Deficiency of the INCL protein Ppt1 results in changes in ectopic F1-ATP synthase and altered cholesterol metabolism

Infantile neuronal ceroid lipofuscinosis (INCL) is a severeneurodegenerative disease caused by deficiency of palmitoylprotein thioesterase 1 (PPT1). INCL results in dramatic lossof thalamocortical neurons, but the disease mechanism has remainedelusive. In the present work we describe the first interactionpartner of PPT1, the F₁-complex of the mitochondrial ATP synthase,by co-purification and in vitro-binding assays. In additionto mitochondria, subunits of F₁-complex have been reported tolocalize in the plasma membrane, and to be capable of actingas receptors for various ligands such as apolipoprotein A-1.We verified here the plasma membrane localization of F₁-subunitson mouse primary neurons and fibroblasts by cell surface biotinylationand TIRF-microscopy. To gain further insight into the Ppt1-mediatedproperties of the F₁-complex, we utilized the Ppt1-deficientPpt1^ex4 mice. While no changes in the mitochondrial functioncould be detected in the brain of the Ppt1^ex4 mice, the levelsof F₁-subunits and β on the plasma membrane were specificallyincreased in the Ppt1^ex4 neurons. Significant changes were alsodetected in the apolipoprotein A-I uptake by the Ppt1^ex4 neuronsand the serum lipid composition in the Ppt1^ex4 mice. These dataindicate neuron-specific changes for F₁-complex in the Ppt1-deficientcells and give clues for a possible link between lipid metabolismand neurodegeneration in INCL.     

Genetics: Preimplantation diagnosis of cystic fibrosis by simultaneous detection of the W1282X and {Delta}F508 mutations

W1282X (W) and F508 () are the two most common mutations ofthe cystic fibrosis Israeli population. Patients who are homozygotes(WW and ) as well as compound heterozygotes (W) present a severephenotype of the disease. In the present study, we have developeda polymerase chain reaction (PCR)-based method for the detectionof both mutations simultaneously in a single blastomere. Unfertilizedhuman oocytes and single polyspermic blastomeres were subjectedto a two-round PCR amplification: a first round of multiplexPCR followed by a second round of nested PCR, done seperatelyat each locus. Clear signals at both loci were obtained in 51%(47/65) of oocytes and 69% (24/35) of blastomeres. The genotypeof the single cell analysed was determined by endonuclease digestionof the W products and by heteroduplex formation of the F products.This diagnostic system will allow the identification of affectedembryos (WW, , W) as well as phenotypically normal carriers(W++), and therefore may be used for cystic fibrosis preimplantationdiagnosis in families who carry either or both mutations     

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc{gamma}R) encoded by an alternatively spliced transcript of the Fc{gamma}RII gene

Low affinity FcR are a heterogeneous group of glycoproteinswhich exist in transmembrane (TM) as well as in soluble forms.Two membrane isoforms of the murine type II FcR, FcRilb1 andFc;Rilb2, have been described. They result from the translationof alternatively spliced premRNA, FcRilb2 lacking sequencesof the first intracytoplasmic domain (IC1). Soluble forms ofFcR (sFcR) have previously been shown to result from proteolysisof membrane receptors. We report here the identification, inmacrophages, of a mRNA derived from the FCRll gene by splicingexons encoding the TM and IC1 domains, i.e. corresponding toa TM-deleted FcRllb2 mRNA. A soluble protein possibly encodedby this mRNA was identified in macrophage supernatants. In accordancewith FcR nomenclature, we propose to name this new FcRll IsoformFcRllb3. It is the most abundant 8FcR present in serum, as comparedwith 8FcR resulting from cleavage of membrane FcR.     

