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G Bonanno

Publications and source records attributed to G Bonanno.

At least 55 records · Page 3Linked to original sources
PubMed1999

Differential effects of zinc on native GABA(A) receptor function in rat hippocampus and cerebellum.

Hippocampal noradrenergic and cerebellar glutamatergic granule cell axon terminals possess GABA(A) receptors mediating enhancement of noradrenaline and glutamate release, respectively. The hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar one is not affected by benzodiazepine agonists, indicating the presence of an alpha6 subunit. We tested here the effects of Zn2+ on these two native GABA(A) receptor subtypes using superfused rat hippocampal and cerebellar synaptosomes. In the cerebellum, zinc ions strongly inhibited (IC50 approximately 1 microM) the potentiation of the K(+)-evoked [3H]D-aspartate release induced by GABA. In contrast, the GABA-evoked release of [3H]noradrenaline from hippocampal synaptosomes was much less sensitive to Zn2+ (IC50 > 30 microM). The effects of Zn2+ were then studied in two rat lines selected for high (ANT) and low (AT) alcohol sensitivity because granule cell GABA(A) receptors in ANT, but not AT, rats respond to benzodiazepine agonists due to a critical mutation in the alpha6 subunit. GABA increased the K(+)-evoked release of [3H]DCNS REGIONS-aspartate from cerebellar synaptosomes of AT and ANT rats, an effect prevented by the GABAA selective antagonist bicuculline. In AT rat cerebellum, the effect of GABA was strongly inhibited by Zn2+ (IC50 < or = 1 microM), whereas in ANT rats, the divalent cation was about 100-fold less potent. Thus, native benzodiazepine-sensitive GABAA receptors appear largely insensitive to functional inhibition by Zn2+ and vice versa. Changes in sensitivity to Zn2+ inhibition consequent to mutations in cerebellar granule cell GABA(A) receptor subunits may lead to changes in glutamate release from parallel fibers onto Purkinje cells and may play important roles in cerebellar dysfunctions.

Animals↗
PubMed1999

Expansion of granulocyte colony-stimulating factor/chemotherapy-mobilized CD34+ hematopoietic progenitors: role of granulocyte-macrophage colony-stimulating factor/erythropoietin hybrid protein (MEN11303) and interleukin-15.

Ex vivo stroma-free static liquid cultures of granulocyte colony-stimulating factor (G-CSF)/chemotherapy-mobilized CD34+ cells were established from patients with epithelial solid tumors. Different culture conditions were generated by adding G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (Flt3), megakaryocyte growth and development factor (Peg-rHuMGDF), GM-CSF/erythropoietin (EPO) hybrid protein (MEN11303), and interleukin-15 (IL-15) to the basic stem cell factor (SCF) + interleukin-3 (IL-3) + EPO combination. This study showed that, among the nine different combinations tested in our 5% autologous plasma-containing cultures, only those containing IL-3/SCF/Flt3/MEN11303 and IL-3/SCF/Flt3/MEN11303/IL-15 significantly expanded colony-forming unit granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), long-term culture-initiating cells (LTC-IC), CD34+, and CD34+/CD38- cells after 14 days of culture. Particularly, the addition of IL-15 to IL-3/SCF/Flt3/MEN11303 combination produced a significant increase of LTC-IC, with an average 26-fold amplification as compared to input cells, without any detrimental effect on CFU-GM and BFU-E expansion. This combination also produced a statistically significant 3.6-fold expansion of primitive CD34+/CD38- cells. Moreover, this study confirms the previously described erythropoietic effect of MEN11303, which, in our experience, was the only factor capable of expanding BFU-E. Compared to equimolar concentrations of GM-CSF and EPO, MEN11303 hybrid protein showed a significantly higher capacity of expanding CFU-GM, BFU-E, LTC-IC, CD34+, and CD34+/CD38- cells when these cytokines were tested in combination with IL-3/SCF/Flt3. These cultures indicated that Peg-rHuMGDF addition to IL-3/SCF/EPO/Flt3 does not affect CFU-GM and BFU-E expansion but, unlike G-CSF or GM-CSF, it does not decrease the ability of Flt3 to expand primitive LTC-IC. These studies indicate that, starting from G-CSF/chemotherapy-mobilized CD34+ cells, concomitant expansion of primitive LTC-IC, CFU-GM, BFU-E, CD34+, and CD34+/CD38- cells is feasible in simple stroma-free static liquid cultures, provided IL-3/SCF/Flt3/MEN11303/IL-15 combination is used as expanding cocktail in the presence of 5% autologous plasma.

