Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain

The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase. We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry. Ser(60), Thr(64), and Thr(68) are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo. This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser(36), is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser(36) are located within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.     

Missense mutations in CRELD1 are associated with cardiac atrioventricular septal defects

Atrioventricular septal defects (AVSD) are common cardiovascular malformations, occurring in 3.5/10,000 births. Although frequently associated with trisomy 21, autosomal dominant AVSD has also been described. Recently we identified and characterized the cell adhesion molecule CRELD1 (previously known as "cirrin") as a candidate gene for the AVSD2 locus mapping to chromosome 3p25. Analysis of the CRELD1 gene from individuals with non-trisomy 21-associated AVSD identified heterozygous missense mutations in nearly 6% of this population, including mutations in isolated AVSD and AVSD associated with heterotaxy syndrome. CRELD1 is the first human gene to be implicated in the pathogenesis of isolated AVSD and AVSD in the context of heterotaxy, which provides an important step in unraveling the pathogenesis of AVSD.     

Age-dependent expression of glucocorticoid- and mineralocorticoid receptors on neural precursor cell populations in the adult murine hippocampus

Steroid hormones are regulators of adult hippocampal neurogenesis and are central to hypotheses regarding adult neurogenesis in age-related and psychiatric disturbances associated with altered hippocampal plasticity--most notably dementias and major depression. Using immunohistochemistry, we examined the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors during adult hippocampal neurogenesis. In young mice only 27% of dividing cells in the subgranular zone expressed GR, whereas 4 weeks after division 87% had become positive for GR and MR. GR was expressed by 50% of the radial glia-like type-1 and type-2a progenitor cells, whereas MR was expressed only by mature calbindin-positive granule cells. Doublecortin-positive neuronal progenitor cells (type-2b) and early postmitotic calretinin-positive neurons were devoid of GR and MR expression. Fifty per cent of the intermediate type-3 cells showed GR expression, possibly reflecting cells terminating maturation. Thus, all subpopulations of dividing precursor cells showed an identical receptor profile (50% GR, no MR), except for type-2b cells, which expressed neither receptor. There was also no overlap between calretinin and GR early postnatally (P8) or after physical activity or exposure to an enriched environment, both of which are potent neurogenic stimuli. In contrast, in old age calretinin-positive young neurons became GR and MR positive, suggesting increased steroid sensitivity. Age also increased the expression of GR in type-1 and type-2a precursor cells. Other intermediates were so rare in old age that they could not be studied. This course and variability of receptor expression in aging might help to explain differential vulnerability of adult neural precursor cells to corticoid-mediated influences.     

Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch

The presenilin (PS)-dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar γ-secretase cleavage of the β-amyloid precursor protein (βAPP) results in the secretion of amyloid β-peptide (Aβ). However, little is known about the corresponding C-terminal cleavage product (CTFγ). We have now identified CTFγ in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFγ is facilitated by PSs, since a dominant-negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, γ-secretase inhibitors, including one that is known to bind to PS, also block CTFγ generation. Sequence analysis revealed that CTFγ is produced by a novel γ-secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.     

Increased blood levels of human S100 in melanoma chick embryo xenografts' circulation

The chorioallantoic membrane (CAM) of the fertilized egg allows grafting of human melanomas for short-term investigations and offers the opportunity to investigate the behavior of metastasizing cells and the release of S100beta into peripheral blood. Tissue from one primary melanoma as well as cutaneous and subcutaneous metastases of 10 melanoma patients with elevated levels of S100 in the peripheral blood before surgery were transplanted onto the CAM of chick embryos at day 5/6 of development. Grafts were nourished by the host blood supply 2 days after transplantation. Histologically, 3 days after grafting, metastasizing melanoma cells could be found near the vessels of the host membrane, penetrating the endothelial layer and entering the blood system. Growth conditions remained stable for 6 days after transplantation. Blood samples were taken from a larger CAM vessel before collecting the xenografts 5 days after grafting. Measurement of human S100 in peripheral blood was performed in a blinded manner. No negative control showed elevated levels of human S100 protein. Samples deriving from melanoma xenografts contained highly elevated levels of S100 protein in 80% of cases. The data strongly support the concept of graft-host interaction concerning adherence of tumors and extravasation of human melanoma cells.     

A New Genus and Species of the Family Anulidentaliidae (Scaphopoda: Dentaliida) and its Systematic Implications

Shell, radula, and anatomy of Epirhabdoides ivanovi new genusand species are described from a sample of the Russian VitjazExpedition from the Japan Trench. It is distinguished from thesimilar Laevidentalium sominium by shell morphometrics and radulamorphology. The radula is almost identical with that of Anulidentaliumbambusa (Anulidentaliidae). The anatomy of the mantle margins,however, including dorsolateral slits at the anterior mantlemargin and a connective tissue bolster at the posterior mantleopening is that of the family Rhabdidae. In a parsimony analysisEpirhabdoides ivanovi takes an intermediate position betweena basal grade of Gadilinidae and the remaining Dentaliida implyingconvergent evolution of mantle characters. An alternative butless parsimonious tree with E. ivanovi as sister taxon to Rhabdusrequires convergences in radula characters. This is the firstdocumented case of convergent anatomical features among Scaphopodaand enhances the need of radula and soft part investigationof the conchologically little informative, smooth-shelled dentaliidgroups. (Received 16 March 1998; accepted 1 June 1998)     

An in vivo assay for the identification of target proteases which cleave membrane-associated substrates

Proteases not only play a fundamental role in numerous physiological processes, but are also involved in several human diseases including Alzheimer's disease (AD). A key protease implicated in AD is the so far unidentified gamma-secretase, which cleaves the membrane-bound beta-amyloid precursor protein (betaAPP) at the C-terminus of its amyloid domain within the membrane to release the neurotoxic amyloid beta-peptide. In order to allow the isolation of proteases, which specifically cleave membrane-bound substrates within or in the vicinity of a transmembrane domain, we developed a reporter gene assay in Saccharomyces cerevisiae. This assay may allow the identification of genes encoding target proteases that specifically cleave membrane bound substrates by transforming expression libraries.