[Terror against the free world--politics and the Norwegian Medical Society].
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Humans↗
Biomedical subjects
O Bakke
Publications and source records attributed to O Bakke.
Mitotic partitioning of endosomes and lysosomes.
BACKGROUND: Some of the mechanisms underlying cell division and partitioning of the cellular components into the daughter cells are well known. Within the endomembrane system, there is a general cessation of membrane traffic, including endocytosis and endosome fusion, at the onset of mitosis. However, the fate of endosomes and lysosomes during mitosis has been less well studied. RESULTS: Using video and confocal microscopy of living cells, we show here that endosomes and lysosomes remain intact and separate during mitosis. The segregation into daughter cells takes place by coordinated movements, and during cytokinesis, these organelles accumulate in the vicinity of the microtubule organization center. However, partitioning into daughter cells is not more accurate than a calculated stochastic distribution, despite the apparent order to the process. CONCLUSION: We conclude that partitioning of endosomes and lysosomes is an ordered, yet imprecise, process, and that the organelle copy number is maintained by the daughter cells.
Animals↗
The MHC class II-associated chicken invariant chain shares functional properties with its mammalian homologs.
The nucleotide sequence of chicken invariant chain (Ii) was determined, and the amino acid sequence similarity with human Ii is 61%. Certain regions important for the biological function of human Ii are highly conserved between chicken and mammals. The cytoplasmic tail of chicken Ii fused to the plasma membrane reporter molecule neuraminidase relocated the protein to endosomes. Moreover, like the mammalian orthologs, the cytoplasmic tail was found to contain two independent leucine-based endosomal sorting signals. Chicken Ii was found to interact with human Ii and crosslinking studies also indicate that chicken Ii assembles as a trimer. The chicken Ii can furthermore bind the human MHC class II (HLA-DR1). Many of the functional properties between the chicken Ii and its mammalian orthologs are thus maintained in spite of their sequence differences.
Animals↗
The cytoplasmic tail of CD1d contains two overlapping basolateral sorting signals.
CD1d is a member of the CD1 polypeptide family that represents a new arm of host defense against invading pathogens. In our previous work (Rodionov, D. G., Nordeng, T. W., Pedersen, K., Balk, S. P., and Bakke, O. (1999) J. Immunol. 162, 1488-1495) we have shown that CD1d contained a classic tyrosine-based internalization signal (YQGV) in its short cytoplasmic tail. CD1d is expressed in polarized epithelial cells, and we found that the cytoplasmic tail of CD1d also contained information for basolateral sorting. Interestingly, a mutation of the critical tyrosine residue of the endosomal sorting signal did not result in the loss of basolateral targeting of the mutant CD1d. To search for a basolateral sorting signal we have constructed a full set of alanine mutants, but no single alanine substitution inactivated the signal. However, deletions or mutations of either the C-terminal valine/leucine pair or the critical tyrosine residue from the internalization signal and either residue from the C-terminal valine/leucine pair inactivated basolateral sorting. Our data thus suggest that the cytoplasmic tail contains two overlapping basolateral signals, one tyrosine- and the other leucine-based, each being sufficient to direct CD1d to the basolateral membrane of polarized Madin-Darby canine kidney cells.
Amino Acid Sequence↗
The leucine-based sorting motifs in the cytoplasmic domain of the invariant chain are recognized by the clathrin adaptors AP1 and AP2 and their medium chains.
Recognition of sorting signals within the cytoplasmic tail of membrane proteins by adaptor protein complexes is a crucial step in membrane protein sorting. The three known adaptor complexes, AP1, AP2, and AP3, have all been shown to recognize tyrosine- and leucine-based sorting signals, which are the most common sorting signals within membrane protein cytoplasmic tails. Although tyrosine-based signals are recognized by the micro-chains of adaptor complexes, the subunit recognizing leucine-based sorting signals is less clear. In this report we show by surface plasmon resonance that the two leucine-based sorting signals within the cytoplasmic tail of the invariant chain bind independently from each other to AP1 and AP2 but not to AP3. We also show that both motifs can be recognized by the micro-chains of AP1 and AP2. Moreover, by using monomeric as well as trimeric invariant chain constructs, we show that adaptor binding does not require trimerization of the invariant chain.
