Dysregulation of PLDN (pallidin) is a mechanism for platelet dense granule deficiency in RUNX1 haplodeficiency.

Essentials Platelet dense granule (DG) deficiency is a major abnormality in RUNX1 haplodeficiency patients. The molecular mechanisms leading to the platelet DG deficiency are unknown. Platelet expression of PLDN (BLOC1S6, pallidin), involved in DG biogenesis, is regulated by RUNX1. Downregulation of PLDN is a mechanism for DG deficiency in RUNX1 haplodeficiency. SUMMARY: Background Inherited RUNX1 haplodeficiency is associated with thrombocytopenia and platelet dysfunction. Dense granule (DG) deficiency has been reported in patients with RUNX1 haplodeficiency, but the molecular mechanisms are unknown. Platelet mRNA expression profiling in a patient previously reported by us with a RUNX1 mutation and platelet dysfunction showed decreased expression of PLDN (BLOC1S6), which encodes pallidin, a subunit of biogenesis of lysosome-related organelles complex-1 (BLOC-1) involved in DG biogenesis. PLDN mutations in the pallid mouse and Hermansky-Pudlak syndrome-9 are associated with platelet DG deficiency. Objectives We postulated that PLDN is a RUNX1 target, and that its decreased expression leads to platelet DG deficiency in RUNX1 haplodeficiency. Results Platelet pallidin and DG levels were decreased in our patient. This was also observed in two siblings from a different family with a RUNX1 mutation. Chromatin immunoprecipitation and electrophoretic mobility shift assays with phorbol ester-treated human erythroleukemia (HEL) cells showed RUNX1 binding to RUNX1 consensus sites in the PLDN1 5' upstream region. In luciferase reporter studies, mutation of RUNX1 sites in the PLDN promoter reduced activity. RUNX1 overexpression enhanced and RUNX1 downregulation decreased PLDN1 promoter activity and protein expression. RUNX1 downregulation resulted in impaired handling of mepacrine and mislocalization of the DG marker CD63 in HEL cells, indicating impaired DG formation, recapitulating findings on PLDN downregulation. Conclusions These studies provide the first evidence that PLDN is a direct target of RUNX1 and that its dysregulation is a mechanism for platelet DG deficiency associated with RUNX1 haplodeficiency.

Intramedullary megakaryocytes internalize released platelet factor 4 and store it in alpha granules.

BACKGROUND: Megakaryocytes express and store platelet factor 4 (PF4) in alpha granules. In vivo, PF4 is a clinically relevant, negative regulator of megakaryopoiesis and hematopoietic stem cell replication. These findings would suggest a regulated source of free intramedullary PF4. OBJECTIVES: Define the source of free intramedullary PF4 and its intramedullary life cycle. METHODS: We interrogated both murine and human bone marrow-derived cells during megakaryopoiesis in vitro by using confocal microscopy and enzyme-linked immunosorbent assay. With immunohistochemistry, we examined in vivo free PF4 in murine bone marrow before and after radiation injury and in the setting of megakaryocytopenia and thrombocytopenia. RESULTS: Exogenously added human PF4 is internalized by murine megakaryocytes. Human megakaryocytes similarly take up murine PF4 but not the related chemokine, platelet basic protein. Confocal microscopy shows that internalized PF4 colocalizes with endogenous PF4 in alpha granules and is available for release on thrombin stimulation. Immunohistochemistry shows free PF4 in the marrow, but not another alphagranule protein, von Willebrand factor. Free PF4 increases with radiation injury and decreases with megakaryocytopenia. Consistent with the known role of low-density lipoprotein receptor-related protein 1 in the negative paracrine effect of PF4 on megakaryopoiesis, PF4 internalization is at least partially low-density lipoprotein receptor-related protein 1 dependent. CONCLUSIONS: PF4 has a complex intramedullary life cycle with important implications in megakaryopoiesis and hematopoietic stem cell replication not seen with other tested alpha granule proteins.

Apoptotic effects of platelet factor VIII on megakaryopoiesis: implications for a modified human FVIII for platelet-based gene therapy.

