Cholinergic Precursors Modulate the Expression of Heme Oxigenase-1, p21 During Astroglial Cell Proliferation and Differentiation in Culture

Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell differentiation. Further, some proteins related to cell cycle including cyclins and p21 are important markers of astrocyte cultures. Aim of investigation was to study the effects of cholinergic precursors (choline, CDP-choline, Acetylcholine and ??-Glyceril-Phosphorylcholine) on HO-1 and p21 expression during astroglial cell proliferation and differentiation in primary cultures at 14 and 35?days in vitro (DIV) treated for 24?h with choline metabolites. Our results showed a slight reduction of HO-1 expression (data not statistical significant) in astroglial cell cultures treated with CDP-choline at 14 DIV and 35 DIV. On the contrary, ACh and choline induced a significant increase of HO-1 expression in 14 DIV astrocyte cultures. Surprisingly, choline and ACh dramatically reduced HO-1 expression at 35 DIV. A slight decrease not statistical significant was detectable for ??-GPC at 14 DIV and particularly significant at 35 DIV. Data concerning p21 expression, a well known protein inhibiting cell cycle, evidenced a significant increase at 14 and 35 DIV after ??-GPC treatment. CDP-choline treatment caused a high increase of p21 expression in 14 DIV astrocyte cultures, but no modification at 35 DIV. Instead, ACh treatment induced a marked increment of p21 expression at 35 DIV. Our data suggest that cholinergic precursors modulate HO-1 and p21 expression during astroglial cell proliferation and differentiation in culture and could be considered a tool to study the induced effects of ischemia and hypoxia diseases in some in vitro models to prevent and reduce its effects after treatment with cholinergic drugs.     

Evidence for an in vivo and in vitro modulation of endogenous cortical GABA release by α-glycerylphosphorylcholine

The effects of α-glycerylphosphorylcholine (α-GPC) on endogenous cortical GABA release were studied both in vivo and in vitro. In freely moving rats, equipped with epidural cups, α-GPC (30–300 mg/kg i.p.) increased GABA release. This effect was potentiated by atropine, both systematically administered (5 mg/kg i.p.) and locally applied (1.4 μM), but not by mecamylamine (4 mg/kg i.p.). The α-GPC-induced increasein GABA release was abolished in rats pretreated with the α₁ receptor antagonist prazosin (14 μg/kg i.p.). In cortical slices α-GPC (0.4 mM) increased the spontaneous GABA efflux. This effectwas abolished by tetrodotoxin (0.5 μM) and prazosin (1 μM), but not by atropine (0.15 μM) ormecamylamine (2.5μM). These results indicate that the facilitatory response by α-GPC on GABArelease does not depend on a direct activation of either muscarinic or nicotinic receptors, but suggest the involvement of the noradrenergic system.     

Effects of Disrupting Calcium Homeostasis on Neuronal Maturation: Early Inhibition and Later Recovery

It has become increasingly clear that agents that disrupt calcium homeostasis may also be toxic to developing neurons. Using isolated primary neurons, we sought to understand the neurotoxicity of agents such as MK801 (which blocks ligand-gated calcium entry), BAPTA (which chelates intracellular calcium), and thapsigargin (TG; which inhibits the endoplasmic reticulum Ca²⁺-ATPase pump). Thus, E18 rat cortical neurons were grown for 1 day in vitro (DIV) and then exposed to vehicle (0.1% DMSO), MK801 (0.01–20 μM), BAPTA (0.1–20 μM), or TG (0.001–1 μM) for 24 h. We found that all three agents could profoundly influence early neuronal maturation (growth cone expansion, neurite length, neurite complexity), with the order of potency being MK801 < BAPTA < TG. We next asked if cultures exposed to these agents were able to re-establish their developmental program once the agent was removed. When we examined network maturity at 4 and 7 DIV, the order of recovery was MK801 > BAPTA > TG. Thus, mechanistically distinct ways of disrupting calcium homeostasis differentially influenced both short-term and long-term neuronal maturation. These observations suggest that agents that act by altering intracellular calcium and are used in obstetrics or neonatology may be quite harmful to the still-developing human brain.     