Integrins are not involved in the process of human sperm-oolemmal fusion

BACKGROUND: We investigated whether integrins are required forthe human sperm–oocyte binding and fusion processes. METHODS:The expression of several integrin subunits at the human oocyteplasma membrane was investigated using immunofluorescence microscopy,and the functional role of integrins expressed at the humanoocyte surface in sperm–oocyte interaction was studiedusing a zona-free human oocyte binding and fusion assay. A totalof 144 unfertilized oocytes were stained with anti-integrinantibodies and 147 zona-free unfertilized oocytes were inseminatedin the presence of various anti-integrin antibodies that wereexpressed in oocyte plasma membrane. RESULTS: The antibodiesof six integrin subunits (₂, ₃, ₅, ₆, _V, _M) and six integrinsubunits (₁, ₂, ₃, ₄, ₅, ₆) were bound to the surface of fixedunfertilized oocytes. In contrast, the presence of ₁ and ₄ subunitscould not be verified. The human sperm–oocyte bindingwas only partially inhibited by blocking antibodies of ₂, ₃,₅, ₆, _V, _M, ₁, ₂ and ₃ with a maximum of 55% inhibition, butantibodies of ₄, ₅ and ₆ showed no effect on sperm–oolemmalbinding. A similar reduction of the number of fused sperm wasobserved. However, the ratio of fused sperm to total sperm (boundand fused) was not impaired by all integrin antibodies, suggestingthat integrins had no role in the sperm–oolemmal fusionprocess. CONCLUSIONS: These results suggest that one of thebinding mechanisms can be inhibited by integrin antibodies butthat this mechanism does not play an essential role in the humansperm–oolemmal binding and fusion processes. The othermechanisms, insensitive to integrins, may involve both bindingand fusion processes in human oocytes.     

Mutation analysis of the ROM1 gene in retinitis pigmentosa

The publishers wish to apologise for the misprinting of thenucleotide changes involved in the ROM1 amino acid substitutionspresented in the above paper. The correct nucleotide changefor each mutation is given below. P60T (CCTACT) T108M (ACGATG) G75D (GGCGAC) R242Q (CGACAA)     

siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells

Most cases of the dominantly inherited movement disorder, earlyonset torsion dystonia (DYT1) are caused by a mutant form oftorsinA lacking a glutamic acid residue in the C-terminal region(torsinAE). TorsinA is an AAA+ protein located predominantlyin the lumen of the endoplasmic reticulum (ER) and nuclear envelopeapparently involved in membrane structure/movement and processingof proteins through the secretory pathway. A reporter proteinGaussia luciferase (Gluc) shows a reduced rate of secretionin primary fibroblasts from DYT1 patients expressing endogenouslevels of torsinA and torsinAE when compared with control fibroblastsexpressing only torsinA. In this study, small interfering RNA(siRNA) oligonucleotides were identified, which downregulatethe levels of torsinA or torsinAE mRNA and protein by over 65%following transfection. Transfection of siRNA for torsinA messagein control fibroblasts expressing Gluc reduced levels of luciferasesecretion compared with the same cells non-transfected or transfectedwith a non-specific siRNA. Transfection of siRNA selectivelyinhibiting torsinAE message in DYT fibroblasts increased luciferasesecretion when compared with cells non-transfected or transfectedwith a non-specific siRNA. Further, transduction of DYT1 cellswith a lentivirus vector expressing torsinA, but not torsinB,also increased secretion. These studies are consistent witha role for torsinA as an ER chaperone affecting processing ofproteins through the secretory pathway and indicate that torsinAEacts to inhibit this torsinA activity. The ability of allele-specificsiRNA for torsinAE to normalize secretory function in DYT1 patientcells supports its potential role as a therapeutic agent inearly onset torsion dystonia.     

Ovary: Immunolocalization of transforming growth factor-{beta}1 and transforming growth factor-{beta}2 in the mouse ovary during gonadotrophin-induced follicular maturation

An immunohistochemical approach was utilized to evaluate thecellular distribution of transforming growth factor-₁ (TGF₁)and transforming growth factor ₂ (TGF₂) at different stagesof follicle development in the prepubertal mouse ovary underthe following conditions: (i) after pregnant mare's serum gonadotrophin(PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin(HCG) treatment; (iii) after PMSG and HCG treatment plus mating.In the immature ovary, TGFF₁ and TGF₂ immunoreactivities arelocalized in theca and granulosa cells and in oocytes. AfterPMSG treatment, TGF₁ and TGF₂ immunoreactivities are localizedin granulosa cells; in addition, TGF₂ staining is noted in thematrix surrounding antral cells. Staining for both TGF₁ andTGF₂ drops in the theca but persists in the oocyte. PMSG plusHCG treatment results in a significant increase in TGF₁ andTGF₂ immunoreactivity in the theca and in the maintenance ofTGF₁ staining in both basal granulosa cells and cumulus cellswhereas TGF₂ immunoreactivity is essentially localized in thematrix surrounding cumulus cells. Staining for TGF₁ and TGF₂persists in the oocyte. Following PMSG plus HCG treatment andmating, TGF₁ immunoreactivity is localized in the luteal cellsof corpora lutea and TGF₂ shows a similar localization pattern.This study provides evidence that TGF₁ and TGF₂ peptides areexpressed in specific cell types during induced follicular maturationin the mouse ovary.     