Adolescent↗
PubMed1999

Evidence for calcium-dependent vesicular transmitter release insensitive to tetanus toxin and botulinum toxin type F.

Whether exocytosis evoked by a given releasing stimulus from different neuronal families or by different stimuli from one neuronal population occurs through identical mechanisms is unknown. We studied the release of [3H]noradrenaline, [3H]acetylcholine and [3H]dopamine induced by different stimuli from superfused rat brain synaptosomes pretreated with tetanus toxin or botulinum toxin F, known to block exocytosis by cleaving VAMP/synaptobrevin. The external Ca2(+)-dependent [3H]transmitter overflows evoked by KCl were similarly inhibited by tetanus toxin or botulinum toxin F; the toxins cleaved similar amounts of synaptosomal synaptobrevin, as determined by western blot analysis, suggesting prevalent involvement of synaptobrevin-II. GABA uptake-mediated release of the three [3H]transmitters was that differentially sensitive to the toxins: only the release of [3H]noradrenaline, which is dependent on external Ca2+, but not of [3H]acetylcholine and [3H]dopamine was blocked. Neither toxin affected the [3H]transmitter overflows evoked by the Ca2(+) ionophore ionomycin. Cadmium blocked the K(+)-evoked release of all [3H]transmitters and the GABA-evoked release of [3H]noradrenaline; the GABA-evoked releases of [3H]acetylcholine and [3H]dopamine and those elicited by ionomycin were insensitive to cadmium. The results suggest that tetanus toxin and botulinum toxin F selectively affect exocytosis linked to activation of voltage-sensitive Ca2(+) channels; the Ca2(+)-dependent, exocytotic-like release induced by stimuli not leading to activation of voltage-sensitive Ca2+ channels seems insensitive to these clostridial toxins.

Animals↗
PubMed1999

Cytotoxic effects toward human hematopoietic progenitor cells and tumor cell lines of paclitaxel, docetaxel, and newly developed analogues IDN5109, IDN5111, and IDN5127.

The growth inhibitory effect of paclitaxel, docetaxel, and newly developed taxanes IDN5109, IDN5111, and IDN5127 was assessed on peripheral blood (PB) CD34+ maintained in liquid culture and on three human cancer cell lines (MDA-MB231, MCF-7 ADRr, CEM VBLr). Concomitantly, DNA analysis was also performed. For unfractionated peripheral blood progenitor cells (PBPC) toxicity was also assessed by clonogenic assay. The cytotoxic effects induced by taxanes toward PBPC as measured by clonogenic assay were correlated with those found for multidrug resistance (MDR)-positive cell lines (IDN5109 > IDN5111 > IDN5127 > docetaxel > paclitaxel). We established a therapeutic index (TI) between the antitumor activity in MDR-positive cells and the toxicity toward PBPC. Paclitaxel and IDN5109, as determined by TI, showed the best value in MDR-negative and MDR-positive cells, respectively. The ranking of the cytotoxic effects observed in PB CD34+ was not correlated with that obtained in clonogenic assay and in cancer cells (IDN5127 > IDN5109 > docetaxel > IDN5111). Remarkably, in DNA analysis docetaxel induced the maximal cell cycle blocking activity. Newly developed taxanes IDN5109 and IDN5111 are endowed of a profile of anticancer activity in MDR-bearing cells and toxicity toward hematopoietic progenitors better than that of docetaxel. However, mechanism(s) underlying toxicity toward hematopoietic progenitors could be, at least in part, different from that of docetaxel and likely dependent on the interaction with P-glycoprotein function in PB CD34+ cells.

Antigens, CD34↗
PubMed1998

GABA(B) receptors as potential targets for drugs able to prevent excessive excitatory amino acid transmission in the spinal cord.

