Metalloprotease meprin beta in rat kidney: glomerular localization and differential expression in glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprin beta in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprin beta in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprin beta expression. The glomerular meprin beta expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprin beta staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprin beta is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprin beta in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprin beta in this form of glomerulonephritis.

Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle

The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland biopsies and blood samples were taken from 10 animals at 7 stages of the pregnancy-lactation cycle. Expression levels of the specific mRNAs were determined by quantitative reverse transcription-PCR, whereas ABCA1 was localized by immunohistochemistry. Blood serum metabolites were determined by common enzymatic chemistries. Elevated mRNA profiles of ABCA1 and ABCA7 were found during DP as compared with lactation and were inversely associated with blood cholesterol levels. Elevated levels of ABCG2, NPC1, SREBP1, SREBP2, LXR alpha, and PPAR gamma were found postpartum, whereas ABCG1 did not differ between the functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis.

In vivo nuclear translocation of mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of corticosteroids along the distal tubule

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that inactivates 11β-hydroxy glucocorticoids. High expression levels of 11β-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11β-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11β-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.

Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development

Normal placentation involves the development of an utero-placental circulation following the migration of the extravillous cytotrophoblasts into the decidua and invasion of the spiral arteries, which are thereby transformed into large vessels of low resistance. Given the documented role of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 in the establishment of the embryonal vascular network, we hypothesized that these molecules are also instrumental in the development of the human placenta. Monitoring the expression during placental development revealed that in first trimester and term placentae both molecules are equally expressed at the RNA level. In contrast, the protein levels were significantly reduced during gestation. Immunohistochemistry revealed a distinct localization of the EphB4 and ephrin-B2 proteins. EphB4 was predominantly expressed in the villous syncytial trophoblast layer and in a subset of intravillous capillaries. Prominent expression was also observed in the extravillous cytotrophoblast giant cells. In contrast, ephrin-B2 expression was detected in the villous cytotrophoblast and syncytial trophoblast cell layers, as well as initially in all intravillous capillaries. Strong expression was also observed in extravillous anchoring cytotrophoblast cells. Hypoxia is a major inducer of placental development. In vitro studies employing trophoblast-derived cell lines revealed that predominantly ephrin-B2 expression is induced by hypoxia, however, in an Hif-1alpha independent manner. These experiments suggest that EphB4 and ephrin-B2 are instrumental in the establishment of a functional placental structure and of the utero-placental circulation.

Plasmodium berghei MAPK1 displays differential and dynamic subcellular localizations during liver stage development

Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite's nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization.

The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns

Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K-m = 0.1-0.3 mM), lower for Pro (K-m = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K-m = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.

A Time-based Passive Source Localization System for Narrow-band Signal

Time-based indoor localization has been investigated for several years but the accuracy of existing solutions is limited by several factors, e.g., imperfect synchronization, signal bandwidth and indoor environment. In this paper, we compare two time-based localization algorithms for narrow-band signals, i.e., multilateration and fingerprinting. First, we develop a new Linear Least Square (LLS) algorithm for Differential Time Difference Of Arrival (DTDOA). Second, fingerprinting is among the most successful approaches used for indoor localization and typically relies on the collection of measurements on signal strength over the area of interest. We propose an alternative by constructing fingerprints of fine-grained time information of the radio signal. We offer comprehensive analytical discussions on the feasibility of the approaches, which are backed up by evaluations in a software defined radio based IEEE 802.15.4 testbed. Our work contributes to research on localization with narrow-band signals. The results show that our proposed DTDOA-based LLS algorithm obviously improves the localization accuracy compared to traditional TDOA-based LLS algorithm but the accuracy is still limited because of the complex indoor environment. Furthermore, we show that time-based fingerprinting is a promising alternative to power-based fingerprinting.

Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans.

The adjustment of X-linked gene expression to the X chromosome copy number (dosage compensation [DC]) has been widely studied as a model of chromosome-wide gene regulation. In Caenorhabditis elegans, DC is achieved by twofold down-regulation of gene expression from both Xs in hermaphrodites. We show that in males, the single X chromosome interacts with nuclear pore proteins, while in hermaphrodites, the DC complex (DCC) impairs this interaction and alters X localization. Our results put forward a structural model of DC in which X-specific sequences locate the X chromosome in transcriptionally active domains in males, while the DCC prevents this in hermaphrodites.