TCR isoform containing the Fc receptor {gamma} chain exhibits structural and functional differences from isoform containing CD3{xi}

The structure and function of the TCR-CD3 complex containinga homodimer of the gamma chain of the high affinity receptorfor IgE (FcR) (FcR⁺ TCR) was investigated by transfecting theFcR gene into a CD3^–, CD3^–, FcR^– T cell line.Introduction of FcR, as well as CD3, induced a high expressionof the TCR-CD3 complex on the cell surface. Transfected FCRformed a homodimer and associated firmly with the TCRßdimer but only weakly with the CD3. Stimulation of both FcRand CD3 transfectants by antibodies against TCR or CD3 inducedaccumulation of inositol phosphates, the Ca²⁺ response, IL-2production, and growth inhibition. On the other hand, antigenstimulation of transfectants expressing FcR as well as CD3 inducedIL-2 production, but only the latter exhibited the antigen-inducedgrowth inhibition. In vitro kinase assay suggested that theCD3 dimer but not the FcR dimer associates with the Fyn kinase.These results indicate that the FcR homodlmer Is able to forma functional TCR complex but that the mode of assembly and thesignaling function of FcR⁺ TCR, including its association withtyrosine klnase(s), may differ from the TCR-CD3 complex containingCD3 homodimers (⁺ TCR). This provides an example which illustratesthat different TCR isoforms mediate distinct signals and functions.     

{gamma}{delta} T cells promote CD4 and CD8 expression by SCID thymocytes

Molecular studies of the TCR, which is expressed by a minorsubpopulatlon of T lymphocytes in all vertebrate species, havedefined a subset which expresses a receptor with extreme junctionaldiversity and a second subset, most commonly found in eplthella,which expresses a receptor of very limited diversity. In thedeveloping murine thymus, T cells appear in an ordered sequenceof specific v rearrangements, V3V, 1 on day 14, V2V1 on day17, and subsequently V4V5, V6, or V7. We demonstrate that thetransfer of expanded populations of cells from newborn thymusand cell lines expressing the invariant V3V1 receptor into SCIDmice, which lack T and B cells, results in the appearance ofCD3^–CD4⁺CD8⁺ thymocytes. Thus, one role of the early appearingV3V1 T cells in thymlc development in vivo is to promote CD4and CO8 surface expression on precursor cells.     

Molecular aspects of implantation: Human trophoblast adhesion to matrix proteins: inhibition and signal transduction

At the time of implantation, the extracellular matrix proteinslaminin and fibronectin are abundant in the decidua and aredistributed pericellularly around each individual stromal cell.First trimester human trophoblast expresses both laminin andfibronectin receptors, specifically the ₁₁, ₅₁, ₆₁ and ₆₄ integrinheterodimers. In this study we have demonstrated that in-vitroadhesion of first trimester human trophoblast to purified extracellularmatrix proteins and to purified decidual stromal cell monolayerscan be inhibited by monoclonal antibodies directed against appropriateintegrin subunits and by synthetic peptides containing an arginine-glycine-asparticacid sequence. Monoclonal antibodies (mAbs) to the ₅ and ₁ integrinsubunits and a synthetic peptide significantly inhibited adhesionto fibronectin. Binding of trophoblast to laminin was blockedwith mAbs to the ₆ and ₁ but not ₁ and ₄ integrin subunits.Similarly, integrin-mediated adhesion to monolayers of decidualstromal cells could be blocked with mAbs to the ₅, ₆, ₁ and₄ integrin subunits. Integrin-mediated signal transduction innormal and malignant trophoblast was investigated by Westernblotting. A 115 kDa protein was the major tyrosine phosphorylatedprotein detected in trophoblast after binding to laminin orfibronectin. The profile of tyrosine phosphorylated proteinsdiffered for malignant trophoblast.     

