Conditional control of selectin ligand expression and global fucosylation events in mice with a targeted mutation at the FX locus

Glycoprotein fucosylation enables fringe-dependent modulation of signal transduction by Notch transmembrane receptors, contributes to selectin-dependent leukocyte trafficking, and is faulty in leukocyte adhesion deficiency (LAD) type II, also known as congenital disorder of glycosylation (CDG)-IIc, a rare human disorder characterized by psychomotor defects, developmental abnormalities, and leukocyte adhesion defects. We report here that mice with an induced null mutation in the FX locus, which encodes an enzyme in the de novo pathway for GDP–fucose synthesis, exhibit a virtually complete deficiency of cellular fucosylation, and variable frequency of intrauterine demise determined by parental FX genotype. Live-born FX(−/−) mice exhibit postnatal failure to thrive that is suppressed with a fucose-supplemented diet. FX(−/−) adults suffer from an extreme neutrophilia, myeloproliferation, and absence of leukocyte selectin ligand expression reminiscent of LAD-II/CDG-IIc. Contingent restoration of leukocyte and endothelial selectin ligand expression, general cellular fucosylation, and normal postnatal physiology is achieved by modulating dietary fucose to supply a salvage pathway for GDP–fucose synthesis. Conditional control of fucosylation in FX(−/−) mice identifies cellular fucosylation events as essential concomitants to fertility, early growth and development, and leukocyte adhesion

Phosphorylation State of Olig2 Regulates Proliferation of Neural Progenitors

SummaryThe bHLH transcription factors that regulate early development of the central nervous system can generally be classified as either antineural or proneural. Initial expression of antineural factors prevents cell cycle exit and thereby expands the pool of neural progenitors. Subsequent (and typically transient) expression of proneural factors promotes cell cycle exit, subtype specification, and differentiation. Against this backdrop, the bHLH transcription factor Olig2 in the oligodendrocyte lineage is unorthodox, showing antineural functions in multipotent CNS progenitor cells but also sustained expression and proneural functions in the formation of oligodendrocytes. We show here that the proliferative function of Olig2 is controlled by developmentally regulated phosphorylation of a conserved triple serine motif within the amino-terminal domain. In the phosphorylated state, Olig2 maintains antineural (i.e., promitotic) functions that are reflected in human glioma cells and in a genetically defined murine model of primary glioma

Chicken IgL gene rearrangement involves deletion of a circular episome and addition of single nonrandom nucleotides to both coding segments

Chicken immunoglobulin light chain (IgL) gene rearrangement has been characterized. Rearrangement of the single variable (VL) segment with the single joining (JL) segment within the chicken IgL locus results in the deletion of the DNA between VL and JL from the genome. This deletion is accomplished by a molecular mechanism in which a precise joining of the IgL recombination signal sequences leads to the formation of a circular episomal element. The circular episome is an unstable genetic element that fails to be propagated during B cell development. Evidence was obtained that the formation of the circular episome is accompanied by the addition of a single nonrandom base to both the VL and JL coding segments. The subsequent joining of the VL and JL segments appears to occur at random, as we observed at least 25 unique V-J junction sequences, 11 of which are out-of-frame. A novel recombination mechanism that accounts for the observed features of chicken IgL gene rearrangement is discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28019/1/0000455.pd

Regulation of fucosyltransferase-VII expression in peripheral lymph node high endothelial venules

Binding of L-selectin to the highly glycosylated peripheral lymph node addressins (PNAd) plays a central role in the normal recirculation of lymphocytes between the bloodstream and the lymph node. This interaction requires correct fucosylation of the PNAd, mediated by the recently identified fucosyltransferase-VII (Fuc-TVII). Here we show that during ontogeny Fuc-TVII is absent at the day of birth, barely detectable on day 1, and clearly present from day 2 onwards. PNAd expression as detected by the MECA-79 antibody precedes the expression of Fuc-TVII. Furthermore, we demonstrate that in adult mice antigenic stimulation of peripheral lymph nodes leads to a temporary disappearance of Fuc-TVII at days 2 and 3 after stimulation, followed by a complete reappearance by day 4, while expression of MECA-79 is never completely absent during this period. Finally, occlusion of afferent lymphatics to peripheral lymph nodes resulted in a decreased expression of Fuc-TVII in the high endothelial venules by day 5, and complete disappearance within 8 days. We conclude that the activity of Fuc-TVII in cells of high endothelial venules is directly affected by afferent lymph and activation processes that occur in the lymph node after antigenic stimulation. The expression of Fuc-TVII is therefore yet another level at which the function of high endothelial venules, and thus lymphocyte trafficking, can be regulated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48706/1/3040_ftp.pd