The effects of GABA(B) receptor activation on the Ca2+-dependent depolarization-induced overflow of endogenous glutamic acid and gamma-aminobutyric acid (GABA) was studied in rat spinal cord nerve terminals exposed in superfusion to 15 mM KCl. The GABA(B) receptor agonist (-)-baclofen inhibited the K+-evoked overflow of glutamate (EC50=0.098 microM) but was almost inactive against that of GABA. The overflow of both transmitters could be quite similarly inhibited by two other GABA(B) receptor agonists, 3-APPA (3-aminopropylphosphonous acid; EC50=0.087 and 0.050 microM in the case of GABA and glutamate, respectively) and CGP 44532 (3-amino-2(S)-hydroxypropyl)methylphosphinic acid; EC50=0.81 and 0.50 microM). The GABA(B) receptor antagonist CGP 35348 [3-amino-propyl(diethoxymethyl)phosphinic acid] blocked the effect of 3-APPA (1 microM) at the autoreceptors (IC50 approximately = 1 microM), but not at the heteroreceptors. In contrast, the effects of 3-APPA at both autoreceptors and heteroreceptors could be similarly prevented by another GABA(B) receptor antagonist, CGP 52432 [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxymethyl) phosphinic acid (IC50 approximately = 10 microM). The data suggest that, in the spinal cord, GABA(B) autoreceptors on GABA-releasing terminals differ pharmacologically from GABA(B) heteroreceptors on glutamatergic terminals. Selective GABA(B) receptor ligands may be helpful for conditions characterized by excessive glutamatergic transmission in the spinal cord.

Animals↗
PubMed1998

Neurosteroids may differentially affect the function of two native GABA(A) receptor subtypes in the rat brain.

Hippocampal noradrenergic and cerebellar glutamatergic axon terminals are known to possess GABA(A) receptors mediating, respectively, enhancement of noradrenaline (NA) and glutamate release. It has been recently found that the hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar receptor is insensitive to benzodiazepine agonists. We here tested the effects of neurosteroids on these two native GABA(A) receptors using superfused rat hippocampal and cerebellar synaptosomes. Allopregnanolone (3alpha,5alpha-P), at nanomolar concentrations, potentiated the GABA-induced [3H]-NA release from superfused hippocampal synaptosomes; in the absence of GABA, the steroid was ineffective up to 10 microM. The enhancement by GABA of the K+-evoked [3H]-D-aspartate release from cerebellar synaptosomes also was potentiated by nanomolar 3alpha,5alpha-P; in addition, at 1-10 microM, the steroid increased [3H]-D-aspartate release in the absence of GABA. Both in hippocampus and cerebellum the potentiations of the GABA effects produced by nanomolar 3alpha,5alpha-P were abolished by dehydroepiandrosterone sulphate (DHEAS). Added up to 10 microM, DHEAS could not inhibit the effects of GABA alone. The enhancement of [3H]-D-aspartate release elicited by 3 microM 3alpha,5alpha-P in the absence of added GABA was antagonized completely by bicuculline and picrotoxin and halved by DHEAS. To conclude, 3alpha,5alpha-P, at nanomolar concentrations, behaves as a positive allosteric GABA modulator at both the GABA(A) receptors under study. Low micromolar 3alpha,5alpha-P can directly activate the cerebellar receptor, whereas the hippocampal GABA(A) receptor is insensitive to the neurosteroid alone. DHEAS appears to be a pure antagonist at the neurosteroid allosteric sites. Along with the previously observed differential sensitivity to benzodiazepines, the present data strengthen the idea that the two receptors investigated represent native subtypes of the GABA(A) receptor having distinct pharmacology, neuronal localization and function.

Animals↗
PubMed1998

In vitro effect of amifostine on haematopoietic progenitors exposed to carboplatin and non-alkylating antineoplastic drugs: haematoprotection acts as a drug-specific progenitor rescue.

