Assessment of in vitro effects of metyrapone on Leydig cell steroidogenesis

Metyrapone, a specific inhibitor of 11beta-hydroxylase inhibits glucocorticoid production and it is used in the diagnosis/treatment of hypercortisolism and also to test the functional integrity of hypothalamo-pituitary-adrenal axis. To assess the impact of glucocorticoid deficiency, this drug is preferred over adrenalectomy, which eliminates all the hormonal secretions of the adrenal cortex and medulla. However, whether metyrapone has any direct effect on the extra-adrenocortical cellular or tissue functions remains to be resolved. Our previous study showed a depressed testicular Leydig cell testosterone production in rats treated with metyrapone. Therefore, the present study was designed to examine the possible direct effect of metyrapone on testicular Leydig cell steroidogenesis in vitro. Leydig cell viability and the reactive oxygen species (ROS) concentration were not altered by any of the concentration of metyrapone tested. The efficacy of Leydig cell testosterone production under basal as well as LH-stimulated condition was not altered by metyrapone treatment. Further, Leydig cellular (14)C-glucose oxidation, the activity and mRNA levels of cytochrome side chain cleavage (P(450)scc), 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were not altered in metyrapone-treated cells. Therefore, it is concluded from the present study that metyrapone has no direct effect on Leydig cell testosterone production and, therefore, changes recorded in the in vivo studies are exclusively due to corticosterone deficiency.     

Versatile catechol dioxygenases in <Emphasis Type="Italic">Sphingobium scionense</Emphasis> WP01<Superscript>T</Superscript>

The objective was to understand the roles of multiple catechol dioxygenases in the type strain Sphingobium scionense WP01^T (Liang and Lloyd-Jones in Int J Syst Evol Microbiol 60:413–416, 2010a) that was isolated from severely contaminated sawmill soil. The dioxygenases were identified by sequencing, examined by determining the substrate specificities of the recombinant enzymes, and by quantifying gene expression following exposure to model priority pollutants. Catechol dioxygenase genes encoding an extradiol xylE and two intradiol dioxygenases catA and clcA that are highly similar to sequences described in other sphingomonads are described in S. scionense WP01^T. The distinct substrate specificities determined for the recombinant enzymes confirm the annotated gene functions and suggest different catabolic roles for each enzyme. The role of the three enzymes was evaluated by analysis of enzyme activity in crude cell extracts from cells grown on meta-toluate, benzoate, biphenyl, naphthalene and phenanthrene which revealed the co-induction of each enzyme by different substrates. This was corroborated by quantifying gene expression when cells were induced by biphenyl, naphthalene and pentachlorophenol. It is concluded that the ClcA and XylE enzymes are recruited in pathways that are involved in the degradation of chlorinated aromatic compounds such as pentachlorophenol, the XylE and ClcA enzymes will also play a role in degradation pathways that produce alkylcatechols, while the three enzymes ClcA, XylE and CatA will be simultaneously involved in pathways that generate catechol as a degradation pathway intermediate.     

Luminescence studies on RbI:Tb3+ crystals.

Optical absorption, photoluminescence, thermoluminescence (TL) and photostimulated luminescence (PSL) studies on RbI:Tb(3+) crystals irradiated with gamma-rays is reported. Photoluminescence of these crystals exhibits characteristic Tb(3+) emissions, due to transitions from the (5)D(3) and (5)D(4) levels to various levels of the (7)F septet. On F-bleaching the gamma-irradiated crystals, Z(3) centres are observed. The TL glow curve indicates a two-step thermal annihilation process for the radiatively created defects. The presence of the characteristic emissions due to terbium ions in the photostimulation at the F-band, and TL emissions under both glow peaks, confirm the participation of Tb ions in the defect production and recombination processes. Trap parameters for the TL process are calculated and presented. The low temperature glow peak is attributable to Z(3) centres.     

