Summary Current procedures for isolating intestinal epithelial cell surface and intracellular membranes are based on the assumption that each organelle is marked by some unique constitutent. This assumption seemed inconsistent with the dynamic picture of subcellular organization emerging from studies of membrane turnover and recycling. Therefore, we have designed an alternative fractionation which is independent ofa priori marker assignments. We subjected mucosal homogenates to a sequence of separations based on sedimentation coefficient, equilibrium density, and partitioning in aqueous polymer twophase systems. The resulting distributions of protein and enzymatic markers define a total of 17 physically and biochemically distinct membrane populations. Among these are: basal-lateral membranes, with Na,K-ATPase enriched 21-fold; brush-border membranes, with alkaline phosphatase enriched as much as 38-fold; two populations apparently derived from the endoplasmic reticulum; a series of five populations believed to have been derived from the Golgi complex; and a series of five acid phosphatase-rich populations which we cannot identify unequivocally. Each of the five enzymatic markers we have followed is associated with a multiplicity of membrane populations. Basallateral, endoplasmic reticulum, and Golgi membranes contain alkaline phosphatase at the same specific activity as the initial homogenate. Similarly, Na,K-ATPase appears to be associated branes at specific activities two-to seven-fold that of the initial homogenate.     

Large-scale patterns in morphological diversity and species assemblages in Neotropical Triatominae (Heteroptera: Reduviidae)

We analysed the spatial variation in morphological diversity (MDiv) and species richness (SR) for 91 species of Neotropical Triatominae to determine the ecological relationships between SR and MDiv and to explore the roles that climate, productivity, environmental heterogeneity and the presence of biomes and rivers may play in the structuring of species assemblages. For each 110 km x 110 km-cell on a grid map of America, we determined the number of species (SR) and estimated the mean Gower index (MDiv) based on 12 morphological attributes. We performed bootstrapping analyses of species assemblages to identify whether those assemblages were more similar or dissimilar in their morphology than expected by chance. We applied a multi-model selection procedure and spatial explicit analyses to account for the association of diversity-environment relationships. MDiv and SR both showed a latitudinal gradient, although each peaked at different locations and were thus not strictly spatially congruent. SR decreased with temperature variability and MDiv increased with mean temperature, suggesting a predominant role for ambient energy in determining Triatominae diversity. Species that were more similar than expected by chance co-occurred near the limits of the Triatominae distribution in association with changes in environmental variables. Environmental filtering may underlie the structuring of species assemblages near their distributional limits.     

Global 5-Hydroxymethylcytosine Levels Are Profoundly Reduced in Multiple Genitourinary Malignancies

Solid tumors are characterized by a plethora of epigenetic changes. In particular, patterns methylation of cytosines at the 5-position (5mC) in the context of CpGs are frequently altered in tumors. Recent evidence suggests that 5mC can get converted to 5-hydroxylmethylcytosine (5hmC) in an enzymatic process involving ten eleven translocation (TET) protein family members, and this process appears to be important in facilitating plasticity of cytosine methylation. Here we evaluated the global levels of 5hmC using a validated immunohistochemical staining method in a large series of clear cell renal cell carcinoma (n = 111), urothelial cell carcinoma (n = 55) and testicular germ cell tumors (n = 84) and matched adjacent benign tissues. Whereas tumor-adjacent benign tissues were mostly characterized by high levels of 5hmC, renal cell carcinoma and urothelial cell carcinoma showed dramatically reduced staining for 5hmC. 5hmC levels were low in both primary tumors and metastases of clear cell renal cell carcinoma and showed no association with disease outcomes. In normal testis, robust 5hmC staining was only observed in stroma and Sertoli cells. Seminoma showed greatly reduced 5hmC immunolabeling, whereas differentiated teratoma, embryonal and yolk sack tumors exhibited high 5hmC levels. The substantial tumor specific loss of 5hmC, particularly in clear cell renal cell carcinoma and urothelial cell carcinoma, suggests that alterations in pathways involved in establishing and maintaining 5hmC levels might be very common in cancer and could potentially be exploited for diagnosis and treatment.     

Phosphoinositides,ezrin/moesin,and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.     

Activation of TGF-beta by Leishmania chagasi: importance for parasite survival in macrophages

TGF-beta is a potent regulatory cytokine that suppresses expression of inducible NO synthase and IFN-gamma, and suppresses Th1 and Th2 cell development. We examined whether functionally active TGF-beta is present in the local environment surrounding the invading protozoan Leishmania chagasi. Our prior data showed that TGF-beta levels are significantly increased in L. chagasi-infected mice. In the current study, we found TGF-beta was also abundant in bone marrows of humans with acute visceral leishmaniasis but not in those of uninfected controls. Furthermore, L. chagasi infection caused an increase in biologically active TGF-beta in human macrophage cultures without changing the total TGF-beta. Therefore, we investigated the means through which leishmania could augment activated but not total TGF-beta. Incubation of latent TGF-beta with Leishmania sp. promastigotes caused active TGF-beta to be released from the latent complex. In contrast, the nonpathogenic protozoan Crithidia fasciculata could not activate TGF-beta. TGF-beta activation by leishmania was prevented by inhibitors of cysteine proteases and by the specific cathepsin B inhibitor CA074. Physiologic concentrations of TGF-beta inhibited killing of intracellular L. chagasi in macrophages, although the phagocytosis-induced respiratory burst remained intact. In contrast, supraphysiologic concentrations of TGF-beta had no effect on parasite survival. We hypothesize that the combined effect of abundant TGF-beta stores at extracellular sites during infection, and the ability of the parasite to activate TGF-beta in its local environment, leads to high levels of active TGF-beta in the vicinity of the infected macrophage. Locally activated TGF-beta could, in turn, enhance parasite survival through its effects on innate and adaptive immune responses.     

