Biomedical subjects

R Bowser

Publications and source records attributed to R Bowser.

31 records · Page 2Linked to original sources

PubMed1996

Identification and analysis of the complete cDNA sequence for the human FAC1 gene.

The complete cDNA sequence for the FAC1 gene has been isolated from human fetal brain. The FAC1 cDNA encodes for a protein of 810 amino acids that contains a putative Zn(2+)-finger binding domain, nuclear transport signals and a charged acidic domain that may function in protein-protein interactions. Identification of the complete cDNA and protein sequence of FAC1 permits further insight into potential functions for this developmentally regulated gene product.

Amino Acid Sequence↗

PubMed1996

Ironic effects of sleep urgency.

Normal sleepers were instructed either to fall asleep as quickly as they could or to fall asleep whenever they desired, under a high mental load (listening to John Philip Sousa marches) or a low mental load (listening to sleep-conducive new age music). Under low load, participants trying to fall asleep quickly did so faster than those attempting only to fall asleep whenever they desired. Under high load, however, and consistent with the ironic process theory of mental control (Wegner, D. M., 1994, Psychological Review, 101, 34-52), sleep onset latency was greater for participants attempting to fall asleep quickly than for those not attempting to do so.

Female↗

PubMed1996

Immunolocalization and redistribution of the FAC1 protein in Alzheimer's disease.

The presence of senile plaques and neurofibrillary tangles are hallmark neuropathologic features of Alzheimer's disease (AD). Many proteins have previously been immunolocalized to amyloid-containing plaques in AD brain. Using a monoclonal antibody to a recently described developmentally regulated gene product, we demonstrate the presence of FAC1 protein in a subset of diffuse and neuritic plaques in AD brain. FAC1 is not observed in neurofibrillary tangles common in the hippocampus or entorhinal cortex, nor is it localized in diffuse plaques of nondemented elderly control subjects. FAC1 protein is also immunolocalized in swollen dendrites of hippocampal pyramidal cells observed in some cases of early stage AD. Therefore, FAC1 is a novel protein localized in early pathologic features of AD and in a subset of plaques.

Aged↗

PubMed1995

Binding of Alz 50 depends on Phe8 in tau synthetic peptides and varies between native and denatured tau proteins.

Alz 50 is a monoclonal antibody that in Western blotting analysis recognizes both normal tau as well as hyperphosphorylated tau proteins associated with paired helical filaments (PHF-tau) in Alzheimer disease (AD). Within tissue sections of AD brain, however, Alz 50 immunolabels only PHF, which suggests that the antibody recognizes a conformational epitope. Using competitive enzyme-linked immunosorbent assay, we demonstrate that Alz 50 binds to tau synthetic peptides with low affinity (KD between 0.27 to 2.7 x 10(-5) M) and that the binding is specific for the RQEF sequence corresponding to N-terminal residues 5-8 of tau. The Alz 50 epitope appears to be largely dependent on Phe8, a strongly hydrophobic amino acid residue, since the substitution of Phe8 with Ala8 in the synthetic peptide abolishes Alz 50 binding. The effects of tau conformation on Alz 50 binding were studied with various normal tau proteins with either low or high phosphate content (adult vs. fetal) and PHF-tau proteins. The normal tau fractions were isolated from both adult and fetal human brains using affinity chromatography (native form) and heat/perchloric acid treatments (denatured form). PHF-tau was isolated as Sarcosyl-insoluble fraction. With competitive ELISA, the denatured form of normal tau (fetal and adult) bound Alz 50 with the same high affinity as did PHF-tau (KD between 1.3 to 1.8 x 10(-7) M). In contrast, the native form of tau from either brain was unable to fully compete for Alz 50 and at most only 50% of the Alz 50 binding sites in native tau were occupied. These results suggest that native tau may exist either in complexes with other proteins or in a form of dimers/oligomers, in which only some N-termini are available for binding (e.g. head-to-tail assembly). The results also suggest that denaturation rather than phosphorylation of tau has more significant effect on interactions of tau with Alz 50.

Adult↗

PubMed1995

The role of Myo2, a yeast class V myosin, in vesicular transport.

Previous studies have shown that temperature-sensitive, myo2-66 yeast arrest as large, unbudded cells that accumulate vesicles within their cytoplasm (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). In this study we show that myo2-66 is synthetically lethal in combination with a subset of the late-acting sec mutations. Thin section electron microscopy shows that the post-Golgi blocked secretory mutants, sec1-1 and sec6-4, rapidly accumulate vesicles in the bud, upon brief incubations at the restrictive temperature. In contrast, myo2-66 cells accumulate vesicles predominantly in the mother cell. Double mutant analysis also places Myo2 function in a post-Golgi stage of the secretory pathway. Despite the accumulation of vesicles in myo2-66 cells, pulse-chase studies show that the transit times of several secreted proteins, including invertase and alpha factor, as well as the vacuolar proteins, carboxy-peptidase Y and alkaline phosphatase, are normal. Therefore the vesicles which accumulate in this mutant may function on an exocytic pathway that transports a set of cargo proteins that is distinct from those analyzed. Our observations are consistent with a role for Myo2 in transporting a class of secretory vesicles from the mother cell along actin cables into the bud.

Biological Transport↗

PubMed1995

FAC1, a novel gene identified with the monoclonal antibody Alz50, is developmentally regulated in human brain.