Tau Does Not Stabilize Axonal Microtubules but Rather Enables Them to Have Long Labile Domains

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The crystal structures of dystrophin and utrophin spectrin repeats: implications for domain boundaries

Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.     

Prolonged Outbreak of Candida krusei Candidemia in Paediatric Ward of Tertiary Care Hospital

Mycopathologia - A sudden rise of Candida krusei candidemia cases was noticed in our hospital within 1&nbsp;year with maximum cases from paediatric unit. The present study reports the results...     

Cadherin Switching and Activation of ��-Catenin Signaling Underlie Proinvasive Actions of Calcitonin-Calcitonin Receptor Axis in Prostate Cancer

Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic β-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular β-catenin, its translocation in the nucleus, and transactivation of β-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/β-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-β-catenin signaling.Prostate cancer (PC)² is the most commonly diagnosed cancer and the second leading cause of cancer deaths in men in the United States (1, 2). Although androgen ablation therapy is effective in men with advanced disease for some time, the disease subsequently progresses to the androgen-independent stage. The population of prostate cells expressing neuroendocrine factors such as calcitonin (CT) also increases during this progression (3–5). At this stage, the disease is metastatic and chemoresistant. Present evidence suggests that cancer metastasis is usually preceded by the disruption of normal cell-cell adhesion and the loss of integrity of the primary tumor site (6, 7). This process may include several genetic, molecular, and morphological changes characterized by epithelial-to-mesenchymal transition (EMT) (8–10). The EMT is characterized by the loss of cell polarity, altered cell-cell and cell-matrix adhesion, and acquisition of migratory, mesenchymal phenotype. Other reported changes include down-regulation of E-cadherin, induction of N-cadherin, release of β-catenin from junctional complexes, and its translocation to the nucleus (11–13). However, the precise molecular mechanisms associated with this process are obscure.Several growth factors, including hepatocyte growth factor, transforming growth factor-β, vascular endothelial growth factor, and epidermal growth factor, have been reported to induce EMT in tumor cell lines (14–16). We have shown that the expression of CT and its G protein-coupled receptor (CTR) is remarkably higher in advanced PCs, and the CT-CTR autocrine axis is a potent stimulator of PC cell tumorigenicity, invasion, and metastasis (4, 17–19). Although CT-stimulated increase in the motility and invasion of PC cells may be mediated by CT-stimulated secretion of matrix metalloproteinases and urokinase-type plasminogen activator, the precise molecular mechanisms preceding these CTR actions remain to be elucidated (18, 20). We tested the hypothesis that CT induces biochemical and morphological changes associated with EMT to increase the invasiveness of PC cells.Our results indicate that activation of the CT-CTR autocrine axis in prostate cancer cells induced several changes associated with EMT such as remodeling of tight and adherens junctions, cadherin switching, and activation of WNT/β-catenin signaling. In contrast, the silencing of the CT-CTR axis reversed this process. Moreover, cyclic AMP-dependent protein kinase (PKA) plays a key role in this CT-CTR-mediated process. This is the first study demonstrating the action of prostate CTR on junctional complexes and WNT/β-catenin signaling of PC cell lines.     

Directing traffic: Chaperone‐mediated protein transport in malaria parasites

The ability of eukaryotic parasites from the phylum Apicomplexa to cause devastating diseases is predicated upon their ability to maintain faithful and precise protein trafficking mechanisms. Their parasitic life cycle depends on the trafficking of effector proteins to the infected host cell, transport of proteins to several critical organelles required for survival, as well as transport of parasite and host proteins to the digestive organelles to generate the building blocks for parasite growth. Several recent studies have shed light on the molecular mechanisms parasites utilise to transform the infected host cells, transport proteins to essential metabolic organelles and for biogenesis of organelles required for continuation of their life cycle. Here, we review key pathways of protein transport originating and branching from the endoplasmic reticulum, focusing on the essential roles of chaperones in these processes. Further, we highlight key gaps in our knowledge that prevents us from building a holistic view of protein trafficking in these deadly human pathogens.