Adaptor Protein Complex 1↗
Polarized transport of MHC class II molecules in Madin-Darby canine kidney cells is directed by a leucine-based signal in the cytoplasmic tail of the beta-chain.
MHC class II molecules are found on the basolateral plasma membrane domain of polarized epithelial cells, where they can present Ag to intraepithelial lymphocytes in the vascular space. We have analyzed the sorting information required for efficient intracellular localization and polarized distribution of MHC class II molecules in stably transfected Madin-Darby canine kidney cells. These cells were able to present influenza virus particles to HLA-DR1-restricted T cell clones. Wild-type MHC class II molecules were located on the basolateral plasma membrane domain, in basolateral early endosomes, and in late multivesicular endosomes, the latter also containing the MHC class II-associated invariant chain and an HLA-DM fusion protein. A phenylalanine-leucine residue within the cytoplasmic tail of the beta-chain was required for basolateral distribution, efficient internalization, and localization of the MHC class II molecules to basolateral early endosomes. However, distribution to apically located, late multivesicular endosomes did not depend on signals in the class II cytoplasmic tails as both wild-type class II molecules and mutant molecules lacking the phenylalanine-leucine motif were found in these compartments. Our results demonstrate that sorting information in the tails of class II dimers is an absolute requirement for their basolateral surface distribution and intracellular localization.
Amino Acid Sequence↗
Overexpression of proteins containing tyrosine- or leucine-based sorting signals affects transferrin receptor trafficking.
Targeting of many transmembrane proteins to post-Golgi compartments is dependent on cytoplasmically exposed sorting signals. The most widely used signals conform to the tyrosine- or the leucine-based motifs. Both types of signals have been implicated in protein localization to the same intracellular compartments, but previous results from both cell-free experiments and studies of transfected cell lines have indicated that the two types of signals interact with separate components of the sorting machinery. We have overexpressed several transmembrane proteins in stably transfected Madin-Darby canine kidney cells using an inducible promoter system. Overexpression of proteins containing tyrosine- or leucine-based sorting signals resulted in reduced internalization of the transferrin receptor, whereas recycling and polarized distribution was not influenced. Our results indicate that proteins with tyrosine- and leucine-based sorting signals can be transported along common saturable pathways.
Animals↗
Post-replicative base excision repair in replication foci.
Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.
Amino Acid Sequence↗
The Norwegian naturalistic treatment study of depression in general practice (NORDEP)-I: randomised double blind study.
OBJECTIVE: To evaluate the efficacy of emotional support and counselling combined with placebo or antidepressants with single or dual mechanism of action in the treatment of depression in primary care. DESIGN: Randomised double blind study. SETTING: Several locations in Norway. SUBJECTS: 372 patients with depression. MAIN OUTCOME MEASURES: Improvement (clinical remission) reported both by the patient (Montgomery Asberg depression rating scale) and the physician (clinical global improvement and impression scales). RESULTS: Intention to treat analyses showed 47% remission in patients randomised to placebo compared with 61% remission in patients randomised to sertraline (odds ratio 0.56, 95% confidence interval 0.33 to 0.96) and 54% in patients randomised to mianserin (0.75, 0.44 to 1.27). Women responded better than men (1.86, 1.17 to 2.96). Subgroup analyses showed that subjects with recurrent depression (n=273) responded more frequently to sertraline than to placebo (0.43, 0.23 to 0.82) than those having their first episode of depression (1.18, 0.39 to 3.61). Statistically significant interactions between type of drug treatment and history of depression were not shown by logistic regression. CONCLUSION: The combination of active drug and simple psychological treatment (counselling, emotional support, and close follow up over a 24 week period) was more effective than simple psychological treatment alone, in particular for those with recurrent depression. Overall, women may benefit more than men. If confirmed in future studies, the findings should lead to more differentiated treatment guidelines for depression in primary care.
Adolescent↗
A critical tyrosine residue in the cytoplasmic tail is important for CD1d internalization but not for its basolateral sorting in MDCK cells.
