Overexpression of a mutant form of EhRabA, a unique Rab GTPase of Entamoeba histolytica, alters endoplasmic reticulum morphology and localization of the Gal/GalNAc adherence lectin.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/N-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica.

Entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components.

Adhesion is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Lipid rafts, cholesterol-rich domains, function in compartmentalization of cellular processes. In E. histolytica, rafts participate in parasite-host cell interactions; however, their role in parasite-host extracellular matrix (ECM) interactions has not been explored. Disruption of rafts with a cholesterol extracting agent, methyl-beta-cyclodextrin (MbetaCD), resulted in inhibition of adhesion to collagen, and to a lesser extent, to fibronectin. Replenishment of cholesterol in MbetaCD-treated cells, using a lipoprotein-cholesterol concentrate, restored adhesion to collagen. Confocal microscopy revealed enrichment of rafts at parasite-ECM interfaces. A raft-resident adhesin, the galactose/N-acetylgalactosamine-inhibitable lectin, mediates interaction to host cells by binding to galactose or N-acetylgalactosamine moieties on host glycoproteins. In this study, galactose inhibited adhesion to collagen, but not to fibronectin. Together these data suggest that rafts participate in E. histolytica-ECM interactions and that raft-associated Gal/GalNAc lectin may serve as a collagen receptor.

Entamoeba histolytica: comparison of the role of receptors and filamentous actin among various endocytic processes.

Entamoeba histolytica is the causative agent of amoebic dysentery. Uptake of iron is critical for E. histolytica growth and iron-bound human transferrin (holo-transferrin) has been shown to serve as an iron source in vitro. Although a transferrin-binding protein has been identified in E. histolytica, the mechanism by which this iron source is taken up by this pathogen is not well understood. To gain insight into this process, the uptake of fluorescent-dextran, -holo-transferrin, and human red blood cells (hRBCs) was compared. Both dextran and transferrin were taken up in an apparent receptor-independent fashion as compared to hRBCs, which were taken up in a receptor-mediated fashion. Interestingly, the uptake of FITC-dextran and FITC-holo-transferrin differentially relied on an intact actin cytoskeleton suggesting that their internalization routes may be regulated independently.

Entamoeba histolytica: FYVE-finger domains, phosphatidylinositol 3-phosphate biosensors, associate with phagosomes but not fluid filled endosomes.

Endocytosis is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Although a number of E. histolytica proteins that regulate this process have been identified, less is known about the role of lipids. In other systems, phosphatidylinositol 3-phosphate (PI3P), a product of phosphatidylinositol 3-kinase (PI 3-kinase), has been shown to be required for endocytosis. FYVE-finger domains are protein motifs that bind specifically to PI3P. Using a PI3P biosensor consisting of glutathione-S-transferase (GST) fused to two tandem FYVE-finger domains, we have localized PI3P to phagosomes but not fluid-phase pinosomes in E. histolytica, suggesting a role for PI3P in phagocytosis. Treatment of cells with PI 3-kinase inhibitors impaired GST-2 x FYVE-phagosome association supporting the authenticity of the biosensor staining. However, treatment with PI 3-kinase inhibitors did not inhibit E. histolytica-particle interaction, indicating that PI3P is not required for the initial step, but is required for subsequent steps of phagocytosis.

[Influence of endoleaks on aneurysm volume and hemodynamics after endovascular aneurysm repair]. / Einfluss von Typ-II-Endoleaks auf die Grössenänderung und Hämodynamik experimenteller Aortenaneurysmen nach endovaskulärer Therapie.

PURPOSE: To evaluate the aneurysm volume and the intra-aneurysmatic pressure and maximal pressure pulse (dp/dtmax) in completely excluded aneurysms and cases with endoleaks. MATERIALS AND METHODS: In 36 mongrel dogs, experimental autologous aneurysms were treated with stent-grafts. All aortic side branches were ligated in 18 cases (group I) but were preserved in group II (n = 18). Aneurysm volumes were calculated from CT scans before and after intervention, and from follow-up CT scans at 1 week, 6 weeks and 6 months. Finally, for hemodynamic measurements, manometer-tipped catheters were introduced into the excluded aneurysm sac (group I and II), selectively in endoleaks (group II), and intraluminally for aortic reference measurement. Systemic hypertension was induced by volume load and pharmacologic stress. Pressure curves and dp/dt were simultaneously recorded and the ratios of aneurysm pressure to systemic reference pressure calculated. RESULTS: At follow-up, type-II. endoleaks were excluded in all cases of group I by selective angiography. In contrast, endoleaks were evident in all cases of group II. Volumetric analysis of the aneurysms showed a benefit for group I with an improved aneurysm shrinkage: DeltaVolume + 0.08 %, - 1.62 % and -9.76 % at 1 week, 6 weeks and 6 months follow-up (median, group I), compared to + 1.43 %, + 0.67 %, and - 4.04 % (group II), p < 0.05. In case of complete aneurysm exclusion the ratio of systolic aneurysm pressure to systemic reference pressure was 0.662, 0.575 and 0.385 (median) at 1 week, 6 weeks and 6 months. The corresponding dp/dtmax ratios were 0.12, 0.07 and 0.04, respectively. However, within endoleaks selective measurements showed significantly increased pressure load: the ratios of systolic endoleak pressure to systemic reference pressure and the corresponding ratios for dp/dtmax were 0.882 and 0.913 (median), respectively. These hemodynamic findings were linear from hypotension, physiologic blood pressure to hypertension. CONCLUSION: Occlusion of all aortic side branches of an aneurysm prior to stent-grafting reduces endoleaks and promotes aneurysm shrinkage. Complete aneurysm exclusion significantly reduces systolic pressure and dp/dt max. In contrast, endoleaks showed nearly systemic pressure load and undamped pulsatility.