BACKGROUND: Ectopically expressed B-domainless factor VIII in megakaryocytes is stored in α-granules, is effective in a number of murine hemostatic models, and is protected from circulating inhibitors. However, this platelet (p) FVIII has different temporal-spatial availability from plasma FVIII, with limited efficacy in other murine hemostatic models. OBJECTIVES AND METHODS: We sought to improve pFVIII hemostatic efficacy by expressing canine (c) FVIII, which has higher stability and activity than human (h) FVIII in FVIII(null) mice. RESULTS AND CONCLUSIONS: We found that pcFVIII was more effective than phFVIII at restoring hemostasis, but peak pcFVIII antigen levels were lower and were associated with greater megakaryocyte apoptosis than phFVIII. These new insights suggest that pFVIII gene therapy strategies should focus on enhancing activity rather than levels. We previously showed that modification of the PACE/furin cleavage site in hFVIII resulted in secretion of hFVIII primarily as a single-chain molecule with increased biological activity. In megakaryocytes, this variant was expressed at the same level as phFVIII with a lentiviral bone marrow transplant approach to reconstitute FVIII(null) mice, but was more effective, resulting in near-normal hemostasis in the cremaster laser injury model. These studies may have implications for pFVIII gene therapy in hemophilia A.

Vaccination with soluble Abeta oligomers generates toxicity-neutralizing antibodies.

In recent studies of transgenic models of Alzheimer's disease (AD), it has been reported that antibodies to aged beta amyloid peptide 1-42 (Abeta(1-42)) solutions (mixtures of Abeta monomers, oligomers and amyloid fibrils) cause conspicuous reduction of amyloid plaques and neurological improvement. In some cases, however, neurological improvement has been independent of obvious plaque reduction, and it has been suggested that immunization might neutralize soluble, non-fibrillar forms of Abeta. It is now known that Abeta toxicity resides not only in fibrils, but also in soluble protofibrils and oligomers. The current study has investigated the immune response to low doses of Abeta(1-42) oligomers and the characteristics of the antibodies they induce. Rabbits that were injected with Abeta(1-42) solutions containing only monomers and oligomers produced antibodies that preferentially bound to assembled forms of Abeta in immunoblots and in physiological solutions. The antibodies have proven useful for assays that can detect inhibitors of oligomer formation, for immunofluorescence localization of cell-attached oligomers to receptor-like puncta, and for immunoblots that show the presence of SDS-stable oligomers in Alzheimer's brain tissue. The antibodies, moreover, were found to neutralize the toxicity of soluble oligomers in cell culture. Results support the hypothesis that immunizations of transgenic mice derive therapeutic benefit from the immuno-neutralization of soluble Abeta-derived toxins. Analogous immuno-neutralization of oligomers in humans may be a key in AD vaccines.

Extensive gastric heterotopia of the small intestine resulting in massive gastrointestinal bleeding, bowel perforation, and death: report of a case and review of the literature.

Gastric heterotopia of the small intestine is a rare occurrence outside of Meckel's diverticulum and intestinal duplication. The vast majority of cases of gastric heterotopia occur as polypoid or tumorous lesions in the duodenum. These lesions have been associated with clinical symptoms including diarrhea, obstruction, dyspepsia, ulceration, and gastrointestinal bleeding. We present a case of gastric heterotopia that is unique because the lesions occurred as multiple, carpet-like, nonpolypoid areas throughout a large portion of the small intestine. A review of the literature is included.

Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.

Abeta1-42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer's pathogenesis. Neurotoxicity of amyloid beta protein (Abeta) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Abeta oligomers (referred to as ADDLs, for Abeta-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer's disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer's disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Abeta-derived diffusible ligands acting upon particular neural signal transduction pathways.

[The Pfannenstiel incision in colorectal surgery]. / Incision de Pfannenstiel en chirurgie colo-rectale.