Alterations in mitochondrial membrane potential during preimplantation stages of mouse and human embryo development

Mitochondria are cellular organelles regulating metabolism andcell death pathways. This study examined changes in mitochondrialmembrane potential (m) throughout the stages of preimplantationdevelopment in mouse embryos conceived either in vivo or invitro and human embryos donated to research from IVF. Embryosstained with the m-sensitive dye (JC-1) were quantified forthe ratio of high- to low-polarized mitochondria using a deconvolutionmicroscope. Overall, mouse zygotes and early embryos containa subset of high-polarized mitochondria with a progressive increasein the ratio of m observed with increasing cleavage. A transientincrease in the ratio of high to low m was observed in in vivofertilized 2-cell stage embryos, coincident with embryonic genomeactivation in the mouse, but not in 2-cell embryos obtainedthrough IVF. We further observed that arrested mouse 2-cellembryos possessed an increased ratio of m compared with non-arrestedembryos. In human 8-cell embryos we observed an increased ratioof high- to low-polarized mitochondria with increasing degreesof embryo fragmentation. We concluded that the pattern of mitochondrialmembrane potential progressively changes throughout preimplantationdevelopment, and that an aberrant shift in m could contributeto, or is associated with, decreased developmental potential.     

Emigration of selected subsets of {gamma}{delta}+ T cells from the adult murine thymus

Cells bearing the form of the TCR make up only 1–3% ofT cells in the adult murine thymus and peripheral lymphold organs.Evidence from studies of nude mice suggests that the developmentof at least some T cells is thymus dependent; however, untilnow it has not been directly demonstrated that cells are exportedfrom the thymus. In this paper we have used the technique oflabelling thymocytes in vivo with FITC, followed by flow cytometrlcanalysis to trace cells emigrating from the thymus to the spleen.Using this approach we have been able to demonstrate for thefirst time that T cells are exported from the adult murinethymus to the spleen. We also demonstrate that the cells emigratingto the spleen are a selected subset of thymocytes being heatstable antigen positive, Thy-1⁺, and expressing low levels ofCD44 (Pgp-1). In addition, investigation of TCR V; gene usageamong adult ⁺ thymocytes, recent emigrants, and spleen cells,indicated a selective emigration of cells expressing certainVgenes.     

Peripheral lymphoid development and function in TCR mutant mice

We describe the development and function of the peripheral lymphoidsystem of mutant mice rendered deficient in either ßor T cells via targeting of TCR genes In embryonic stem cells.In the spleen of ß T cell-deficient mice, T cellsdo not compensate in numbers for the lack of ß Tcells, but B cells do. ß T cell-deficient mice areunable to mount an antibody response to ovalbumln and do notreject skin allografts. Natural killer cell function is notimpaired in any of the mutant mice. TCR mutant mice will proveuseful in dissecting differential functions of ßand T cells in vivo.     

Immunology: {beta}2-Glycoprotein I-dependent and -independent anticardiolipin antibodies in healthy pregnant women

The purpose of this study was to determine the association between₂-glycoprotein I (₂GPI)-dependent anticardiolipin antibodies(aCL) and ₂GPI-independent aCL and their respective relevanceto adverse pregnancy outcomes. Therefore, we prospectively studied210 normal pregnant women, utilizing a modified enzyme-linkedimmunosorbent assay method for ₂GPI-dependent and -independentaCL. Seven of the 210 pregnant women (3.3%) demonstrated evidencefor ₂GPI-independent immunoglobulin G (IgG)-aCL. Two patients,who also appeared positive for ₂GPI-dependent IgG-aCL, wereproven to be false positives. Amongst the 210 patients, notone was thus positive for ₂GPI-dependent aCL. Women with ₂GPI-independentaCL demonstrated no adverse pregnancy outcomes. These resultssuggest that the presence of ₂GPI-independent aCL is not associatedwith the presence of 2GPI-dependent aCL, though it may giverise to false positive results. Since the presence of 2GPI-independentaCL does not appear to be associated with adverse pregnancyoutcomes, ₂GPI-dependent assays may represent better markersof miscarriage risk.     

The development of dermatitis infiltrated by {gamma}{delta} T cells in IL-7 transgenic mice

Transgenic mice constitutively expressing IL-7 developed severedermatitis with erythroderma and alopecia. The skin lesionswere characterized by massive infiltration of mononuclear cells.Immunofluorescence staining showed that most of the infiltratingcells were T cells with the majority bearing the TCR otherthan the V5 moiety. Furthermore, the number of T cells hadincreased in the lymphold organs of the dermatitis animals.These findings idicate the strong relationship between the expressionof IL-7 and the development of T cells in vivo and the pathologicalinvolvement of proliferated and/or activated T cells in skindisease.