A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease

Immunohistochemical detection of macrophage migration inhibitory factor in fetal and adult bovine epididymis: Release by the apocrine secretion mode?

Originally defined as a lymphokine inhibiting the random migration of macrophages, the macrophage migration inhibitory factor (MIF) is an important mediator of the host response to infection. Beyond its function as a classical cytokine, MIF is currently portrayed as a multifunctional protein with growth-regulating properties present in organ systems beyond immune cells. In previous studies, we detected substantial amounts of MIF in the rat epididymis and epididymal spermatozoa, where it appears to play a role during post-testicular sperm maturation and the acquisition of fertilization ability. To explore its presence in other species not yet examined in this respect, we extended the range of studies to the bull. Using a polyclonal antibody raised against MIF purified from bovine eye lenses, we detected MIF in the epithelium of the adult bovine epididymis with the basal cells representing a prominently stained cell type. A distinct accumulation of MIF at the apical cell pole of the epithelial cells and in membranous vesicles localized in the lumen of the epididynnal duct was obvious. In the fetal bovine epididymis, we also detected MIF in the epithelium, whereas MIF accumulation was evident at the apical cell surface and in apical protrusions. By immuno-electron microscopy of the adult bovine epididymis, we localized MIF in apical protrusions of the epithelial cells and in luminal membrane-bound vesicles that were found in close proximity to sperm cells. Although the precise origin of the MIF-containing vesicles remains to be delineated, our morphological observations support the hypothesis that they become detached from the apical surface of the epididymal epithelial cells. Additionally, an association of MIF with the outer dense fibers of luminal spermatozoa was demonstrated. Data obtained in this study suggest MIF release by an apocrine secretion mode in the bovine epididymis. Furthermore, MIF localized in the basal cells of the epithelium and in the connective tissue could be responsible for regulating the migration of macrophages in order to avoid contact of immune cells with spermatozoa that carry a wide range of potent antigens. Copyright (c) 2006 S. Karger AG, Basel

Multicenter evaluation of the vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of gram-positive aerobic bacteria

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting

Iminosugar-Based Inhibitors of Glucosylceramide Synthase Increase Brain Glycosphingolipids and Survival in a Mouse Model of Sandhoff Disease

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects

Genome-wide DNA methylation analysis in blood cells from patients with Werner syndrome

Werner syndrome is a progeroid disorder characterized by premature age-related phenotypes. Although it is well established that autosomal recessive mutations in the WRN gene is responsible for Werner syndrome, the molecular alterations that lead to disease phenotype remain still unidentified

Genetic aberrations in glioblastoma multiforme: translocation of chromosome 10 in an O-2A-like cell line

We have examined the genetic aberrations in two near-diploid glioblastoma multiforme cell lines that appear to have arisen from different glial lineages. One cell line, Hu-O-2A/Gb1, expresses antigens and metabolic profiles characteristic of the oligodendrocyte-type-2 astrocyte (0-2A) lineage of the rat central nervous system. This line generates, in vitro, cells with characteristics of 0-2A progenitor cells, oligodendrocytes and astrocytes. The second cell line, IN1434, is derived from an astrocyte or a precursor cell restricted to astrocytic differentiation. In Hu-O-2A/Gb1 the sole homologue of chromosome 10 is disrupted at band 10p11–12.1 by translocation with chromosomes X and 15. The translocation breakpoint is localized between genetic markers D10S2103 and [D10S637, D10S1962, D10S355]. Other aberrations include a 5;14 translocation, deletion of the long and short arms of chromosome 16 and loss of one copy of the CDKN2 gene. IN1434 cells share some cytogenetic abnormalities with Hu-O-2A/Gb1 cells, despite their apparent derivation from a different biological origin, but also have translocations involving the long and short arms of chromosome 1 and the long arm of chromosome 7, and deletion of chromosome 13 at bands 13q12–21. © 1999 Cancer Research Campaig