The CD1 family of polypeptides is divided into two groups, the CD1b and CD1d group. Both groups are involved in stimulation of T cell response. Molecules of the CD1b group can present Ag derived from bacterial cell walls to T cells; the process of Ag acquisition is thought to take place in endosomes. Little is known about Ag presentation by CD1d. We therefore studied the intracellular trafficking of human CD1d in Madin-Darby canine kidney (MDCK) and COS cells. CD1d was found in endosomal compartments after its internalization from the plasma membrane. It is therefore possible that CD1d acquires its yet unidentified exogenous ligand in the same compartments as the MHC class II and CD1b molecules. CD1d contains a tyrosine-based sorting signal in its cytoplasmic tail that is necessary for internalization. Furthermore, the cytoplasmic tail of CD1d also contains a signal for basolateral sorting that is, however, different from the internalization signal.
Amino Acid Sequence↗
Estimation of offspring production from a limited number of stage-structured censuses.
We propose a procedure for maximum likelihood estimation of the number of animals or offspring in a closed population where the individuals counted go through stages or age-groups. Application of the procedure requires knowledge of the distributions of the stage durations. A procedure for maximum likelihood estimation of those based on marked animals is also given. The procedures are illustrated by applying them to gray seal (Halichoerus grypus) data from Froan Nature Reserve, Central Norway, from the breeding seasons 1990-1999.
Animals↗
Intracellular traffic to compartments for MHC class II peptide loading: signals for endosomal and polarized sorting.
In this review we focus on the traffic of MHC class II and endocytosed antigens to intracellular compartments where antigenic peptides are loaded. We also discuss briefly the nature of the peptide loading compartment and the sorting signals known to direct antigen receptors and MHC class II and associated molecules to this location. MHC class II molecules are expressed on a variety of polarized epithelial and endothelial cells, and polarized cells are thus potentially important for antigen presentation. Here we review some cell biological aspects of polarized sorting of MHC class II and the associated invariant chain and the signals that are involved in the sorting process to the basolateral domain. The molecules involved in sorting and loading of peptide may modulate antigen presentation, and in particular we discuss how invariant chain may change the cellular phenotype and the kinetics of the endosomal pathway.
Amino Acid Motifs↗
Culture characterization of differentiated high endothelial venule cells from human tonsils.
High endothelial venules (HEV) are specialized vessels that support abundant lymphocyte emigration from peripheral blood into secondary lymphoid organs. HEV endothelial cells (HEVEC) exhibit particular structural and functional features, including secretion of the HEV-specific extracellular matrix protein hevin and an array of uniquely glycosylated counter-receptors for L-selectin expressed on lymphocytes. These ligands are collectively called the peripheral lymph node addressin (PNAd), originally defined by the monoclonal antibody MECA-79. PNAd expression was used to purify HEVEC by positive immunoselection from enzyme-digested human tonsils after negative immunoselection for other cells. Purified HEVEC maintained secretion of hevin and homogenous expression of intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2 (CD102), and CD31, at high levels following 8 days in culture. Expression of functional PNAd was maintained during the first 4 to 5 days of culture but decreased gradually and disappeared on day 8, while the expression of CD34 remained strong. However, the CD34 glycoform shifted toward the in situ phenotype of flat-walled vessels, suggesting that the observed loss of L-selectin binding determinants and MECA-79 antigen was due to down-regulation of the glycosyl- and sulfo-transferases essential for their expression. Our rapid and reproducible method to establish HEVEC cultures provides a useful mechanistic tool for identification of the factors that induce and maintain the HEV phenotype, as well as a source for isolation of HEV-specific genes.
Calcium-Binding Proteins↗
DR/CLIP (class II-associated invariant chain peptides) and DR/peptide complexes colocalize in prelysosomes in human B lymphoblastoid cells.