We evaluated the protective ability of amifostine on peripheral blood mononuclear cell (PBMC)-derived colony-forming unit (CFU) and PB CD34+ cells which were previously exposed in vitro to etoposide, carboplatin, doxorubicin and taxotere. Amifostine pretreatment protected PBMC-derived CFU from the toxic effect of etoposide, carboplatin and taxotere. A significant detrimental effect was exerted by amifostine on the growth of doxorubicin-treated PBMC-derived CFU. Liquid cultures of PB CD34+ cells reproduced faithfully the effects observed on growth of PBMC-derived CFU and confirmed amifostine chemoprotection against etoposide and carboplatin with its detrimental effect on doxorubicin-treated progenitors. Combining the data of viable cell count, cytometric estimation of apoptosis, cell cycle and viable cell replication rate, we found that amifostine protects from etoposide and carboplatin toxicity mainly through a mechanism of cell rescue. Conversely, the detrimental effect of amifostine on the growth of doxorubicin-treated PB CD34+ cells is apparently due to an increased G2/M arrest. In conclusion, amifostine protects haematopoietic progenitors from etoposide, carboplatin and taxotere. Progenitor rescue is the mechanism through which amifostine reduced etoposide and carboplatin toxicity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

NK-3 receptors mediate enhancement of substance P release from capsaicin-sensitive spinal cord afferent terminals.

1. The effects of NK-3 receptor agonists on the release of substance P-immunoreactivity (SP-LI) have been investigated using superfused rat spinal cord synaptosomes. 2. The Ca2+-dependent overflow of SP-LI evoked by 35 mM KCl was concentration-dependently enhanced by senktide (EC50 = 52 nM; maximal effect = 70%) or [MePhe7]NKB (EC50 = 5.5 nM; maximal effect 125%), both selective agonists at receptors of the NK-3 type. 3. The potentiation of the SP-LI overflow elicited by 100 nM senktide or [MePhe7]NKB was prevented by the NK-3 receptor antagonist (+)-SR142801. The antagonist halved, at 10 nM, and almost abolished, at 100 nM, the effect of both agonists. The effect of senktide or [MePhe7]NKB was insensitive to antagonists at NK-1 or NK-2 receptors. 4. Capsaicin (0.1-1 microM) stimulated SP-LI release in a concentration-dependent manner from spinal cord synaptosomes. The SP-LI overflow elicited by 1 microM capsaicin was completely dependent on external Ca2+. Senktide could not affect the capsaicin-evoked release of SP-LI. 5. Senktide failed to potentiate the K+-evoked overflow of SP-LI from synaptosomes previously exposed for 15 min in superfusion to capsaicin. 6. The results show that release-enhancing NK-3 receptors are located on axon terminals of capsaicin-sensitive primary afferent neurones in the spinal cord. Antagonists at NK-3 receptors might help controlling pain transmission.

Animals↗
PubMed1998

Functional, phenotypic and molecular characterization of cytokine low-responding circulating CD34+ haemopoietic progenitors.

Circulating CD34+ cell populations characterized by a low rate (up to five) or high rate (more than five) of cell divisions were isolated from 8 d cultures in the presence of stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), Flt3 ligand and Peg-rHu megakaryocyte growth and development factor (Peg-rHuMGDF) using the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometric cell sorting. Phenotypic characterization of cells which had experienced up to five divisions (CFDA-SEbright) showed a similar surface antigen expression to starting, freshly isolated CD34+ cells. Conversely, cells which experienced more than five divisions (CFDA-SEdim) showed a differentiating behaviour, down-regulating CD34 antigen and acquiring differentiation markers. CFDA-SEbright cells were significantly enriched in CD105 (endoglin) positive precursors as compared to both freshly isolated CD34+ and CFDA-SEdim cells. Functional analysis indicated that CFDA-SEbright had a 3-fold and 10-fold greater cumulative cloning efficiency as compared to freshly isolated CD34+ cells and CFDA-SEdim cells, respectively. CFDA-SEbright cells retained the vast majority of LTC-IC and showed a LTC-IC frequency 2.8-fold higher than that found in freshly isolated CD34+ cells. RT-PCR and Western blot analyses showed significantly higher bcl-2 RNA and protein levels in CFDA-SEbright cells as compared to freshly isolated CD34+ and CFDA-SEdim cells. This study indicates that cytokine low-responding circulating CD34+ cells (CFDA-SEbright cells) represent a functionally, phenotypically and molecularly distinct multipotent progenitor population with biological properties associated with primitive precursors.

Antigens, CD↗
PubMed1998

Nerve growth factor and brain-derived neurotrophic factor increase neurotransmitter release in the rat visual cortex.