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 μmol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 μmol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 μmol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld’s staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 μmol/L rutin. Enhanced TNFα release was observed in astrocyte primary cultures treated with 100 μmol/L rutin and in mixed primary cultures treated with 50 and 100 μmol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFα production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.     

Cornel Iridoid Glycoside Inhibits Inflammation and Apoptosis in Brains of Rats with Focal Cerebral Ischemia

The capacity of cornel iridoid glycoside (CIG) to suppress the manifestations of ischemic stroke was investigated. CIG was administered to rats by the intragastric route once daily for 7 days. Focal cerebral ischemia was induced by 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. In non-treated rats large infarct areas were observed within 24 h of reperfusion. Examination of the ischemic cerebral cortex revealed microglia and astrocyte activation, increased interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) concentrations, increased DNA fragmentation in the ischemia penumbra, elevated Bax expression, increased caspase-3 cleavage, and decreased Bcl-2 expression. Pretreatment with CIG decreased the infarct area, DNA fragmentation, IL-1β and TNF-α concentrations, microglia and astrocyte activation, Bax expression, and caspase-3 cleavage while increasing Bcl-2 expression. CIG exerts anti-neuroinflammatory and anti-apoptotic effects which should prove beneficial for prevention or treatment of stroke.     

Cytosine arabinofuranoside-induced activation of astrocytes increases the susceptibility of neurons to glutamate due to the release of soluble factors

Activation of astrocytes occurs during many forms of CNS injury, but its importance for neuronal survival is poorly understood. When hippocampal cultures of neurons and astrocytes were treated from day 2–4 in vitro (DIV 2–4) with 1 μM cytosine arabinofuranoside (AraC), we observed a stellation of astrocytes, an increase in glial fibrillary acidic protein (GFAP) level as well as a higher susceptibility of the neurons to glutamate compared with cultures treated from DIV 2–4 with vehicle. To find out whether factors released into the culture medium were responsible for the observed differences in glutamate neurotoxicity, conditioned medium of AraC-treated cultures (MCMAraC) was added to vehicle-treated cultures and conditioned medium of vehicle-treated cultures (MCMvh) was added to AraC-treated cultures 2 h before and up to 18 h after the exposure to 1 mM glutamate for 1 h. MCMAraC increased glutamate neurotoxicity in vehicle-treated cultures and MCMvh reduced glutamate neurotoxicity in AraC-treated cultures. Heat-inactivation of MCMvh increased, whereas heat-inactivation of MCMAraC did not affect glutamate toxicity suggesting that heat-inactivation changed the proportion of factors in MCMvh inhibiting and exacerbating the excitotoxic injury. Similar findings were obtained using conditioned medium of pure astrocyte cultures of DIV 12 treated from DIV 2–4 with vehicle or 1 μM AraC suggesting that heat-sensitive factors in MCMvh were mainly derived from astrocytes. Treatment of hippocampal cultures with 1 mM dibutyryl-cAMP for 3 days induced an activation of the astrocytes similar to AraC and increased neuronal susceptibility to glutamate. Our findings provide evidence that activation of astrocytes impairs their ability to protect neurons after excitotoxic injury due to changes in the release of soluble and heat-sensitive factors.     

Expression of Cholinergic,Insulin, Vitamin D Receptors and GLUT 3 in the Brainstem of Streptozotocin Induced Diabetic Rats: Effect of Treatment with Vitamin D3

Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B_max of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, α7 nicotinic acetylcholine, insulin, Vitamin D₃ receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and α7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D₃ treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D₃ in managing neurological disorders associated with diabetes.     

The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster

The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca²⁺]_i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine (10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid. Although imidacloprid acts via nAChRs, increases in [Ca²⁺]_i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced increases in [Ca²⁺]_i.     