The monoclonal antibody Alz50 recognizes both neurofibrillary pathology associated with Alzheimer's disease and subplate neurons in the developing human brain. To attempt to identify Alz50 antigens expressed during development, a human fetal brain cDNA library was immunoscreened. A positive clone was isolated and sequenced. The clone represents a novel gene named FAC1 (Fetal Alz-50-Reactive Clone 1). The FAC1 gene is located on human chromosome 17 and is conserved across species. In the human fetal brain, the FAC1 gene product is abundantly expressed and the protein is located both in the nucleus and the cytoplasm of cells throughout the developing cortex. Decreased levels of FAC1 protein are observed in adult brain by immunoblot analysis. By immunocytochemistry, the FAC1 protein is almost exclusively localized in the nucleus of neurons in the adult neocortex. Therefore, expression of the FAC1 gene is developmentally regulated and the cellular localization of the protein product is altered during development.

Adolescent↗

PubMed1993

The cycle of SEC4 function in vesicular transport.

Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast. Unlike Ras, it is the ability of Sec4 to cycle between the GTP- and GDP-bound forms rather than the absolute levels of the GTP-bound form that is critical for function. This cycle may be coupled to an observed cycle of Sec4 localization within the cell. Sec4 binds to secretory vesicles which then fuse with the plasma membrane in exocytosis. Sec4 can recycle from the plasma membrane through a soluble pool to rebind to a new round of vesicles. We have found an activity in yeast (Saccharomyces cerevisiae) comparable to that of the GDP dissociation inhibitor protein isolated from mammalian cells that releases GDP-bound Sec4 from membranes. DSS4-1, a dominant suppressor of the sec4-8 temperature-sensitive mutation, encodes a nucleotide exchange protein. The cycle of Sec4 may function to allow the assembly and subsequent disassembly of a set of proteins necessary for exocytosis. Candidates for members of this set of proteins are encoded by sec genes which show strong genetic interactions with sec4-8. Two of these (SEC8 and SEC15) encode large proteins which form a complex that is peripherally associated with the plasma membrane.

Biological Transport↗

PubMed1992

SEC6 encodes an 85 kDa soluble protein required for exocytosis in yeast.

The SEC6 gene encodes a protein required for an event leading to fusion of post-Golgi vesicles with the plasma membrane in Saccharomyces cerevisiae cells. The gene was cloned by complementation of the temperature-sensitive growth defect of a sec6-4 strain. The nucleotide sequence was determined and the longest open reading frame was found to encode an 85 kDa protein of 733 amino acids. The Sec6 protein is predicted to be hydrophilic and is found predominantly in the soluble fraction of a yeast lysate, in a species that sediments with a coefficient of 14S. No extensive homology was found with known proteins of the database. Gene disruption and marker rescue experiments indicate that SEC6 is a single copy gene essential for growth. Overproduction of Sec6p does not suppress any of the other late-acting sec mutants, yet sec6-4 does display synthetic lethality with sec8-9, suggesting that the two gene products may fulfill inter-related functions.

Amino Acid Sequence↗

PubMed1992

Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis.

The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily. The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon. The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness. Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p. This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles. Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle. We propose that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane.

Amino Acid Sequence↗

PubMed1991

Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle.

SEC15 encodes a 116-kD protein that is essential for vesicular traffic from the Golgi apparatus to the cell surface in yeast. Although the sequence predicts a largely hydrophilic protein, a portion (23%) of Sec15p is found in association with the plasma membrane. The remainder is not associated with a membrane but is found in a 19.5S particle which is not dissociated by 0.5 M NaCl. Sec15p may attach directly to the plasma membrane since it is not found on the Golgi apparatus nor on the secretory vesicle precursors to the plasma membrane. Loss of function of most of the late-acting sec gene products does not alter the distribution of Sec15p. However, the sec8-9 mutation and to a lesser extent the sec10-2 mutation result in a shift of Sec15p to the plasma membrane, suggesting a role for these gene products in the regulation of the Sec15p membrane attachment/detachment processes. Depletion of Sec15p by repression of synthesis indicates that the plasma membrane bound pool is the most stable. During the course of these studies we have found that two activities associated with the yeast Golgi apparatus, Kex2 endopeptidase and GDPase, are in separable subcompartments.

Cell Fractionation↗

PubMed1990

Kinetics of hydrolysis of endocytosed substrates by mammalian cultured cells: early introduction of lysosomal enzymes into the endocytic pathway.

The kinetics of exposure of endocytosed material to two lysosomal enzymes were determined for a number of cultured cell lines using fluorogenic substrates. Hydrolysis of endocytosed substrates for cathepsin B and acid phosphatase was observed to begin within 3-10 min of substrate addition and to proceed linearly for up to 60 min thereafter. Hydrolysis of the cathepsin B substrate was not affected by inhibition of protein synthesis with cycloheximide, indicating that the enzymes present in early endosomes are not exclusively newly synthesized. As had been observed previously for a cathepsin B substrate (Roederer, M., Bowser, R., and Murphy, R. F., J. Cell. Physiol., 131:200-209, 1987), hydrolysis of the acid phosphatase substrate was not blocked at temperatures below 20 degrees C. The results suggest that the endosome is the primary site of initial exposure of endocytosed material to hydrolytic enzymes.

Acid Phosphatase↗

PubMed1987

Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes.

The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.

Animals↗

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