A number of experiments have shown that neurotrophins are involved in the development and plasticity of the visual cortex (Bonhoeffer, T., Curr. Op. Neurobiol., 6, 119, 1996). A possible mechanism underlying these effects is the neurotrophin modulation of synaptic transmission. We investigated whether nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) can modulate the release of neurotransmitter in the rat visual cortex at the peak of the critical period for plasticity (P23). The release of glutamate, acetylcholine and gamma-aminobutyric acid (GABA) from visual cortical synaptosomes was analysed in continuous perfusion conditions. We found that NGF enhances the depolarization-evoked release of glutamate (approximately 90%) and acetylcholine (approximately 35%) but not that of GABA. By contrast, BDNF enhances the depolarization-evoked release of all three neurotransmitters investigated (approximately 30%). BDNF and NGF were ineffective on basal release of neurotransmitters. The effect of NGF was not blocked by cholinergic antagonists atropine and mecamylamine. NGF and BDNF potentiation of transmitter release was strongly but not completely blocked by K252a, a tyrosine kinase inhibitor. The role of TrkA and p75NTR receptors was investigated in NGF-induced potentiation of glutamate release. Block of NGF binding to p75NTR using specific blocking antibodies (REX-IgG) slightly but significantly reduced the effect of NGF. Activation of TrkA in isolation by RTA-IgG, an antibody that specifically activates TrkA, was less effective than activation of both receptors by NGF. These results show that neurotrophin action on neurotransmitter release was mostly mediated by Trk receptors with p75NTR having a little but significant positive role. Antigen blot analysis showed the presence of TrkA, TrkB and p75NTR receptors in the visual cortex.

Animals↗
PubMed1998

Time- and region-specific variations in somatostatin release following amygdala kindling in the rat.

Somatostatin biosynthesis is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon translates into enhanced release of the peptide and whether it is involved in kindling maintenance. A marked increase in somatostatin-like immunoreactivity (somatostatin-LI) was observed in hilar interneurons of the hippocampus and in their presumed projections to the outer molecular layer 1 week, but not 1 month, after the last kindled seizure. No overt changes were observed in the striatum or in the cortex. Compared with sham-stimulated controls, (a) in the hippocampus, high-K+-evoked somatostatin-LI release was unchanged in synaptosomes taken from rats killed 7 days after the last kindled seizure but was bilaterally reduced after 30 days; (b) in the striatum, it was increased (mainly ipsilaterally to stimulation) 7, but not 30, days after the last seizure; and (c) in the cortex, somatostatin-LI release was bilaterally increased in synaptosomes taken from kindled rats 30, but not 7, days after the last seizure. This study shows that distinct changes occur in synaptosomal somatostatin-LI release after kindling acquisition, depending on the brain area analyzed and on the time elapsed from the last generalized seizure.

Amygdala↗
PubMed1997

Pharmacologically distinct GABAB receptors that mediate inhibition of GABA and glutamate release in human neocortex.

1. The release of endogenous gamma-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours. 2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K(+)-evoked overflows of both amino acids were almost totally dependent on the presence of Ca(2+) in the superfusion medium. 3. The GABAB receptor agonist (-)-baclofen (1, 3 or 10 microM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 microM of the agonist ranged from 45-50%. 5. The effect of three selective GABAB receptor antagonists on the inhibition of the K(+)-evoked GABA and glutamate overflows elicited by 10 microM (-)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 microM; almost totally at 300 microM) the effect of (-)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (-)-baclofen on the K(+)-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 microM); however, CGP 35348 (10 or 100 microM) antagonized (-)-baclofen (complete blockade at 100 microM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 microM, blocked the GABAB autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52423 was lost at 30 microM, as the compound, at this concentration, inhibited completely the (-)-baclofen effect on both GABA and glutamate release. 5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABAB receptors.

Aged↗
PubMed1997

Generation of multinuclear tartrate-resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors.

Circulating CD34+ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34 contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3+ T-lymphocytes (range 2-12%), CD3+/CD16+/CD56+ natural killer cells (range 2-11%) and rare mature CD15+/ CD11b+ granulocytes (range 1-2%). CD34+ cells were cultured in liquid medium in the presence of interleukin-3, granulocyte-macrophage colony stimulating factor. stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate-resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34+ cells until day 35. These observations suggest that circulating progenitors have a multilineage potential in vitro and contribute to the clarification of osteoclast development in humans: additionally, they provide the basis for the future development of optimized osteoclast culture techniques in liquid medium and the basic culture system, to test the distinct activity of 1,25(OH)2D3. parathyroid hormone interleukin-11 and of other cytokines on osteoclast development in humans.

