The Schizosaccharomyces pombe rad1 gene consists of three exons and the cDNA sequence is partially homologous to the Ustilago maydis REC1 cDNA.

We show that the rad1 gene of Schizosaccharomyces pombe is comprised of three exons and encodes a protein of 37 kDa. A cDNA clone containing these three exons complements the sensitivity of the rad1-1 mutant to ultraviolet and gamma-radiation and to hydroxyurea. The newly identified ORF of the rad1 gene was found to exhibit partial homology to the REC1 gene of Ustilago maydis. These two genes share putative functional similarities in their respective organisms.

Incomplete ecdysis is an indicator of ecdysteroid exposure in Daphnia magna.

Daphnia magna were exposed for 21 d to the ecdysteroids, 20-hydroxyecdysone (20-E), the accepted molting hormone, and ponasterone A (PoA), an ecdysteroid found in some crustaceans and many plants. Daphnids were monitored for alterations in molting, fecundity, and survival time. The 20-E elicited no significant effects on molting frequency, and its significant effects on reproduction were only at concentrations (260 nM) associated with premature death caused by incomplete ecdysis. We also examined PoA, which has been reported to have 10x higher affinity for the ecdysone receptor than 20-E. Ponasterone A elicited effects similar to those of 20-E at approximately 10x lower concentrations. This suggests that affinity for the receptor is the major parameter determining activity in vitro and in vivo and that differences in metabolism and elimination in vivo were not significant. The effects of PoA on daphnids mimicked those of 20-E except PoA reduced fecundity in the second generation and 20-E had no effect. Last, both exogenous 20-E and PoA show similar effects, including premature death associated with incomplete ecdysis, and the overall difference in toxicity is mostly likely due to receptor affinity.

The role of endocytosis in regulating L1-mediated adhesion.

L1 is a neural cell adhesion molecule critical for neural development. Full-length L1 (L1(FL)) contains an alternatively spliced cytoplasmic sequence, RSLE, which is absent in L1 expressed in nonneuronal cells. The RSLE sequence follows a tyrosine, creating an endocytic motif that allows rapid internalization via clathrin-mediated endocytosis. We hypothesized that L1(FL) would internalize more rapidly than L1 lacking the RSLE sequence (L1(Delta)(RSLE)) and that internalization might regulate L1-mediated adhesion. L1 internalization was measured by immunofluorescence microscopy and by uptake of (125)I-anti-rat-L1 antibody, demonstrating that L1(FL) is internalized 2-3 times faster than L1(Delta)(RSLE). Inhibition of clathrin-mediated endocytosis slowed internalization of L1(FL) but did not affect initial uptake of L1(Delta)(RSLE). To test whether L1 endocytosis regulates L1 adhesion, cell aggregation rates were tested. L1(Delta)(RSLE) cells aggregated two times faster than L1(FL) cells. Inhibition of clathrin-mediated endocytosis increases the aggregation rate of the L1(FL) cells to that of L1(Delta)(RSLE) cells. Our results demonstrate that rapid internalization of L1 dramatically affects L1 adhesion.

Time-dependent loss of surface complement regulatory activity during storage of donor blood.

The survival of transfused red cells (RBCs) diminishes with time of in vitro storage in blood banks, but the molecular mechanisms underlying the slow but incessant deterioration are incompletely understood. To investigate the possibility that impaired resistance to autologous complement attack could play a role in this phenomenon, packed RBCs stored for variable periods were assayed for decay-accelerating factor (DAF) and CD59, two glycoinositol-phospholipid (GPI)-anchored, membrane-associated complement regulatory proteins that function physiologically to protect blood cells from autologous complement activation on their surfaces. Immunoradiometric and flow cytometric assays employing DAF and CD59 monoclonal antibodies showed that levels of both surface proteins gradually declined over 6 weeks. Digestion analyses with phosphatidylinositol-specific phospholipase C, an enzyme that releases GPI-anchored proteins from cell surfaces, showed that DAF and CD59 molecules with GPI anchors containing unacylated inositol were preferentially lost. These findings suggest: 1) that DAF and CD59 molecules with acylated GPI anchors are more stable in RBC membranes than are molecules with unacylated GPI anchors, and 2) that DAF and CD59 loss may participate with other membrane alterations that occur during in vitro storage in compromising the survival of transfused cells.

Effect of glycoinositolphospholipid anchor lipid groups on functional properties of decay-accelerating factor protein in cells.