Validation of organotypical hippocampal slice cultures as an ex vivo model of brain ischemia: different roles of NMDA receptors in cell death signalling after exposure to NMDA or oxygen and glucose deprivation

N-Methyl-D-aspartate receptors (NMDARs) are essential mediators of synaptic plasticity under normal physiological conditions. During brain ischemia, these receptors are excessively activated due to glutamate overflow and mediate excitotoxic cell death. Although organotypical hippocampal slice cultures are widely used to study brain ischemia in vitro by induction of oxygen and glucose deprivation (OGD), there is scant data regarding expression and functionality of NMDARs in such slice cultures. Here, we have evaluated the contribution of NMDARs in mediating excitotoxic cell death after exposure to NMDA or OGD in organotypical hippocampal slice cultures after 14 days in vitro (DIV14). We found that all NMDAR subunits were expressed at DIV14. The NMDARs were functional and contributed to cell death, as evidenced by use of the NMDAR antagonist MK-801 (dizocilpine). Excitotoxic cell death induced by NMDA could be fully antagonized by 10 μM MK-801, a dose that offered only partial protection against OGD-induced cell death. Very high concentrations of MK-801 (50–100 μM) were required to counteract cell death at long delays (48–72 h) after OGD. The relative high dose of MK-801 needed for long-term protection after OGD could not be attributed to down-regulation of NMDARs at the gene expression level. Our data indicate that NMDAR signaling is just one of several mechanisms underlying ischemic cell death and that prospective cytoprotective therapies must be directed to multiple targets.     

Astroglial-Conditioned Media and Growth Factors Modulate Proliferation and Differentiation of Astrocytes in Primary Culture

Astroglial conditioned media (ACM) influence the development and maturation of cultured nerve cells and modulate neuron-glia interaction. To clarify mechanisms of astroglial cell proliferation/differentiation in culture, incorporation of [methyl-³H]-thymidine or [5,6-³H]-uridine in cultured astrocytes was assessed. Cultures were pre-treated with epidermal growth factor (EGF), insulin (INS), insulin-like growth factor-I (IGF-I), and basic fibroblast growth factor (bFGF) and subsequently with ACM. DNA labeling revealed a marked stimulatory effect of ACM from 15 days in vitro (DIV) cultures in 30 DIV astrocytes after12 h pre-treatment with growth factors. The main effects were found after INS or EGF pre-treatment in 30 DIV cultures. ACM collected from 15 or 60 or 90 DIV increased RNA labeling of 15 and 30 DIV astrocyte cultures, being the highest value that of 30 DIV cultures added with ACM from 90 DIV. The findings of increased DNA labeling after EGF or INS pre-treatment in 30 DIV cultures, followed by addition of ACM from 15 DIV cultures, suggest that these phenomena may depend by extra cellular signal-regulated kinase 1 (ERK1) activation.     

Effect of 24-epibrassinolide on biomass, growth and free proline concentration in Spirulina platensis (Cyanophyta) under NaCl stress

In this study, biomass, growth and free proline concentration were investigated in Spirulina platensis treated with different concentrations of NaCl (50, 100, 150, and 200 mM) and 24-epibrassinolide (24-epiBL) hormone (0.5, 1.0, and 3.0 μM) over 5 days. As a result of analysing the cultures under salinity stress, it was determined that biomass and growth rate decreased significantly, while proline concentration increased considerably under salinity stress. The increase in the concentration of proline suggests a role in response to NaCl stress. Among the cultures treated with different concentrations of 24-epiBL, maximum growth was determined at the cultures at 1.0 μM 24-epiBL. Algal growth was also greater at the 0.5 and 3.0 μM concentrations of 24-epiBL with respect to control cultures. With respect to control, 24-epiBL affected growth rate and biomass positively, but proline concentration did not change. Among the cultures supplied with different combinations of NaCl and 24-epiBL, growth rate increased in 150/0.5 and 150/3.0 mM/μM concentrations, but was maximal for the culture containing 150/1.0 mM/μM combination. The increase in algal growth suggests a role for 24-epiBL in partially alleviated to NaCl stress. These results suggest that 24-epiBL may have a protective role for S. platensis reducing the inhibitor effects of salinity stress.     