A hidden pseudogap under the 'dome' of superconductivity in electron-doped high-temperature superconductors.

The ground state of superconductors is characterized by the long-range order of condensed Cooper pairs: this is the only order present in conventional superconductors. The high-transition-temperature (high-T(c)) superconductors, in contrast, exhibit more complex phase behaviour, which might indicate the presence of other competing ground states. For example, the pseudogap--a suppression of the accessible electronic states at the Fermi level in the normal state of high-T(c) superconductors-has been interpreted as either a precursor to superconductivity or as tracer of a nearby ground state that can be separated from the superconducting state by a quantum critical point. Here we report the existence of a second order parameter hidden within the superconducting phase of the underdoped (electron-doped) high-T(c) superconductor Pr2-xCe(x)CuO4-y and the newly synthesized electron-doped material La2-xCe(x)CuO4-y (ref. 8). The existence of a pseudogap when superconductivity is suppressed excludes precursor superconductivity as its origin. Our observation is consistent with the presence of a (quantum) phase transition at T = 0, which may be a key to understanding high-T(c) superconductivity. This supports the picture that the physics of high-T(c) superconductors is determined by the interplay between competing and coexisting ground states.

Presence of leptin in breast cell lines and breast tumors.

Leptin is the product of the ob gene, reported to be secreted exclusively from adipocytes and thought to control satiety by providing information to the central nervous system. However, the function of leptin appears to be more complex because multiple studies demonstrate its role in hematopoiesis, reproduction, and immunity. In addition, several nonadipose sources of leptin have been reported. The purpose of this study was to examine several breast cancer cell lines and ductal carcinomas of the breast for expression of leptin messenger RNA (mRNA) and protein. For tumor studies, specimens were preassayed for contaminating adipose tissue. Northern blot analyses demonstrated leptin mRNA in several breast cancer cell lines (MCF-7, T47D, and MDA-MB-231), a normal breast epithelial cell line (MCF10A), and four breast tumors. Leptin protein was identified in T47D breast cancer cells by indirect immunofluorescent staining and in samples of the same breast tumors used for Northern studies by enzyme-linked immunosorbent assays (ELISA). This preliminary study suggests that leptin is expressed in malignant epithelial cells of the breast. Further investigation is needed to determine whether this protein plays a role in breast carcinogenesis.

Identification of progesterone receptor in human subcutaneous adipose tissue.

Sex steroids are postulated to play a role in adipose tissue regulation and distribution, because the amount and location of adipose tissue changes during puberty and menopause. Because of the nature of adipose tissue, receptors for the female sex steroids have been difficult to demonstrate. To date, estrogen receptor messenger RNA and protein have been identified in human subcutaneous adipose tissue, but the presence of progesterone receptor (PR) has not been reported. In this study, we demonstrate PR message by Northern blot analysis in RNA isolated from the abdominal subcutaneous adipose tissue of premenopausal women. These preliminary studies revealed that PR messenger RNA levels are higher in the stromal-vascular fraction as opposed to the adipocyte fraction. Western blot analysis demonstrates both PR protein isoforms (human PR-A and human PR-B) in human subcutaneous adipose tissue. Using an enzyme-linked immunosorbent assay, total PR could be quantitated. These studies substantiate that sex steroid receptors are present in human adipose tissue, thereby providing a direct route for regulation of adipose tissue by female sex steroids.

Estrogen regulation of adipose tissue lipoprotein lipase--possible mechanism of body fat distribution.

OBJECTIVE: The purpose of this study was to evaluate the regulation of lipoprotein lipase activity, protein mass, and messenger ribonucleic acid by estradiol. STUDY DESIGN: Premenopausal women not taking exogenous sex steroids had transdermal 17 beta-estradiol and placebo patches placed in the gluteal region during the early follicular phase of the menstrual cycle. Adipose biopsies were performed from beneath the patches. Adipose tissue lipoprotein lipase activity was determined by a radiometric assay, protein mass was determined by enzyme-linked immunosorbent assay, and messenger ribonucleic acid level was determined by Northern analysis. Comparisons between the treated and placebo sides were analyzed by nonparametric statistics. RESULTS: Adipose tissue from beneath the 17 beta-estradiol patch had significantly decreased lipoprotein lipase activity and extracellular protein mass than did adipose tissue from beneath the placebo patch. There was no difference in lipoprotein lipase messenger ribonucleic acid levels. CONCLUSION: Estrogen decreases lipoprotein lipase activity by a posttranscriptional modification of protein levels. A hypothesis of sex steroid regulation of body fat distribution is proposed.