Liposome retention in size exclusion chromatography

Background  

Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void.     

Calcium as an extracellular signalling molecule: perspectives on the Calcium Sensing Receptor in the brain

Calcium acts as a universal signal that is responsible for controlling a spectrum of cellular processes ranging from fertilization to apoptosis. For a long time, calcium was regarded solely as an intracellular second messenger. However, the discovery that calcium can also act as an external ligand together with the molecular cloning of its cell surface receptor, the Calcium Sensing Receptor (CaSR), demonstrated that calcium also acts as an important extracellular or first messenger. Here, we give an overview of the main structural, pharmacological and physiological features of the CaSR and provide an assessment of its functions and cellular and molecular mechanisms of action. In addition, we propose possible avenues for future research into the trafficking of CaSR and the role(s) of this receptor in the central nervous system.     

AMP-activated protein kinase beta subunit tethers alpha and gamma subunits via its C-terminal sequence (186-270)

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable alphabetagamma heterotrimer comprising a catalytic alpha and two non-catalytic subunits, beta and gamma. The beta subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the beta subunit, beta1-(186-270), is sufficient to form an active AMP-dependent heterotrimer alpha1beta1-(186-270)-gamma1, whereas the 25-residue beta1 C-terminal (246-270) sequence is sufficient to bind gamma1, gamma2, or gamma3 but not the alpha subunit. Deletion of the beta C-terminal Ile-270 precludes betagamma association in the absence of the alpha subunit, but the presence of the alpha subunit or substitution of Ile-270 with Ala or Glu restores betagamma binding. Truncation of the alpha subunit reveals that beta1 binding requires the alpha1-(313-473) sequence. The conserved C-terminal 85-residue sequence of the beta subunit (90% between beta1 and beta2) is the primary alphagamma binding sequence responsible for the formation of the AMPK alphabetagamma heterotrimer.     

Homo- and hetero-oligomerization of beta-arrestins in living cells

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous beta-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of beta-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged beta-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, beta-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of beta-arrestin2 and beta-arrestin1 prevented beta-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that beta-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions.     

Identification of zebrafish insertional mutants with defects in visual system development and function

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.     

Proteomic analysis of mouse growth plate cartilage

Cartilage is a highly specialized load-bearing tissue with a small number of cells and a high proportion of extracellular matrix (ECM). The abundance of heavily sulfated proteoglycans and a poorly soluble collagenous ECM presents a major technical challenge to 2-DE. Here we report proteomic analysis of mouse growth plate cartilage using novel methodology for tissue dissection and sample prefractionation. We have successfully resolved cartilage tissue extracts by 2-DE for the first time and identified cartilage ECM proteins by Western blotting and MS/MS.     

Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron-specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6-8 weeks and the ratio of human amyloid (A)beta42 to Abeta40 is 1.5 and 5 in pre-depositing and amyloid-depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid-associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau-positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross-breeding to other genetically engineered mouse models.     

Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas

Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 "specimens" collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.