Julia R. Currie

Rod outer segments elongate in constant light: Darkness is required for normal shedding

Abstract Shedding of large rod outer segment fragments is greatly reduced when Rana pipiens are maintained under moderate levels of continuous light. This inhibition of shedding results in an increase in outer segment length, since disc addition continues even as shedding stops. Some shedding will occur when constant-light frogs are subjected to a brief period of darkness, suggesting that darkness alone may stimulate shedding. Massive shedding, resulting in a reduction to normal outer segment length, occurs when constant-light animals are maintained for as little as 30 min in darkness and are then returned to constant light. This shedding response will occur even after both optic nerves are severed, and after pinealectomy or hypophysectomy.

The fragile X mental retardation protein interacts with U-rich RNAs in a yeast three-hybrid system ☆

We recently identified several ESTs that bind to the fragile X mental retardation protein (FMRP) in vitro. To determine whether they interacted in vivo we performed three-hybrid screens in a Saccharomyces cerevisiae histidine auxotroph. We demonstrate that two of the ESTs support growth on histidine and transduce beta-galactosidase activity when co-expressed with FMRP under selective growth conditions. In contrast, the iron response element (IRE) RNA does not. Likewise, the ESTs do not support growth or transduce beta-galactosidase activity when co-expressed with the iron response element binding protein (IRP). Each EST is relatively small and has 40% identity with a sequence in FMR1 mRNA harboring FMRP binding determinants. Interestingly, while neither the ESTs contain a G-quartet structural motif they do contain U-rich sequences that are found in mRNA with demonstrated in vitro binding and in vivo association with FMRP. This indicates that U-rich elements comprise another motif recognized by FMRP.

Developmentally regulated plasmalemmal glycoconjugates of the surface and neural ectoderm

The plasmalemmal glycoconjugates of the ectoderm surrounding the rat embryos caudal neuropore were mapped at the ultrastructural level, using various lectin probes. These included the agglutinins of wheat germ, soybean, Ricinus communis, Lotus tetragonolobus, and Canavalia ensiformis. Each lectin produced a characteristic binding pattern. Comparison of precursor cells of surface ectoderm, neural crest, and neural tube revealed that, even prior to neural tube formation, these three cell types can be distinguished by the sets of lectin receptors they express on their apical plasmalemma. The high density of lectin receptors found at the open neural groove level decreases dramatically during neurulation. Further changes in surface glycoconjugates must occur during neuronal differentiation because sprouting neurons exhibit yet another lectin binding pattern (K.H. Pfenninger, M.-F. Maylié-Pfenninger, L. B. Friedman, and P. Simkowitz, 1984, Dev. Biol. 106, 97-108). These results indicate that the commitment of ectodermal cells to diverging lineages (epidermis, neural crest, and tube) is reflected in their surface carbohydrates and occurs while they are still part of a continuous epithelial sheet. Furthermore, the plasmalemmal glycoconjugates of the ectoderm are developmentally regulated, and particularly dramatic changes in glycoconjugates expression are linked to neurulation.

KH domain-containing proteins of yeast: absence of a fragile X gene homologue.

The KH domain is a region defined by its homology to the RNA-binding domains of the heterogeneous nuclear ribonucleoprotein K (hnRNPK). There are two such domains in the FMR1 protein which is underexpressed in the fragile X syndrome. We developed a computer method to search the S. cerevisiae protein sequences as they became available for the KH domain of the FMR1 protein. Using our motif and FINDPATTERNS of the Wisconsin Package of GCG, nine proteins were identified in the completed yeast ORF database that contain KH domains. Five proteins have known or predicted functions; four await functional analysis. Using GeneWorks and GeneJockeyII alignments, we found that the yeast protein KH domain showing the most similarity to either FMR1P KH domain was a KH domain in HX/SCP160. Its sequence is 50% identical to the second KH domain of FMR1P. However, SCP160 contains eight conserved and six degenerate KH domains. Further analysis showed that SCP160 is a better match overall to the vertebrate and C. elegans protein Vigilin, which also contains 14 KH domains. The next most similar yeast KH domain was found in YB83, a protein shorter than FMR1P and containing three KH domains, one of which shares 45% identity with the second KH domain in FMR1P. There is no significant overall sequence similarity between this yeast protein and FMR1P. Thus, while several proteins in yeast contain KH domains, no apparent yeast homologue exists for the FMR1 protein of the fragile X gene family.

Reduction of histone cytotoxicity by the Alzheimer β-amyloid peptide precursor

Abstract In a search for Alzheimer β-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little β-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD50=1.5 μM). Extracellular Aβ precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD50=5.4 μM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD50=3.2 μM) than cells that expressed the vector alone. Histone H4 binds to cell-associated Aβ precursor. Cells expressing the Aβ precursor-directed ribozyme bound less 125I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface Aβ precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.