Impact of siRNA-Mediated Cofilin-1 Knockdown and Obesity Associated Microenvironment on the Motility of Natural Killer Cells.

The anti-tumor capacity of natural killer (NK) cells heavily relies on their ability to migrate towards their target cells. This process is based on dynamic actinrearrangement, so-called actin treadmilling, andis tightly regulated by proteins such as cofilin-1. The aim of the present study was to identify the role of cofilin-1 (CFL-1) in the migratory behavior of NK cells and to investigate a possible impact of an obesity-associated micromilieu on these cells, as it is known that obesity correlates with various impaired NK cell functions. CFL-1 was knocked-down via transfection of NK-92 cells with respective siRNAs. Obesity associated micromilieu was mimicked by incubation of NK-92 cells with adipocyte-conditioned medium from human preadipocyte SGBS cells or leptin. Effects on CFL-1 levels, the degree of phosphorylation to the inactive pCFL-1 as well as NK-92 cell motility were analyzed. Surprisingly, siRNA-mediated CFL-1 knockdown led to a significant increase of migration, as determined by enhanced velocity and accumulated distance of migration. No effect on CFL-1 nor pCFL-1 expression levels, proportion of phosphorylation and cell migratory behavior could be demonstrated under the influence of an obesity-associated microenvironment. In conclusion, the results indicate a significant effect of a CFL-1 knockdown on NK cell motility.

Modulation of transcriptional mineralocorticoid receptor activity by casein kinase 1.

The mineralocorticoid receptor (MR) with its ligand aldosterone (aldo) physiologically regulates electrolyte homeostasis and blood pressure but it can also lead to pathophysiological effects in the cardiovascular system. Previous results show that posttranslational modifications (PTM) can influence MR signaling and function. Based on in silico and in vitro data, casein kinase 1 (CK1) was predicted as a candidate for MR phosphorylation. To gain a deeper mechanistic insight into MR activation, we investigated the influence of CK1 on MR function in HEK cells. Co-immunoprecipitation experiments indicated that the MR is located in a protein-protein complex with CK1α and CK1ε. Reporter gene assays with pharmacological inhibitors and MR constructs demonstrated that especially CK1ε acts as a positive modulator of GRE activity via the C-terminal MR domains CDEF. CK1 enhanced the binding affinity of aldosterone to the MR, facilitated nuclear translocation and DNA interaction of the MR, and led to expression changes of pathophysiologically relevant genes like Per-1 and Phlda1. By peptide microarray and site-directed mutagenesis experiments, we identified the highly conserved T800 as a direct CK1 phosphorylation site of the MR, which modulates the nuclear import and genomic activity of the receptor. Direct phosphorylation of the MR was unable to fully account for all of the CK1 effects on MR signaling, suggesting additional phosphorylation of MR co-regulators. By LC/MS/MS, we identified the MR-associated proteins NOLC1 and TCOF1 as candidates for such CK1-regulated co-factors. Overall, we found that CK1 acts as a co-activator of MR GRE activity through direct and indirect phosphorylation, which accelerates cytosolic-nuclear trafficking, facilitates nuclear accumulation and DNA binding of the MR, and increases the expression of pathologically relevant MR-target genes.

Importance of Micromilieu for Pathophysiologic Mineralocorticoid Receptor Activity-When the Mineralocorticoid Receptor Resides in the Wrong Neighborhood.

The mineralocorticoid receptor (MR) is a member of the steroid receptor family and acts as a ligand-dependent transcription factor. In addition to its classical effects on water and electrolyte balance, its involvement in the pathogenesis of cardiovascular and renal diseases has been the subject of research for several years. The molecular basis of the latter has not been fully elucidated, but an isolated increase in the concentration of the MR ligand aldosterone or MR expression does not suffice to explain long-term pathologic actions of the receptor. Several studies suggest that MR activity and signal transduction are modulated by the surrounding microenvironment, which therefore plays an important role in MR pathophysiological effects. Local changes in micromilieu, including hypoxia, ischemia/reperfusion, inflammation, radical stress, and aberrant salt or glucose concentrations affect MR activation and therefore may influence the probability of unphysiological MR actions. The surrounding micromilieu may modulate genomic MR activity either by causing changes in MR expression or MR activity; for example, by inducing posttranslational modifications of the MR or novel interaction with coregulators, DNA-binding sites, or non-classical pathways. This should be considered when developing treatment options and strategies for prevention of MR-associated diseases.

Variability in the distance from the end of the gray matter to the end of the conus medullaris: a case-triggered histological investigation.

OBJECTIVE: The background for this investigation was the dramatic course of a 14-year-old girl with a spontaneous hemorrhage in the area of the conus medullaris resulting in a complete cross-sectional syndrome with bladder and bowel dysfunction. Despite immediate surgical treatment, the patient showed close to no postoperative improvement. Subsequent histopathological examination of the removed masses revealed a cavernoma. To better understand the link between the site and symptoms of conus medullaris lesions, the authors performed a literature search and then histological examination of the conus medullaris of 18 cadaveric specimens from body donors. METHODS: After a literature search regarding the histological features of the structure of the conus medullaris did not lead to satisfying results, the authors performed histological examination of the conus medullaris in 18 cadaveric specimens from body donors. The largest (a) and smallest (b) diameters of the conus medullaris were measured, noting individual variations in the distance from the caudal ending of the gray matter to the macroscopically visible end of the conus medullaris. Correlations of these differences with sex, body height, gray matter transverse diameter, and cross-sectional area at the end of the gray matter were analyzed. RESULTS: Gray matter displayed in the form of a butterfly figure was found along almost the entire length of the conus medullaris. The specific slide containing the end of the gray matter was noted. The distance between the caudal ending of the gray matter in the conus and the macroscopical end of the conus medullaris was defined as the gray matter to cone termination (GMCT) distance. There were great individual variations in the distance from the caudal ending of the gray matter to the macroscopically visible end of the conus medullaris. Analysis of the correlations of these differences with sex, body height, gray matter transverse diameter, and cross-sectional area at the end of the gray matter showed no significant sex-specific differences in the GMCT distance. Patient body height and transverse diameter at the end of the gray matter were found to be correlated positively with the GMCT distance. Moreover, greater height also correlated positively with the cross-sectional area at the end of the gray matter. CONCLUSIONS: This report is, to the authors' knowledge, the first published description of the histological structure of the conus medullaris and can serve as the basis for a better understanding of neurological deficits in patients with a conus medullaris syndrome. Findings that gray matter can be detected far into the conus medullaris, with large individual differences in the endpoint of the gray matter, are important for operative care of intramedullary masses and vascular malformations in this area. It is therefore important to use electrophysiological monitoring during these operations.