Retinol-binding protein-deficient mice: biochemical basis for impaired vision

We reported previously that mice lacking plasma retinol-binding protein (RBP) are phenotypically normal except that they display impaired vision at the time of weaning. This visual defect is associated with greatly diminished eyecup levels of retinaldehyde and is reversible if the mutants are maintained for several months on a vitamin A-sufficient diet. Here we provide a biochemical basis for the visual phenotype of RBP-deficient mice. This phenotype does not result from inadequate milk total retinol levels since these are not different for RBP-deficient and wild-type mice. The eye, unlike all other tissues that have been examined, takes up dietary retinol very poorly. Moreover, compared to other tissues, the eye displays a strong preference for retinol uptake when retinol is delivered bound to RBP. The poor uptake of dietary retinol by the eye coupled with its marked ability to take up retinol from RBP, we propose, provides a basis for the impaired vision observed in weanling RBP-deficient mice. Further study of the mutants suggests that the impaired vision is reversible because the eyes of mutant mice slowly acquire sufficient retinol from the low levels of retinol present in their circulation either bound to albumin or present in lipoprotein fractions. Thus, the eye is unlike other tissues in the body in that it shows a very marked preference for acquiring retinol needed to support vision from the retinol-RBP complex and is unable to meet adequately its retinol need through uptake of recently absorbed dietary retinol. This provides an explanation for the impaired vision phenotype of RBP-deficient mice.     

Novel inhibitors for murine and human leukemia inhibitory factor based on fused soluble receptors

Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases.     

White matter differences between healthy young ApoE4 carriers and non-carriers identified with tractography and support vector machines

The apolipoprotein E4 (ApoE4) is an established risk factor for Alzheimer's disease (AD). Previous work has shown that this allele is associated with functional (fMRI) changes as well structural grey matter (GM) changes in healthy young, middle-aged and older subjects. Here, we assess the diffusion characteristics and the white matter (WM) tracts of healthy young (20-38 years) ApoE4 carriers and non-carriers. No significant differences in diffusion indices were found between young carriers (ApoE4+) and non-carriers (ApoE4-). There were also no significant differences between the groups in terms of normalised GM or WM volume. A feature selection algorithm (ReliefF) was used to select the most salient voxels from the diffusion data for subsequent classification with support vector machines (SVMs). SVMs were capable of classifying ApoE4 carrier and non-carrier groups with an extremely high level of accuracy. The top 500 voxels selected by ReliefF were then used as seeds for tractography which identified a WM network that included regions of the parietal lobe, the cingulum bundle and the dorsolateral frontal lobe. There was a non-significant decrease in volume of this WM network in the ApoE4 carrier group. Our results indicate that there are subtle WM differences between healthy young ApoE4 carriers and non-carriers and that the WM network identified may be particularly vulnerable to further degeneration in ApoE4 carriers as they enter middle and old age.     

Nedd4 family-interacting protein 1 (Ndfip1) is required for the exosomal secretion of Nedd4 family proteins

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50-90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress.     

Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition

CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.     

Induced Endocytosis of the Receptor Kinase FLS2

Receptor-like kinases (RLKs) that function as pattern-recognition receptors (PRRs) play a key role in plant immune responses. The receptor recognizing flagellin in Arabidopsis, FLS2, is encoded by a membrane resident RLK. FLS2 is involved in preinvasive immunity against bacterial infection. Recent observations revealed that upon flagellin perception FLS2 accumulates in intracellular mobile vesicles and is then degraded. Reminiscent of ligand-induced receptor endocytosis in animals, FLS2 internalization is Wortmannin-sensitive. Mutation of the potentially phosphorylated residue threonine-867 impaired FLS2 endocytosis and flagellin-triggered responses. Furthermore, mutation of a PEST-motif abolished FLS2 endocytosis and downstream flagellin-elicited responses were affected. Thus, FLS2 endocytosis likely involves phosphorylation and ubiquitination events and appears to be interconnected with flagellin signaling. Similarly, TLR4, the mammalian PRR recognizing bacterial lipopolysaccharides (LPS) is internalized in a ligand specific manner. In this addendum, we discuss endocytic processes of plant RLKs focussing on FLS2 and provide a brief comparison with TLR4 endocytosis.Key words: Endocytosis, RLK, FLS2, flagellin, TLR4, LPS     

The human immunodeficiency virus type 1 nef gene can to a large extent replace simian immunodeficiency virus nef in vivo.

The nef gene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1) nef alleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of the nef alleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency. nef long terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6 nef allele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1 nef can, to a large extent, functionally replace SIVmac nef in vivo.