Activation of group IV cytosolic phospholipase A2 in human eosinophils by phosphoinositide 3-kinase through a mitogen-activated protein kinase-independent pathway

Activation of group IV cytosolic phospholipase A(2) (gIV-PLA(2)) is the essential first step in the synthesis of inflammatory eicosanoids and in integrin-mediated adhesion of leukocytes. Prior investigations have demonstrated that phosphorylation of gIV-PLA(2) results from activation of at least two isoforms of mitogen-activated protein kinase (MAPK). We investigated the potential role of phosphoinositide 3-kinase (PI3K) in the activation of gIV-PLA(2) and the hydrolysis of membrane phosphatidylcholine in fMLP-stimulated human blood eosinophils. Transduction into eosinophils of Deltap85, a dominant negative form of class IA PI3K adaptor subunit, fused to an HIV-TAT protein transduction domain (TAT-Deltap85) concentration dependently inhibited fMLP-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. FMLP caused increased arachidonic acid (AA) release and secretion of leukotriene C(4) (LTC(4)). TAT-Deltap85 and LY294002, a PI3K inhibitor, blocked the phosphorylation of gIV-PLA(2) at Ser(505) caused by fMLP, thus inhibiting gIV-PLA(2) hydrolysis and production of AA and LTC(4) in eosinophils. FMLP also caused extracellular signal-related kinases 1 and 2 and p38 MAPK phosphorylation in eosinophils; however, neither phosphorylation of extracellular signal-related kinases 1 and 2 nor p38 was inhibited by TAT-Deltap85 or LY294002. Inhibition of 1) p70 S6 kinase by rapamycin, 2) protein kinase B by Akt inhibitor, or 3) protein kinase C by Ro-31-8220, the potential downstream targets of PI3K for activation of gIV-PLA(2), had no effect on AA release or LTC(4) secretion caused by fMLP. We find that PI3K is required for gIV-PLA(2) activation and hydrolytic production of AA in activated eosinophils. Our data suggest that this essential PI3K independently activates gIV-PLA(2) through a pathway that does not involve MAPK.     

Identification of an equilibrium intermediate in the unfolding process of galectin-1, which retains its carbohydrate-binding specificity

The unfolding process of galectin-1 (Gal-1) in the presence of a denaturing agent was examined using fluorescence and far-UV circular dichroism (CD) spectroscopy determinations, and was found to be completely reversible. The data showed that the transitions of guanidine hydrochloride (GdnHCl)-induced lectin unfolding, in the absence of ligand, were biphasic in nature, clearly showing the existence of at least one stable intermediate. On the other hand, the unfolding in the presence of disaccharide yielded data that could fit very well to a two-state model, indicating a stabilizing effect of the ligand. The folding intermediate was further characterized by size exclusion chromatography, near-UV CD and anilinonaphtalene sulfonate binding, and shown to belong to the molten globule type. Strikingly, this intermediate retained its carbohydrate-binding specificity, as evidenced by the tryptophan fluorescence changes detected upon its interaction with lactose.     

Group V phospholipase A2 induces leukotriene biosynthesis in human neutrophils through the activation of group IVA phospholipase A2

We reported previously that exogenously added human group V phospholipase A(2) (hVPLA(2)) could elicit leukotriene B(4) (LTB(4)) biosynthesis in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism of the hVPLA(2)-induced LTB(4) biosynthesis in neutrophils, we thoroughly examined the effects of hVPLA(2) and their lipid products on the activity of group IVA cytosolic PLA(2) (cPLA(2)) and LTB(4) biosynthesis under different conditions. As low as 1 nm exogenous hVPLA(2) was able to induce the release of arachidonic acid (AA) and LTB(4). Typically, AA and LTB(4) were released in two phases, which were synchronized with a rise in intracellular calcium concentration ([Ca(2+)](i)) near the perinuclear region and cPLA(2) phosphorylation. A cellular PLA(2) assay showed that hVPLA(2) acted primarily on the outer plasma membrane, liberating fatty acids and lysophosphatidylcholine (lyso-PC), whereas cPLA(2) acted on the perinuclear membrane. Lyso-PC and polyunsaturated fatty acids including AA activated cPLA(2) and 5-lipoxygenase by increasing [Ca(2+)](i) and inducing cPLA(2) phosphorylation, which then led to LTB(4) biosynthesis. The delayed phase was triggered by the binding of secreted LTB(4) to the cell surface LTB(4) receptor, which resulted in a rise in [Ca(2+)](i) and cPLA(2) phosphorylation through the activation of mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2. These results indicate that a main role of exogenous hVPLA(2) in neutrophil activation and LTB(4) biosynthesis is to activate cPLA(2) and 5-lipoxygenase primarily by liberating from the outer plasma membrane lyso-PC that induces [Ca(2+)](i) increase and cPLA(2) phosphorylation and that hVPLA(2)-induced LTB(4) production is augmented by the positive feedback activation of cPLA(2) by LTB(4).