The α(1,3)fucosyltransferases FucT-IV and FucT-VII Exert Collaborative Control over Selectin-Dependent Leukocyte Recruitment and Lymphocyte Homing

AbstractE-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by α(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV−/−/FucT-VII−/− mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII−/− mice is revoked in FucT-IV−/−/FucT-VII−/− mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing

Expression analysis of carbohydrate antigens in ductal carcinoma in situ of the breast by lectin histochemistry

<p>Abstract</p> <p>Background</p> <p>The number of breast cancer patients diagnosed with ductal carcinoma <it>in situ </it>(DCIS) continues to grow. Laboratory and clinical data indicate that DCIS can progress to invasive disease. Carbohydrate-mediated cell-cell adhesion and tumor-stroma interaction play crucial roles in tumorigenesis and tumor aggressive behavior. Breast carcinogenesis may reflect quantitative as well as qualitative changes in oligosaccharide expression, which may provide a useful tool for early detection of breast cancer. Because tumor-associated carbohydrate antigens (TACA) are implicated in tumor invasion and metastasis, the purpose of this study was to assess the expression of selected TACA by lectin histochemistry on DCIS specimens from the archival breast cancer tissue array bank of the University of Arkansas for Medical Sciences.</p> <p>Methods</p> <p>For detection of TACA expression, specimens were stained with <it>Griffonia simplicifolia </it>lectin-I (GS-I) and <it>Vicia vilosa </it>agglutinin (VVA). We studied associations of lectin reactivity with established prognostic factors, such as tumor size, tumor nuclear grade, and expression of Her-2/neu, p53 mutant and estrogen and progesterone receptors.</p> <p>Results</p> <p>We observed that both lectins showed significant associations with nuclear grade of DCIS. DCIS specimens with nuclear grades II and III showed significantly more intense reactivity than DCIS cases with nuclear grade I to GS-1 (Mean-score chi-square = 17.60, DF = 2; <it>P </it>= 0.0002) and VVA (Mean-score chi-square = 15.72, DF = 2; <it>P </it>= 0.0004).</p> <p>Conclusion</p> <p>The results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk.</p

Directing Astroglia from the Cerebral Cortex into Subtype Specific Functional Neurons

Forced expression of single defined transcription factors can selectively and stably convert cultured astroglia into synapse-forming excitatory and inhibitory neurons

Elevated intracellular calcium concentration increases secretory processing of the amyloid precursor protein by a tyrosine phosphorylation-dependent mechanism.

Secretory cleavage of the amyloid precursor protein (APP), a process that releases soluble APP derivatives (APPs) into the extracellular space, is stimulated by the activation of muscarinic receptors coupled to phosphoinositide hydrolysis. The signalling pathways involved in the release process exhibit both protein kinase C- and protein tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337-8344]. The possibility that elevations in intracellular Ca2+ concentration initiate the tyrosine phosphorylation-dependent release of APPs was examined in human embryonic kidney cells expressing muscarinic m3 receptors. Inhibition of protein kinase C with the bisindolylmaleimide GF 109203X decreased the carbachol-evoked release of APPs by approx. 30%, as shown previously. The residual response was further decreased, in an additive manner, by the Ca2+ chelator EGTA, or by the tyrosine kinase inhibitor tyrphostin A25. The Ca2+ ionophore, ionomycin, like carbachol, stimulated both the release of APPs and the tyrosine phosphorylation of several proteins, one of which was identified as paxillin, a component of focal adhesions. The effects of ionomycin on APPs release and on protein tyrosine phosphorylation were concentration-dependent, and occurred over similar concentration ranges; both effects were inhibited only partly by GF 109203X, but were abolished by EGTA or by tyrosine kinase inhibitors. The results demonstrate for the first time that ionophore-induced elevations in intracellular Ca2+ levels elicit APPs release via increased tyrosine phosphorylation. Part of the increase in APPs release evoked by muscarinic receptor activation might be attributable to a similar mechanism