In APCs, MHC class II molecules (MHC class II) bind antigenic peptides after HLA-DM mediated removal of CLIP. To characterize intracellular sites of peptide loading in human B lymphoblastoid cell lines, we conducted immunoelectron microscopy studies with Abs recognizing MHC class II associated with CLIP or bound peptide, respectively, together with Abs to HLA-DM and endocytic markers. The distribution of these molecules indicates that peptide binding occurs in compartments with characteristics of normal late endosomes, and in compartments that show characteristics of late endosomes, but are not detectably accessed by endocytosed BSA-gold. The latter compartments may represent or give rise to recycling vesicles that deliver peptide-loaded class II molecules to the cell surface. In addition, we have compared cells in which HLA-DM and HLA-DR interaction is defective with cells in which this interaction is intact, and find that DM/DR interaction is not required for the proper localization of either molecule to peptide-loading compartments.
Antigens, Differentiation, B-Lymphocyte↗
A region from the medium chain adaptor subunit (mu) recognizes leucine- and tyrosine-based sorting signals.
Tyrosine-based sorting signals in the cytosolic tails of membrane proteins have been found to bind directly to the medium chain subunit (mu) of the adaptor complexes AP-1 and AP-2. For the leucine-based signals, an interaction with AP-1 and AP-2 has been reported, but no specific interacting subunit has been demonstrated. After searching for molecules interacting with the leucine-based sorting signals within the cytosolic tail of the major histocompatibility complex class II-associated invariant chain using a phage display approach, we identified phage clones with homology to a conserved region of the AP-1 and AP-2 mu chains. To investigate the relevance of these findings, we have expressed regions of mouse mu1 and mu2 chains on phage gene product III and investigated the binding to tail sequences from various transmembrane proteins with known endosomal targeting signals. Enzyme-linked immunosorbent binding assays showed that these phages specifically recognized peptides containing functional leucine- and tyrosine-based sorting signals, suggesting that these regions of the mu1 and mu2 chains interact with both types of sorting signals.
Amino Acid Sequence↗
Medium chains of adaptor complexes AP-1 and AP-2 recognize leucine-based sorting signals from the invariant chain.
Interactions between tyrosine- and leucine-based sorting signals in the cytoplasmic tails of transmembrane proteins and adaptor complexes AP-1 and AP-2 are believed to be the first step in the formation of clathrin-coated vesicles that deliver these proteins to their destination. Medium chains of AP-1 and AP-2 have been reported to interact with tyrosine-based sorting signals in a number of in vitro assays. In the present study we found that recombinant medium chains could interact with leucine-based sorting signals from the cytoplasmic tail of the invariant chain. Medium chains may therefore be responsible for the proper recognition of both tyrosine and leucine sorting signals by AP-1 and AP-2 complexes.
Adaptor Protein Complex alpha Subunits↗
Intracellular transport of molecules engaged in the presentation of exogenous antigens.
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Animals↗
Major histocompatibility complex class II-dependent antigen presentation by human intestinal endothelial cells.
BACKGROUND & AIMS: In the normal gut, human intestinal microvascular endothelial cells (HIMECs) express major histocompatibility complex (MHC) class II molecules. Enhanced expression is found in chronic inflammation. We examined the cytokine regulation of MHC class II molecules and the associated invariant chain (Ii) in HIMECs and investigated whether such cells can process and present a complex protein antigen to T cells. METHODS: Enzyme-linked immunosorbent assay, flow cytometry, immunoelectron microscopy, as well as T-cell activation assay with HIMECs and HLA-DR-restricted T-cell clones were employed. RESULTS: In unstimulated HIMEC monolayers, HLA-DR, -DP, and -DQ and Ii were undetectable at the protein level, but interferon gamma (IFN-gamma) (100 U/mL) induced expression that peaked for DR after 2-3 days, for DP after 4-6 days, for DQ after 10-12 days, and for Ii after 2-3 days. Tumor necrosis factor alpha had no effect alone but enhanced class II expression in combination with IFN-gamma, most notably for DQ and DP. HLA-DR3-restricted and Mycobacterium tuberculosis heat shock 65-kilodalton-specific T-cell clones were activated to produce IFN-gamma in response to relevant antigen presented by IFN-gamma-treated HIMECs. This response was inhibited by blocking monoclonal antibody to HLA-DR and by chloroquine when compared to professional antigen-presenting cells, HIMECs activated T-cell clones quite efficiently. CONCLUSIONS: These data suggest that microvascular endothelial cells can present complex protein antigens in the human gut.
Antigen Presentation↗