A basidomycetous hydroxynaphthalene-prenylating enzyme exhibits promiscuity toward prenyl donors.

The fungal prenyltransferase ShPT from Stereum hirsutum was believed to prenylate 4-hydroxybenzyl alcohol and thereby be involved in the vibralactone biosynthesis. In this study, we demonstrate that hydroxynaphthalenes instead of benzyl alcohol or aldehyde were accepted by ShPT for regular C-prenylation in the presence of both dimethylallyl and geranyl diphosphate. Although the natural substrate of ShPT remains unknown, our results provide one additional prenyltransferase from basidiomycetes, which are less studied, in comparison to those from other sources.

Neuroprotective effects of Ginkgo biloba extract.

Ginkgo biloba extract has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of dementia. The extract contains multiple compounds such as flavonoids and terpenoids that are thought to contribute to its neuroprotective and vasotropic effects. In this review, we summarize the experimental results on the mechanism of neuroprotection induced by standardized extract of Ginkgo biloba leaves (EGb 761) and its constituents.

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 microM 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 1mM glutamate for 1 h.

Increase in glutamate-induced neurotoxicity by activated astrocytes involves stimulation of protein kinase C.

Activation of astrocytes is a common feature of neurological disorders, but the importance of this phenomenon for neuronal outcome is not fully understood. Treatment of mixed hippocampal cultures of neurones and astrocytes from day 2-4 in vitro (DIV 2-4) with 1 micro m cytosine arabinofuranoside (AraC) caused an activation of astrocytes as detected by a stellate morphology and a 10-fold increase in glial fibrillary acidic protein (GFAP) level compared with vehicle-treated cultures. After DIV 12, we determined 43% and 97% damaged neurones 18 h after the exposure to glutamate (1 mm, 1 h) in cultures treated with vehicle and AraC, respectively.

Stimulation of beta-adrenoceptors activates astrocytes and provides neuroprotection.

Our previous studies established that induction of growth factor synthesis and neuroprotection by the beta(2)-adrenoceptor agonist clenbuterol in vitro and in vivo was associated with the activation of astrocytes, the major source of trophic factors in the brain. In the present study, we further investigated the specificity of beta(2)-adrenoceptor-mediated effects on astrocyte activation and neuroprotection. In mixed hippocampal cultures neuroprotection against glutamate-induced cell death by clenbuterol (1 microM) was blocked by the beta(1/2)-adrenoceptor antagonist propranolol and the specific beta(2)-adrenoceptor antagonists 1-[2,3-(Dihydro-7-methyl-1H-inden-4-yl)-oxy]-3-[(1-methylethyl)-amino]-2-butanol (ICI 118,551, 10 microM) and butoxamine (10 microM), while the beta(1)-adrenoceptor-selective antagonist metoprolol (10 microM) showed no effect.

Release of cytochrome c into the extracellular space contributes to neuronal apoptosis induced by staurosporine.

The mitochondrial protein cytochrome c has been identified as one of the key signalling molecules of apoptosis. In the present study, we used primary neuronal cultures to investigate whether cytochrome c was released from the mitochondria into the cytosol and subsequently into the culture medium during staurosporine-induced apoptosis and whether extracellular cytochrome c modulates the degree of damage caused by staurosporine. We found the cytochrome c content in the mitochondria decreased 24 h after and increased in the cytosol 8 h after staurosporine was added to the culture medium.

Preconditioning-induced protection against cyanide-induced neurotoxicity is mediated by preserving mitochondrial function.

The central nervous system is one of the main target organs in cyanide toxicity. In this study, primary cultures of chick embryonic neurons were used to characterize sodium cyanide (NaCN)-induced cell death and to investigate the mechanism of NaCN-mediated preconditioning. After treatment of the cells with 1mM NaCN for 1h followed by a NaCN-free incubation period of 23 h, we observed features of apoptosis such as a reduction in nuclear size, chromatin condensation and nuclear fragmentation as evaluated by nuclear staining with Hoechst 33258 and electron microscopy.

Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C.

The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-kappaB.

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor-kappaB (NF-kappaB) and how this activation contributes to preconditioning-induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine-induced (200 nM, 24 h) apoptosis.

Retinoic acid reduces apoptosis and oxidative stress by preservation of SOD protein level.

Retinoic acid (RA) has already been shown to exert antiapoptotic and antioxidative activity in various cells. In this study, we determined the effect of RA on the mRNA and protein levels of the Cu-,Zn-superoxide dismutase (SOD-1) and Mn-superoxide dismutase (SOD-2) during staurosporine-induced apoptosis in primary cultures from neonatal rat hippocampus. Exposure to staurosporine (300 nM, 24 h) increased the percentage of apoptotic neurons to 62% compared with 18% in controls.

Neuroprotective effects of NV-31, a bilobalide-derived compound: evidence for an antioxidative mechanism.

In previous studies we have already shown that the extract of Ginkgo biloba, and some of its constituents, such as ginkgolide B and bilobalide, protected cultured neurons against apoptotic and excitotoxic damage and reduced the infarct volume after focal cerebral ischemia in mice and rats. In this work, we determined the neuroprotective and antioxidative effects of 4-hydroxy-4-tert-butyl-2,3,5,6-tetrahydrothiopyran-1-oxide (NV-31), a stable compound which was synthesized to mimic the pharmacological activity profile of bilobalide. In pure neuronal cultures from chick embryo telencephalon, damage was induced by serum deprivation (24 h) and exposure to staurosporine (200 nM, 24 h) which caused an increase in the percentage of apoptotic neurons from 14 (controls) to 30 and 55%, respectively.

Staurosporine-induced apoptosis in cultured chick embryonic neurons is reduced by polyethylenimine of low molecular weight used as a coating substrate.