A parietal incision derived from Pfannenstiel's incision has been used since 1983 for surgery of the left colon and rectum. We conducted a prospective study in 100 patients with a mean age of 61 years who underwent surgery from 1984 to 1987 to assess the parietal outcome, postoperative pain and respiratory impact. The most frequent procedures were: sigmoidectomy (n = 48), anterior resection of the rectum (n = 24), rectopexia (n = 12), amputation of the rectum (n = 5) and total colectomy (n = 5). The splenic angle was mobilized in 23 cases. The operation also included a procedure to relieve occlusion in 4 patients. Operative mortality was nil. Parietal complications were bleeding (n = 4) or infection (n = 8). Seven early reoperations used the same access. Mean follow-up for 61 patients was 75 months. No cases of eventration were observed despite factors predictive of failure. Ventilatory impact, as measured by spirometry showed ventilatory peak-flow and blood gases comparable to those observed after a medial hypogastric incision. Pain, assessed on the bases of a visual analog scale and use of antalgesics, was considered to be mild on day 1, low on day 2 and absent on day 5. Sixty-five percent of the patients did not require antalgesics. In conclusion, this incision creates a hypogastric minilaparotomy allowing midline sub- and peri-umbilical, or even xyphoidial access with little pain and operative risk as the parietal risk is eliminated. Oral nutrition and activity can be resumed rapidly, reducing surgical stress in a protocol for minimally-invasive surgery.

CNS neuronal focal adhesion kinase forms clusters that co-localize with vinculin.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that appears to play a central role in integrin-mediated signal transduction in non-neuronal cells, linking the extracellular matrix to the actin-based cytoskeleton at focal adhesion contacts. Biochemical analysis has revealed the presence of FAK immunoreactivity in cells of neuronal lineage (Zhang et al., 1994) and in the CNS (Burgaya et al. 1995; Grant et al., 1995). In the current work, we have examined the immunodistribution of FAK in nerve cell cultures and tissue sections from the rat CNS. Cultures of B103 CNS neuroblastoma cells and primary cultures of hippocampal neurons both showed abundant FAK immunoreactivity in nerve cell bodies. Immunoreactivity also extended into neurites and growth cones. The most striking feature of FAK distribution was the presence of short, punctate clusters of high FAK concentration. These FAK clusters were maintained in triton-extracted cell ghosts, indicating association with the cytoskeleton. Double-label confocal imaging showed that clusters of FAK coincided with clusters of vinculin, another actin-associated signal transduction molecule implicated in control of growth cone motility. Data from hippocampal sections verified the presence of FAK in adult neurons where it was enriched in somato-dendritic domains and showed a non-uniform distribution. Quantitative FAK immunoprecipitation to compare adult with embryonic brain showed a 7-fold developmental down-regulation of FAK and a 21-fold down-regulation of FAK TyrP. The data suggest that neuronal FAK may participate in signal transduction complexes relevant to neuronal morphogenesis and plasticity.

Delivery of membrane-impermeant fluorescent probes into living neural cell populations by lipotransfer.

Use of fluorescent probes to monitor f-actin in living cells typically relies on difficult microinjection procedures. The current work has developed cationic lipotransfer of membrane-impermeant probes as an alternative to microinjection. BODIPY FL-phallacidin, a fluorescent f-actin probe, was packaged into 40-50 nm cationic liposomes. Packaging, verified by gel filtration, enabled delivery of the probe into living nerve cells and provided an image of f-actin that was identical to that seen in fixed, permeabilized cells. Phallacidin alone did not enter living cells, nor was its uptake stimulated by the presence of empty liposomes. All predicted f-actin structures were fluorescent in living cells, indicating a high efficacy of delivery. Cationic lipotransfer of fluorescent probes was rapid, not disruptive to cells, and delivered a probe en masse to a large sample population. Lipotransfer appears to be a promising alternative to microinjection for introducing membrane-impermeant probes and reagents into living cells.

Constitutive Alzheimer's-type tau epitopes in a neuritogenic rat CNS cell line.

