Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality.
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Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice. Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function. (+info)
Morphological domains of Lewis-X/FORSE-1 immunolabeling in the embryonic neural tube are due to developmental regulation of cell surface carbohydrate expression.
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The Lewis-X (LeX) carbohydrate epitope, recognized by the FORSE-1 monoclonal antibody (mAb), shares expression boundaries with neural regulatory genes and may be involved in patterning the neural tube by creating domains of differential cell adhesion. The present experiments focus on the question of what determines the expression pattern of LeX in embryonic rat brain. Comparisons of FORSE-1-positive glycolipid and protein antigens in embryonic, early postnatal, and adult tissues show that the LeX epitope is carried primarily by glycolipids during embryonic development and by a proteoglycan and glycoproteins in postnatal and adult tissue. Immunohistochemistry using FORSE-1 and an antibody to the proteoglycan phosphacan, which carries LeX, shows that the distribution of LeX is more restricted than phosphacan. These observations suggest that the precise spatial regulation of FORSE-1 binding in the embryonic forebrain is due to the expression pattern of the LeX carbohydrate on glycolipids, rather than to the transcriptional regulation of a carrier protein. (+info)
Overexpression of ptc1 inhibits induction of Shh target genes and prevents normal patterning in the neural tube.
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Patched (Ptc) is a human tumor suppressor protein and a candidate receptor for Hedgehog (Hh) proteins, which regulate growth and patterning in embryos. Ptc represses expression of Hh target genes such as Gli1 and ptc1 itself. Localized secretion of Hh appears to induce transcription of target genes in specific patterns by binding to Ptc and preventing it from functioning in recipient cells. People who are heterozygous for PTC1 exhibit a range of developmental defects, suggesting that some genes are inappropriately expressed when there is not enough Ptc protein. To test the idea that a balance between Hh and Ptc activities is essential for normal development, we overexpressed Ptc in the neural tube. We find that excess Ptc is sufficient to inhibit expression of Gli1 and ptc1, suggesting that Sonic hedgehog (Shh) cannot signal effectively. This leads to partial dorsalization of the neural tube and a wide spectrum of neural defects, ranging from embryonic lethality to hydrocephaly. (+info)
Tissue-specific expression of dominant negative mutant Drosophila HSC70 causes developmental defects and lethality.
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The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins' dominant negative effects in vivo. (+info)
UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles.
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The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly. (+info)
Protein synthesis-dependent and mRNA synthesis-independent intermediate phase of memory in Hermissenda.
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The conditioned stimulus pathway in Hermissenda has been used to examine the time-dependent mechanisms of memory consolidation following one-trial conditioning. Here we report an intermediate phase of memory consolidation following one-trial conditioning that requires protein synthesis, but not mRNA synthesis. In conditioned animals, enhanced excitability normally expressed during an intermediate phase of memory was reversed by the protein synthesis inhibitor anisomycin, but not by the mRNA synthesis inhibitor 5, 6-dichloro-1-beta-D-ribobenzimidazole (DRB). Associated with the intermediate phase of memory is an increase in the phosphorylation of a 24-kDa protein. Anisomycin present during the intermediate phase blocked the increased phosphorylation of the 24-kDa phosphoprotein, but did not block the increased phosphorylation of other proteins associated with conditioning or significantly change their baseline phosphorylation. DRB did not reverse enhanced excitability or decrease protein phosphorylation expressed during the intermediate phase of memory formation, but it did reverse enhanced excitability 3.5 h after conditioning. Phosphorylation of the 24-kDa protein may support enhanced excitability during the intermediate phase, in the transition period between short- and long-term memory. (+info)
Different contributions of pannier and wingless to the patterning of the dorsal mesothorax of Drosophila.
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In Drosophila, the GATA family transcription factor Pannier and the Wnt secreted protein Wingless are known to be important for the patterning of the notum, a part of the dorsal mesothorax of the fly. Thus, both proteins are necessary for the development of the dorsocentral mechanosensory bristles, although their roles in this process have not been clarified. Here, we show that Pannier directly activates the proneural genes achaete and scute by binding to the enhancer responsible for the expression of these genes in the dorsocentral proneural cluster. Moreover, the boundary of the expression domain of Pannier appears to delimit the proneural cluster laterally, while antagonism of Pannier function by the Zn-finger protein U-shaped sets its limit dorsally. So, Pannier and U-shaped provide positional information for the patterning of the dorsocentral cluster. In contrast and contrary to previous suggestions, Wingless does not play a similar role, since the levels and vectorial orientation of its concentration gradient in the dorsocentral area can be greatly modified without affecting the position of the dorsocentral cluster. Thus, Wingless has only a permissive role on dorsocentral achaete-scute expression. We also provide evidence indicating that Pannier and U-shaped are main effectors of the regulation of wingless expression in the presumptive notum. (+info)
Improvement in specific aspects of neurocognitive performance in children after renal transplantation.
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BACKGROUND: Chronic renal failure in childhood is considered to affect neurocognitive function adversely, and kidney transplantation may ameliorate the deficits. However, previous studies have suffered from the use of poorly matched control groups, comparison of transplant with uncorrected uremia, lack of standardization of dialysis, and insufficiently sensitive neuropsychological tests. METHODS: We studied nine medically stable children and adolescents age 14.2 +/- 3.5 years with end-stage renal disease prior to and again one year after successful renal transplant. At baseline, the Wechsler Intelligence Scale for Children-III (WISC-III) or the Wechsler Adult Intelligence Scale-Revised (WAIS-R) was performed. Repeatable tests used before and after transplant included the Paced Auditory Serial Addition Test (PASAT) or the Children's Paced Auditory Serial Addition Test (CHIPASAT), the Stroop Color-Word Naming Test, the Buschke Selective Reminding Test, the Meier Visual Discrimination Test, the Grooved Pegboard Test, the WISC-III or the WAIS-R Coding subtests and the Trailmaking Test. Computer-based measures of mental processing speed, reaction time, and discrimination sensitivity included the Cognitive Abilities Test (CAT) and the Connors Continuous Performance Test (CPT). Formal kinetic modeling of dialysis delivery ensured adequate renal replacement therapy. Transplant function was good on stable doses of immunosuppressives, without recent rejections at the time of testing. RESULTS: Within-subject comparison showed statistically significant improvement in mental processing speed by CAT, reaction time and discrimination sensitivity by CPT, and working memory by PASAT/CHIPASAT after renal transplant. Other measures were unchanged. CONCLUSION: Mental processing speed and sustained attention improved in children after renal transplantation in a carefully controlled prospective cross-over study. (+info)