PubMed Health⌕ Search

Biomedical subjects

N Melamed-Book

Publications and source records attributed to N Melamed-Book.

6 recordsLinked to original sources
PubMed2001

Interactions between adaptor protein-1 of the clathrin coat and microtubules via type 1a microtubule-associated proteins.

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5'-adenylylimidodiphosphate. An overexpressed gamma-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The gamma-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubule-associated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.

Adaptor Protein Complex gamma Subunits↗
PubMed2001

Complex host cell responses to antisense suppression of ACHE gene expression.

3'-End-capped, 20-mer antisense oligodeoxynucleotides (AS-ODN) protected with 2'-O-methyl (Me) or phosphorothioate (PS) substitutions were targeted to acetylcholinesterase (AChE) mRNA and studied in PC12 cells. Me-modified AS-ODN suppressed AChE activity up to 50% at concentrations of 0.02-100 nM. PS-ODN was effective at 1-100 nM. Both AS-ODN displayed progressively decreased efficacy above 10 nM. In situ hybridization and confocal microscopy demonstrated dose-dependent decreases, then increases, in AChE mRNA. Moreover, labeling at nuclear foci suggested facilitated transcription or stabilization of AChE mRNA or both under AS-ODN. Intracellular concentrations of biotinylated oligonucleotide equaled those of target mRNA at extracellular concentrations of 0.02 nM yet increased only 6-fold at 1 microM ODN. Above 50 nM, sequence-independent swelling of cellular, but not nuclear, volume was observed. Our findings demonstrate suppressed AChE expression using extremely low concentrations of AS-ODN and attribute reduced efficacy at higher concentrations to complex host cell feedback responses.

Acetylcholinesterase↗
PubMed2001

ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities.

BACKGROUND: Psychological stress induces rapid and long-lasting changes in blood cell composition, implying the existence of stress-induced factors that modulate hematopoiesis. Here we report the involvement of the stress-associated "readthrough" acetylcholinesterase (AChE-R) variant, and its 26 amino acid C-terminal domain (ARP) in hematopoietic stress responses. MATERIALS AND METHODS: We studied the effects of stress, cortisol, antisense oligonucleotides to AChE, and synthetic ARP on peripheral blood cell composition and clonogenic progenitor status in mice under normal and stress conditions, and on purified CD34 cells of human origin. We employed in situ hybridization and immunocytochemical staining to monitor gene expression, and 5-bromo-2-deoxyuridine (BrdU), primary liquid cultures, and clonogenic progenitor assays to correlate AChE-R and ARP with proliferation and differentiation of hematopoietic progenitors. RESULTS: We identified two putative glucocorticoid response elements in the human ACHE gene encoding AChE. In human CD34+ hematopoietic progenitor cells, cortisol elevated AChE-R mRNA levels and promoted hematopoietic expansion. In mice, a small peptide crossreacting with anti-ARP antiserum appeared in serum following forced swim stress. Ex vivo, ARP was more effective than cortisol and equally as effective as stem cell factor in promoting expansion and differentiation of early hematopoietic progenitor cells into myeloid and megakaryocyte lineages. CONCLUSIONS: Our findings attribute a role to AChE-R and ARP in hematopoietic homeostasis following stress, and suggest the use of ARP in clinical settings where ex vivo expansion of progenitor cells is required.

Acetylcholinesterase↗
PubMed2000

Antisense prevention of neuronal damages following head injury in mice.

Closed head injury (CHI) is an important cause of death among young adults and a prominent risk factor for nonfamilial Alzheimer's disease. Emergency intervention following CHI should therefore strive to improve survival, promote recovery, and prevent delayed neuropathologies. We employed high-resolution nonradioactive in situ hybridization to determine whether a single intracerebro-ventricular injection of 500 ng 2'-O-methyl RNA-capped antisense oligonucleotide (AS-ODN) against acetylcholinesterase (AChE) mRNA blocks overexpression of the stress-related readthrough AChE (AChE-R) mRNA splicing variant in head-injured mice. Silver-based Golgi staining revealed pronounced dendrite outgrowth in somatosensory cortex of traumatized mice 14 days postinjury that was associated with sites of AChE-R mRNA overexpression and suppressed by anti-AChE AS-ODNs. Furthermore, antisense treatment reduced the number of dead CA3 hippocampal neurons in injured mice, and facilitated neurological recovery as determined by performance in tests of neuromotor coordination. In trauma-sensitive transgenic mice overproducing AChE, antisense treatment reduced mortality from 50% to 20%, similar to that displayed by head-injured control mice. These findings demonstrate the potential of antisense therapeutics in treating acute injury, and suggest antisense prevention of AChE-R overproduction to mitigate the detrimental consequences of various traumatic brain insults.

Acetylcholinesterase↗
PubMed1999

Neuronal calcium sparks and intracellular calcium "noise".

Intracellular calcium ions are involved in many forms of cellular function. To accommodate so many control functions, a complex spatiotemporal organization of calcium signaling has developed. In both excitable and nonexcitable cells, calcium signaling was found to fluctuate. Sudden localized increases in the intracellular calcium concentration-or calcium sparks-were found in heart, striated and smooth muscle, Xenopus Laevis oocytes, and HeLa and P12 cells. In the nervous system, intracellular calcium ions were found important in key processes such as transmitter release, repetitive firing, and gene expression. Hence, we examined whether calcium sparks also exist in neurons. Using confocal laser-scanning microscopy and fluorescent probes, we found that calcium sparks exist in two types of neuronal preparations: the presynaptic boutons of the lizard neuromuscular junction and rat hippocampal neurons in cell culture. Control experiments exclude the possibility that these calcium sparks originate from instrumental or biological artifacts. Calcium sparks seem to be just the tip of the iceberg of a more general phenomenon of intracellular calcium "noise." We speculate that calcium sparks and calcium noise may be of key importance in calcium signaling in the nervous system.

Animals↗