Paired helical filaments (PHFs) of Alzheimer's disease (AD) largely comprise hyperphosphorylated forms of the cytoskeletal protein tau. AD-type tau phosphoepitopes, detected by various monoclonal antibodies, are absent from normal adult neurons, but recent studies have shown that their expression may contribute to neuritogenesis and axon differentiation in the developing nervous system. Therefore, we have examined a brain nerve cell line that is spontaneously neuritogenic for possible expression of AD-type tau epitopes. The neuritogenic rat brain cell line B103 was found to constitutively produce two-AD related epitopes of tau, detected by cellular immunofluorescence studies with the PHF-1 and Alz-50 monoclonal antibodies. Biochemical studies showed that the antibodies bound to proteins within the molecular, weight range expected for phosphorylated tau isoforms. Further verification was established by use of tau antisense oligomers, which eliminated cellular immunofluorescence due to the AD-related monoclonals and polyclonal anti-tau but did not eliminate fluorescence due to anti-tubulin. Cells treated with tau antisense were not neurite-free. Neurites that remained, however, were abnormal, generally short and wavy in appearance. Cellular distribution of the tau epitopes was found to be particularly interesting. Alz-50 recognized only cytoplasmic tau whereas PHF-1 recognized nuclear tau as well as cytoplasmic. Thus, the two epitopes, are morphologically segregated within the cell. Because subcellular segregation of tau is compromised in Alzheimer's disease, mechanisms that segregate AD-type phosphotau epitopes in B103 cells may have relevance to this neurodegenerative disorder.

Alzheimer's-associated phospho-tau epitope in human neuroblastoma cell cultures: up-regulation by fibronectin and laminin.

Alzheimer's-afflicted neurons contain phosphorylated forms of tau that are not present in healthy adults. these can be recognized with great specificity by monoclonal antibodies such as paired helical filament-1 (PHF-1) [Greenberg S. G. and Davies P. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 5827-5831; Greenberg S. G. et al. (1992) J. biol. Chem. 267, 564-569]. The PHF-1 phospho-tau epitope is also present in immature neurons undergoing axodendritic differentiation [Pope W. B. et al. (1993) Expl Neurol. 120, 106-113]. Analogous to its presence in immature neurons, we report here that the PHF-1 tau epitope spontaneously occurs in the human neuroblastoma cell line SHSY5Y, where its level can be regulated by differentiation and by molecules found in the extracellular matrix. Confocal immunofluorescence studies showed PHF-1 epitope to be constitutively expressed in the somatic cytoplasm as well as in short neurites typical of undifferentiated SHSY5Y cells. Induction of differentiation with retinoic acid produced cells with a neuronal morphology and a redistribution of the expression of PHF-1 tau in the long neurites. Protracted exposure to retinoic acid decreased the levels of PHF-1 immunofluorescence without a loss of neurites, similar to the developmental down-regulation seen in situ. The effects of retinoic acid on PHF-1 immunofluorescence were modifiable by fibronectin, which can be released by some neuroblastoma cell lines [Ciccarone V. et al. (1989) Cancer Res. 49, 219-225; Yoshihara T. et al. (1992) Int. J. Cancer 51, 620-626]. Exogenous human fibronectin caused a marked up-regulation of PHF-1 immunofluorescence. Quantitative analysis of 15 multicellular areas, from six different cultures, per experimental condition showed a 16-fold increase compared to untreated controls. Up-regulation by fibronectin was also evident in undifferentiated cells. Cell counts indicated no proliferative effects of the fibronectin under the conditions used. Laminin also caused an increase of PHF-1 tau in retinoic acid-treated cells. Data obtained from immunoblots verified the results observed with immunofluorescence. The data show that the PHF-1 tau epitope is spontaneously expressed by non-degenerating human neuroblastoma cells, down-regulated by cellular differentiation, induced by retinoic acid and up-regulated by the extracellular matrix components fibronectin and laminin. One explanation of the data is that fibronectin maintains a population of SHSY5Y cells in a biochemical state of differentiation in which PHF-1 tau is expressed.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of beta-amyloid peptides with integrins in a human nerve cell line.