The inositol ring in the glycoinositolphospholipid (GPI) anchor of human decay-accelerating factor (DAF) is unmodified in nucleated cells, whereas it is fatty acid acylated in erythrocytes (Ehu). To assess the effect of this and of the glycerol sn-2-associated acyl substituent on the abilities of DAF to cell membrane incorporate and function, 1) endogenous (physiologically anchored) DAF proteins bearing three- and two-"footed" GPI anchors were purified from Ehu and HeLa cells and 2) synthetic DAF variants bearing alternative one- "footed" anchors (retaining either the sn-1 glycerol- or inositol-associated lipid) were prepared by alkaline hydroxylamine treatment and phosphatidylinositol-specific phospholipase D digestion of Ehu DAF, respectively. The different DAF species were added to antibody-sensitized sheep erythrocytes (EshA) and their abilities to insert into the plasma membranes of the cells and control subsequent complement activation on their surfaces were compared. DAF proteins bearing all four GPI anchor structures adhered to the Esh hemolytic intermediates and inhibited expression of C3 convertase (C4b2a) activity. However, mixing of DAF-treated EshA with untreated EshAC142 and stripping of cell-associated DAF proteins with vesicles showed that only the physiologically anchored proteins remained stably associated with the lipid bilayer and functioned intrinsically. Both three- and two-"footed" Ehu and HeLa DAF proteins exhibited comparable ability to incorporate and function in the intermediates as well as to accumulate to levels 1000-fold higher/cell in Schistosoma mansoni schistosomula. These findings indicate that 1) an intact inositolphospholipid-containing GPI anchor is necessary for stable membrane integration and intrinsic function, 2) endogenous GPI anchors (with either unsubstituted and acylated inositol) incorporate and function with comparable efficiency, and 3) the transfer of either endogenous DAF form can account for the previously described circumvented uptake of human C3b by blood stage schistosomula.

The neural cell adhesion molecule L1 interacts with the AP-2 adaptor and is endocytosed via the clathrin-mediated pathway.

Cell-cell interactions mediated via cell adhesion molecules (CAMs) are dynamically regulated during nervous system development. One mechanism to control the amount of cell surface CAMs is to regulate their recycling from the plasma membrane. The L1 subfamily of CAMs has a highly conserved cytoplasmic domain that contains a tyrosine, followed by the alternatively spliced RSLE (Arg-Ser-Leu-Glu) sequence. The resulting sequence of YRSL conforms to a tyrosine-based sorting signal that mediates clathrin-dependent endocytosis of signal-bearing proteins. The present study shows that L1 associates in rat brain with AP-2, a clathrin adaptor that captures plasma membrane proteins with tyrosine-based signals for endocytosis by coated pits. In vitro assays demonstrate that this interaction occurs via the YRSL sequence of L1 and the mu 2 chain of AP-2. In L1-transfected 3T3 cells, L1 endocytosis is blocked by dominant-negative dynamin that specifically disrupts clathrin-mediated internalization. Furthermore, endocytosed L1 colocalizes with the transferrin receptor (TfR), a marker for clathrin-mediated internalization. Mutant forms of L1 that lack the YRSL do not colocalize with TfR, indicating that the YRSL mediates endocytosis of L1. In neurons, L1 is endocytosed preferentially at the rear of axonal growth cones, colocalizing with Eps15, another marker for the clathrin endocytic pathway. These results establish a mechanism by which L1 can be internalized from the cell surface and suggest that an active region of L1 endocytosis at the rear of growth cones is important in L1-dependent axon growth.

Amyloid beta protein (A beta) in Alzheimer's disease brain. Biochemical and immunocytochemical analysis with antibodies specific for forms ending at A beta 40 or A beta 42(43).

Biochemical and immunocytochemical analyses were performed to evaluate the composition of the amyloid beta protein (A beta) deposited in the brains of patients with Alzheimer's disease (AD). To quantitate all A beta s present, cerebral cortex was homogenized in 70% formic acid, and the supernatant was analyzed by sandwich enzyme-linked immunoabsorbent assays specific for various forms of A beta. In 9 of 27 AD brains examined, there was minimal congophilic angiopathy and virtually all A beta (96%) ended at A beta 42(43). The other 18 AD brains contained increasing amounts of A beta ending at A beta 40. From this set, 6 brains with substantial congophilic angiopathy were separately analyzed. In these brains, the amount of A beta ending at A beta 42(43) was much the same as in brains with minimal congophilic angiopathy, but a large amount of A beta ending at A beta 40 (76% of total A beta) was also present. Immunocytochemical analysis with monoclonal antibodies selective for A beta s ending at A beta 42(43) or A beta 40 confirmed that, in brains with minimal congophilic angiopathy, virtually all A beta is A beta ending at A beta 42(43) and showed that this A beta is deposited in senile plaques of all types. In the remaining AD brains, A beta 42(43) was deposited in a similar fashion in plaques, but, in addition, widely varying amounts of A beta ending at A beta 40 were deposited, primarily in blood vessel walls, where some A beta ending at A beta 42(43) was also present. These observations indicate that A beta s ending at A beta 42(43), which are a minor component of the A beta in human cerebrospinal fluid and plasma, are critically important in AD where they deposit selectively in plaques of all kinds.