Dedifferentiation of leaf explants and cytotoxic activity of an aqueous extract of cell cultures of Lantana camara L.

Several secondary metabolites are present in Lantana camara L. as its leaves serve as reservoirs for various bioactive compounds. Callus cultures of L. camara were induced from leaf discs incubated on Murashige and Skoog medium supplemented with 5 μM 6-benzyladenine, 1 μM 2,4-dichlorophenoxyacetic acid, and 1 μM α-naphthalene acetic acid (NAA). An aqueous extract (0.23%), obtained from these calli (50 g dry mass), had an apparent cytotoxic effect on HeLa cells with an IC₅₀ value of 1,500 μg/ml in 36 h. A dose-time dependent activity of the extract was established wherein higher dosage exhibited increased activity; however, over time cell necrosis was observed.     

Resveratrol Increases Glutamate Uptake, Glutathione Content, and S100B Secretion in Cortical Astrocyte Cultures

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenol present in grapes and red wine, which has antioxidant properties and a wide range of other biological effects. In this study, we investigated the effect of resveratrol, in a concentration range of 10–250 μM, on primary cortical astrocytes; evaluating cell morphology, parameters of glutamate metabolism such as glutamate uptake, glutamine synthetase activity and glutathione total content, and S100B secretion. Astrocyte cultures were prepared of cerebral cortex from neonate Wistar rats. Morphology was evaluated by phase-contrast microscopy and immunocytochemistry for glial fibrillary acidic protein (GFAP). Glutamate uptake was measured using l-[2,3-³H]glutamate. Glutamine synthetase and content of glutathione were measured by enzymatic colorimetric assays. S100B content was determined by ELISA. Typical polygonal morphology becomes stellated when astrocyte cultures were exposed to 250 μM resveratrol for 24 h. At concentration of 25 μM, resveratrol was able to increase glutamate uptake and glutathione content. Conversely, at 250 μM, resveratrol decreased glutamate uptake. Unexpectedly, resveratrol at this high concentration increased glutamine synthetase activity. Extracellular S100B increased from 50 μM upwards. Our findings reinforce the protective role of this compound in some brain disorders, particularly those involving glutamate toxicity. However, the underlying mechanisms of these changes are not clear at the moment and it is necessary caution with its administration because elevated levels of this compound could contribute to aggravate these conditions.     

Transglutaminase 2 silencing reduced the beta-amyloid-effects on the activation of human THP-1 cells

The aberrant expression and activation of transglutaminase 2 (TG2), the ubiquitous enzyme which catalyzes calcium-dependent protein cross-linking reactions, has been reported in many inflammatory diseases. Chronic inflammation, mediated by prolonged activation of brain-resident immunocompetent cells, appears to be involved in the pathogenesis of several age-related diseases, such as Alzheimer’s disease. Given that increased TG2 expression has been observed in AD brains, this study was aimed to characterize the role of TG2 in THP-1 monocytes stimulated with amyloid-beta (Aβ). Aβ_1–42 treatment dose-dependently increased TG2 expression in THP-1 cells. In particular, a fivefold up-regulation of TG2, compared with control cells, was observed in the presence of 0.5 μM Aβ_1–42. At the same concentration, Aβ_1–42 was able to promote monocyte maturation as suggested by increased expression of the cell surface antigen CD14 as well as the adhesion-promoting factor fibronectin. The stimulation of THP-1 cells with Aβ_1–42 also led to a significant up-regulation of tumor necrosis factor α (TNF-α) and matrix metalloproteinase 9 (MMP-9). Interestingly, THP-1 cell transfection with small interfering RNA directed against TG2 was able to reduce Aβ_1–42 increased levels of all the examined markers of monocyte maturation (CD14, fibronectin), and activation (TNF-α, MMP-9). These results indicate that TG2 up-regulation is required for the functional THP-1 monocyte activation induced by Aβ_1–42. This work suggests that TG2 inhibition may represent a therapeutic target to ameliorate the inflammation and progression in Alzheimer’s disease.     