Acid Phosphatase↗
PubMed1997

Purified unfractionated G-CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor.

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G-CSF)/chemotherapy mobilized peripheral blood CD34+ cells to selective erythroid differentiation in liquid culture with an average 28-fold increase in the total cell number after 21 d. From day 6 of culture cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34+ cells were culture with EPO plus SCF in serum-free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34+ cells underwent granulocytic/ monocytic differentiation in liquid culture in response to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and SCF, demonstrating that these CD34+ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34+ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34+ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G-CSF/ chemotherapy mobilized CD34+ progenitor cells in liquid culture. The absence of cytokines such as GM-CSF and IL-3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.

Antigens, CD34↗
PubMed1997

Increased presynaptic protein kinase C activity and glutamate release in rats with a prenatally induced hippocampal lesion.

We have previously shown that protein kinase C (PKC) activity is up-regulated in nerve terminals of animals that have been subjected to targeted cellular ablation of cortical and hippocampal neurons by treatment with methylazoxymethanol (MAM), which results in impaired long-term potentiation (LTP) and cognitive deficit. In this study we investigated the consequences of increased membrane-bound PKC in the regulation of release of glutamate, the major excitatory transmitter involved in LTP. We show that nerve terminals of MAM-treated rats show higher PKC activity, as monitored by the in situ phosphorylation of B-50/GAP-43, in both basal and phorbol ester-stimulated conditions. In these animals, hippocampal nerve endings release a greater amount of glutamate than those of controls, both in basal conditions and when synaptosomes are stimulated with KCl or 3,4-diaminopyridine. The potentiation observed in MAM-treated rats was counteracted by the PKC blocker H-7 and the clostridial tetanus toxin. On the contrary, GABA release was not significantly up-regulated, either in basal or in depolarization-evoked conditions. Therefore our data show that the increase in synaptosomal PKC activity is paralleled by increased glutamate but not GABA release in this animal model. Whether this reflects specific up-regulation of membrane PKC activity in glutamatergic terminals or an alteration in the regulation of glutamate release remains to be determined.

Animals↗
PubMed1996

Modulatory effect of tamoxifen and ICI 182,780 on adriamycin resistance in MCF-7 human breast-cancer cells.

In this study the ability of the new pure anti-estrogen ICI 182,780 to modulate the cytotoxic action of adriamycin (ADR) on parental and ADR-resistant MCF-7 (MCF-7 ADRr) human breast-cancer cells was investigated and compared with that of tamoxifen (TAM). TAM enhanced ADR cytotoxicity in MCF-7 ADRr cells in a dose-related manner, but this effect was slight or absent in MCF-7 WT. In contrast, ICI 182,780 was able to enhance ADR toxicity both in MCF-7 ADRr and in the parental cell line. ICI 182,780 was up to 2.5-fold more effective than TAM in reducing the IC50 of ADR in MCF-7 ADRr cells. Analysis of the data by the isobole method showed that the combination ADR/TAM and ADR/ICI 182,780 produced synergistic anti-proliferative activity in MCF-7 ADRr cells. Because ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of anti-estrogens on Pgp expression and activity. Both ICI 182,780 and TAM failed to modulate Pgp expression as assessed by flow cytometry and Western-blot analysis, performed using the monoclonal antibodies MM4.17 and C 219, which are specific for an external or an internal determinant respectively. Pgp activity was investigated by flow cytometry measuring the extrusion of ADR and the cationic dye Rhodamine 123 (Rh 123). ICI 182,780, but not TAM, reduced the activity of Pgp in MCF-7 ADRr cells. Flow cytometry was also used to investigate cell-cycle modifications induced by ADR in MCF-7 ADRr cells, both in the presence and in the absence of anti-estrogens. After 72 hr, higher doses induced an arrest of cells at the G2/M phase. The same effect was visible when lower doses of ADR were combined with ICI 182,780 or TAM. In terms of cell-cycle-blocking activity ICI 182,780 was largely more effective than TAM.

ATP Binding Cassette Transporter, Subfamily B, Mem↗