beta-Amyloid accumulates as extracellular aggregates in Alzheimer's-afflicted brain tissue, but it also is secreted by healthy tissue, for reasons not yet established. One possibility is that beta-amyloid, which contains a sequence (RHDS) homologous to the cell-binding domain of fibronectin, may modulate integrin function, a possibility supported by previous data from non-neuronal cells (Ghiso et al., Biochem. J., 288 (1992) 1053-1059). The current work shows that functional interaction with beta-amyloid peptides is also supported by integrins in neuronal cells. Experiments used the SH-SY5Y human neuroblastoma cell line, which was shown to contain integrins that mediated cell adhesion to substratum-bound fibronectin. Adhesion to fibronectin was partially blocked by synthetic beta-amyloid peptides containing the RHDS sequence. beta-Amyloid sequences adsorbed to substratum themselves were found to mediate cell adhesion, although less effectively than fibronectin. Anti-integrin blocked the peptide-mediated adhesion, at doses commensurate with those blocking fibronectin-mediated adhesion. The data support the hypothesis that beta-amyloid peptides could physiologically, and perhaps pathogenically, modulate the activity of neuronal integrins, important cell surface receptors known to control protein kinase activities, Ca2+ levels, gene expression and organization of the cytoskeleton.

Beta/A4-evoked degeneration of differentiated SH-SY5Y human neuroblastoma cells.

beta/A4 peptides are known to induce neurodegeneration in cultures of rat brain cells and rat neural cell lines (Yankner et al: Science 250:279-282, 1990; Behl et al: Biochem Biophys Res Commun 186:944-950, 1992). The current data show that these peptides induce similar neurodegeneration in SH-SY5Y neuroblastoma cells, extending characterization of beta/A4 toxicity to a human nerve cell line. Human SH-SY5Y cells respond to aggregated beta/A4 with changes in cell shape, membrane blebbing, antigenic modification, loss of attachment to the substrate, and cell death. beta/A4 peptides require aggregation for maximum toxic effects, as cellular degeneration is evoked by aggregated beta/A4 1-42 and 4-41 cysteine but not by monomeric beta/A4 1-40. Aged (pre-aggregated) beta/A4 1-40 also evoked neurodegeneration. Antigenic changes comprise upregulation of Alzheimer's-type tau epitopes, recognized by the PHF-1 and Alz-50 monoclonals. These particular changes in tau support the connectivity between this in vitro model and mechanisms leading to neurodegeneration in Alzheimer's disease. A significant feature of the SH-SY5Y response is that cells must be differentiated before they become sensitive to the degeneration evoked by beta/A4. Signaling pathways leading to beta/A4-evoked neurodegeneration thus are under experimental control, becoming complete only when proliferating cells withdraw from the cell cycle and develop a postmitotic phenotype.

Focal adhesion kinase expressed by nerve cell lines shows increased tyrosine phosphorylation in response to Alzheimer's A beta peptide.

A beta is a 39-43-amino acid peptide that accumulates as extracellular aggregates in Alzheimer's disease-afflicted brain tissue. Contact between these aggregates and neurons is potentially pathogenic, although little is known about the cellular transduction mechanisms. We have investigated the impact of A beta aggregates on the neuronal control of protein tyrosine phosphorylation, which underlies signal transduction for multiple families of growth factor and adhesion receptors. Added to cultures of rat and human nerve cell lines, A beta aggregates evoked a non-desensitizing increase (1.3-3.6-fold) in tyrosine phosphorylation in a band at 118 kDa. The 118-kDa protein was determined by immunoprecipitation to be pp125FAK, not previously documented in cells of neuronal lineage. Immunoblots with anti-focal adhesion kinase (FAK) showed that A beta aggregates had no effect on FAK protein levels. The increase in FAK tyrosine phosphorylation occurred at doses of A beta aggregates that evoked lactate dehydrogenase release; evoked tyrosine phosphorylation preceded the first detectable lactate dehydrogenase release by 4 h. Like degeneration, the FAK response was dependent on A beta aggregation and neuronal differentiation. Since tyrosine phosphorylation of FAK is essential to its activity as a transduction component of integrin-, peptide-, and lysophosphatidic acid-mediated signaling, the data establish a link between A beta aggregates and signal transduction pathways implicated in diverse cell functions including neurite outgrowth, control of the cell cycle, and apoptosis.