Cardiotonic steroid-resistant α1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Na i+ ,K i+ -independent mechanism

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na⁺,K⁺-ATPase α subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the α1 isoform, CTS-sensitive α1S, were stably transfected with a cDNA encoding CTS-resistant α1R-Na⁺,K⁺-ATPase, whose expression was confirmed by RT–PCR. In mock-transfected and α1R-cells, maximal inhibition of ⁸⁶Rb influx was observed at 10 and 1000 μM ouabain, respectively, thus confirming high abundance of α1R-Na⁺,K⁺-ATPase in these cells. Six-hour treatment of α1R-cells with 1000 μM ouabain led to the same elevation of the [Na⁺]_i/[K⁺]_i ratio that was detected in mock-transfected cells treated with 3 μM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 μM ouabain, α1R-cells survived after 24-h incubation with 1000 μM ouabain. Inversion of the [Na⁺]_i/[K⁺]_i ratio evoked by Na⁺,K⁺-ATPase inhibition in K⁺-free medium did not affect survival of α1R-cells but increased their sensitivity to ouabain. Our results show that the α1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na⁺,K⁺-ATPase-mediated Na⁺ and K⁺ fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio.     

Different Mechanisms of NMDA-Mediated Protection Against Neuronal Apoptosis: A Stimuli-Dependent Effect

The mechanisms of protective effect of N-methyl-D-aspartate (NMDA) receptor stimulation on apoptosis of neurons at their early stage of development are poorly understood. In the present study, we investigated the effects of NMDA on staurosporine (St)- and low-potassium (LP)-evoked apoptotic cell death in primary cerebellar granule cell (CGC) cultures at 7 days in vitro (DIV). We found that NMDA (200 μM) attenuated the St (0.5 μM)- and LP (5 mM KCl)-induced neuronal cell death in 7 but not 12 DIV CGC as confirmed by LDH release and MTT reduction assays. Moreover, NMDA attenuated St-and LP-evoked DNA fragmentation and cytosolic apoptosis inducing factor (AIF) protein level but not caspase-3 activation induced by both pro-apoptotic factors. Neuroprotective effects of NMDA on St-induced apoptosis in CGC were attenuated by inhibitors of ERK/MAPK-signaling, PD 98059 and U0126 but not by NMDA receptor antagonists, AP-5 (100 μM) and MK-801 (1 μM) or by inhibitors of PI3-K/Akt pathway (LY 294002 and wortmannin). In contrast to staurosporine model of apoptosis, AP-5 and MK-801 but not inhibitors of PI3-K/Akt and MAPK/ERK1/2 prevented the NMDA-mediated neuroprotection in LP-induced apoptosis of CGC. In separate experiments, we observed also the anti-apoptotic action of NMDA on St (0.5 μM)- and salsolinol (250 μM)-evoked cell death in human neuroblastoma SH-SY5Y cells without its influence on caspase-3 activity, induced by these pro-apoptotic factors. These data indicate that neuroprotection evoked by NMDA in CGC strongly depends on used pro-apoptotic agent and could engage NMDA channel function or be connected with the activation of pro-survival MAPK/ERK1/2 pathway. It is also suggested that anti-apoptotic effects of NMDA is connected with inhibition of fragmentation of DNA via caspase-3-independent mechanism.     

Curcumin modulates eukaryotic initiation factors in human lung adenocarcinoma epithelial cells

Curcumin, a polyphenolic compound, is the active component of Curcuma longa and has been extensively investigated as an anticancer drug that modulates multiple pathways. Eukaryotic initiation factors (eIFs) have been known to play important roles in translation initiation, which controls cell growth and proliferation. Little is known about the effects of curcumin on eIFs in lung cancer. The objective of this study was to exam the curcumin cytotoxic effect and modulation of two major rate-limiting translation initiation factors, including eIF2α and eIF4E protein expression levels in lung adenocarcinoma epithelial cell line A549. Cytotoxicity was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and protein changes were determined by Western blot. A549 cells were treated with 0–240 μM curcumin for 4–96 h. The inhibitory effects of curcumin on cytotoxicity were dose- and time-dependent (P < 0.001). The 50% inhibitory curcumin concentrations (IC50s) at 24, 48, 72, and 96 h were 93, 65, 40, and 24 μM, respectively. Protein expressions of eIF2α, eIF4E, Phospho-4E-BP1 were down-regulated, while Phospho-eIF2α and Phospho-eIF4E were up-regulated after A549 cells were treated with 20 and 40 μM curcumin for 24 h. In addition, the effects of curcumin on these protein expression changes followed a significant dose-response (P < 0.05, trend test). These findings suggest that curcumin could reduce cell viability through prohibiting the initiation of protein synthesis by modulating eIF2α and eIF4E.     

Astrocytes aged in vitro show a decreased neuroprotective capacity

Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100beta increased at 90 days when cells were positive for the senescence beta-galactosidase marker. In long-term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn-superoxide dismutase (SOD-1) and haeme oxygenase-1 (HO-1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90-DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H2O2 exposure. Enhanced glutamate uptake was probably because of up-regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age-related neurodegenerative processes.     

Effects of ethylene on somatic embryogenesis and galanthamine content in <Emphasis Type="Italic">Leucojum aestivum</Emphasis> L. cultures

The influence of ethylene on in vitro morphogenesis of Leucojum aestivum and galanthamine accumulation was studied. Calli were cultivated on Murashige and Skoog (MS) medium supplemented with 25 μM 4-amino-3,5,6-trichloropicolinic acid (picloram) and 0.5 μM benzyladenine (BA). During incubation under these conditions, callus cultures produced ethylene (9.5 nL/g fresh weight: F.W.) whereas no ethylene was found in somatic embryos cultivated on medium supplemented with 0.5 μM α-naphthalene acetic acid (NAA) and 5 μM zeatin. Application of the precursor of ethylene 1-aminocyclopropane-1-carboxylic acid (ACC) increased ethylene production in both cultures, and decreased callus growth by a factor of 1.2, whereas callus growth was enhanced by a factor of 1.1 in the presence of an inhibitor of ethylene silver nitrate (AgNO₃) or by a factor of 1.2 with an absorbent potassium permanganate (KMnO₄). ACC enhanced the induction of somatic embryos and the development of globular embryos. Removal of ethylene by KMnO₄ during somatic embryogenesis led to the development of plants with greater length. Silver thiosulphate (STS) induced galanthamine production in callus cultures (0.1% dry weight), whereas ACC induced galanthamine production in somatic embryo cultures (2% dry weight).     

Phenylpropanoid production in callus and cell suspension cultures of <Emphasis Type="Italic">Buddleja cordata</Emphasis> Kunth

Plant tissue cultures represent a potential source for producing secondary metabolites. In this work, Buddleja cordata tissue cultures were established in order to produce phenylpropanoids (verbascoside, linarin and hydroxycinnamic acids), as these metabolites are credited with therapeutic properties. Highest callus induction (76.4–84.3%) was obtained in five treatments containing 2,4-Dichlorophenoxyacetic acid (2,4-d: 0.45–9.05 μM) with Kinetin (KIN: 2.32, 4.65 μM), whereas highest root induction (79.6%) corresponded to the α-Naphthaleneacetic acid (9.05 μM) with KIN (2.32 μM) treatment. Verbascoside was the major phenylpropanoid produced in in vitro cultures (root, white and green callus) [66.24–86.26 mg g⁻¹ dry weight (DW)], while linarin and hydroxycinnamic acid production was low (0.95–3.01 mg g⁻¹ DW). Verbascoside and linarin production were improved in cell suspension culture (116 mg g⁻¹ DW and 8.12 mg g⁻¹ DW, respectively).