Microtubule-associated protein tau is hyperphosphorylated during mitosis in the human neuroblastoma cell line SH-SY5Y.

A phosphorylated tau epitope specific for paired helical filaments in Alzheimer's disease is recognized by monoclonal antibody PHF-1. Healthy adult brains lack the PHF-1 epitope (PHF-1 tau), but it is transiently expressed by immature neurons during development. We have found that proliferating SH-SY5Y human neuroblastoma cells also express PHF-1 tau. Consistent with the recent finding that cell-cycle-dependent kinases can phosphorylate tau in vitro, flow cytometry showed that mitotic SH-SY5Y cells were up to 18-fold more PHF-1 immunoreactive than nonmitotic cells. On immunoblots, PHF-1 tau in mitotic and nonmitotic cells also was strikingly different. First, mitosis induced a prominent PHF-1 reactive band at 120 kDa, which likely accounted for the large increase in PHF-1 signal seen at mitosis. Although the size of the 120-kDa band is consistent with it being the high-molecular-weight form of tau, other antibodies to tau did not recognize it. Second, mitosis caused a hyperphosphorylation of the PHF-1 immunoreactive tau band normally seen at 50 kDa. In mitotic cells this band had an increased intensity and molecular weight. Alkaline phosphatase treatment abolished tau M(r) heterogeneity, verifying that the variations in mobility were due to phosphorylation. These data show that cell-cycle-dependent hyperphosphorylation of tau occurs in intact cells, and they support the hypothesis that aberrant activity of cell-cycle-dependent kinases may contribute to tau phosphorylation and PHF formation in Alzheimer's disease.

Cholinergic differentiation in neurogenic basal forebrain cultures.

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal forebrain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5-10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating 3H-thymidine (greater than 90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of 3H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.

Soluble proteins from rat olfactory bulb promote the survival and differentiation of cultured basal forebrain neurons.

A previous study of cholinergic development indicated a possible trophic relationship between the olfactory bulb and its afferents from the basal forebrain (Large et al., J. Neurochem., 46 (1986) 671-680). To examine this possibility further, cultured embryonic basal forebrain neurons from rat were used as a test system for trophic factor activity hypothesized to be present in olfactory bulb. Basal forebrain neurons grown in defined medium typically died within 2-3 days. However, survival and differentiation were strikingly enhanced by soluble extracts of olfactory bulb tissue. This trophic effect was noticeable with 2 micrograms/ml olfactory bulb protein, and plateaued at 100 micrograms/ml. The activity was heat- and trypsin-sensitive, non-dialyzable, stable in the cold, resistant to NGF antiserum, and approximately 100-150 kDa in size. Nerve growth factor, bovine serum albumin, laminin and extracts from heart did not mimic the activity. Long-term growth (21 days) in the presence of olfactory bulb proteins resulted in extensive neurite production, formation of thick neurite fascicles, and aggregation of cells. Some glia were present, as evidenced by the presence of glial fibrillary acidic protein, and large numbers of cells were positive for neuron-specific enolase and true acetylcholinesterase. Trophic activity was also present in medium conditioned by olfactory bulb slices, implying secretion of active factors.

Autonomous control of phosphatidylinositol turnover by histamine and acetylcholine receptors in the N1E-115 neuron-like cell line.

Histamine was found to stimulate the turnover of phosphatidylinositol (PI) in cultures of neuron-like NE-115 cells. Turnover was measured by increased production of [3H]inositol phosphates (breakdown) and by accelerated incorporation of 32P into PI (resynthesis). Data were consistent with hydrolysis of polyphosphoinositides being the initial event in receptor-stimulated PI turnover. This response to histamine desensitized within 10 min. Receptor systems for histamine and acetylcholine were tested for possible interactions: PI turnover in response to dual stimulation was approximately equal to the sum of the individual responses while prior desensitization of the acetylcholine receptor system had no effect on subsequent stimulation of the histamine receptor system. These results are consistent with the hypothesis that components of acetylcholine and histamine receptor systems responsible for PI turnover are autonomously organized and regulated. and regulated.