The survival of neurons largely depends on adhesion to extracellular matrix proteins. This study investigated the influence of polycationic macromolecules of different molecular weights used as coating substrates on apoptosis in primary cultures of chick embryonic neurons. Coating of the culture flasks with positively charged polyethylenimine (PEI) of 12, 32 and 1616 kDa led to different susceptibilities of the neurons to apoptosis induced by staurosporine and serum deprivation.

Retinoic acid potentiated the protective effect of NGF against staurosporine-induced apoptosis in cultured chick neurons by increasing the trkA protein expression.

Nerve growth factor (NGF) has already been shown to protect neurons and PC12 cells from cell death induced by different stimuli. When chick embryonic neurons were exposed to staurosporine (200 nM, 24 hr), the percentage of apoptotic neurons increased from 15% in controls to 80%, but the treatment with NGF alone did not show any neuroprotection. In the presence of retinoic acid (RA, 5 microM), however, NGF (20 pg/ml) reduced staurosporine-induced damage to 42% apoptotic neurons compared to 58% in the presence of RA (5 icroM) alone.

S-100beta protects cultured neurons against glutamate- and staurosporine-induced damage and is involved in the antiapoptotic action of the 5 HT(1A)-receptor agonist, Bay x 3702.

The serotonin (5-HT)(1A) receptor agonists have already been shown to protect cultured neurons from excitotoxic as well as from apoptotic damage [B. Ahlemeyer, J. Krieglstein, Stimulation of 5-HT(1A) receptors inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo, Brain Res.

Neuroprotection by estrogens in a mouse model of focal cerebral ischemia and in cultured neurons: evidence for a receptor-independent antioxidative mechanism.

Estrogens have been suggested for the treatment of neurodegenerative disorders, including stroke, because of their neuroprotective activities against various neurotoxic stimuli such as glutamate, glucose deprivation, iron, or beta-amyloid. Here, the authors report that 17beta-estradiol (0.3 to 30 mg/kg) and 2-OH-estradiol (0.

Inhibition of glutathione depletion by retinoic acid and tocopherol protects cultured neurons from staurosporine-induced oxidative stress and apoptosis.

Cellular redox status is an important factor during neuronal apoptosis. In primary cultures of chick embryonic neurons, serum deprivation and treatment with staurosporine (200 nM) for 24 h increased the percentage of apoptotic neurons from 13% in controls to 28%, and 68%, respectively. Both exposure to staurosporine and serum deprivation resulted in a four-fold increase in the mitochondrial reactive oxygen species production 4 h after the onset of the injury.

The 5-HT1A receptor agonist Bay x 3702 inhibits apoptosis induced by serum deprivation in cultured neurons.

We examined whether the highly selective 5-HT1A receptor agonist (-)-(R)-2-[4-[[(3,4-dihydro-2H-1-benzopyran-2-yl)methyl]-amino]butyl]-11 ,2-benz-isothiazol-3(2H)-one 1,1-dioxide monohydrochloride (Bay x 3702) could inhibit neuronal apoptosis induced by serum deprivation. In primary cultures of chick embryonic neurons and in mixed neuronal/glial cultures from neonatal rat hippocampus, Bay x 3702 (1 microM) rescued serum-deprived neurons from apoptosis. The antiapoptotic effect of Bay x 3702 (1 microM) was blocked in chick neurons by the selective 5-HT1A receptor antagonists 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazin]ethyl]-N-2-pyridinyl-be nzamide hydrochloride (p-MPPI, 10 microM) and 4-[3-benzotriazol-1-propyl]-1-(2-methoxyphenyl)-piperazine (BPMP, 10 microM) as well as by anti-nerve growth factor (anti-NGF) antibodies and in rat neurons by N-[2-4-(2-methoxy)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane-carbo xamide trihydrochloride (WAY 100635, 10 microM).

Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents.

Previous studies have already demonstrated that some constituents of an extract of Ginkgo biloba (EGb), such as ginkgolide B and bilobalide, protect cultured neurons from hypoxia- and glutamate-induced damage. This prompted us to investigate whether they were also able to inhibit neuronal apoptosis. We induced apoptosis in cultured chick embryonic neurons as well as in mixed cultures of neurons and astrocytes from neonatal rat hippocampus by serum deprivation and staurosporine.

Retinoic acid reduces staurosporine-induced apoptotic damage in chick embryonic neurons by suppressing reactive oxygen species production.

The effect of all-trans retinoic acid (RA), which is known as a regulator of cell growth and differentiation, was studied during neuronal apoptosis. Apoptosis was induced in primary cultures of chick embryonic neurons by treatment with staurosporine (200 nM) for 24 h which led to a reduction of cellular viability to 40% compared to 83% in untreated cultures as well as to an increase in the number of apoptotic neurons (determined by nuclear staining with Hoechst 33258) to 60% compared to 15% in untreated cultures. RA (1 nM-10 microM) reduced the number of non-viable and apoptotic cells in a concentration-dependent manner and the maximal response was seen at 1 microM RA with 60% cellular viability and 38% apoptotic neurons.

Stimulation of 5-HT1A receptor inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo.

Primary cultures of neurons from chick embryo telencephalons were deprived of serum to induce apoptotic cell death. After 24 h of serum withdrawal, we found a reduction in cell viability from 85% to 72% and an increase in the number of apoptotic cells from 12% to 29%. The 5-HT1A receptor agonist 8-OH-DPAT inhibited the decrease in cell viability and reduced the number of apoptotic cells in a concentration-dependent manner.

[Measurement of free intracellular Ca2+ concentration in cultivated hippocampal neurons].

The cascade of pathobiochemical reactions caused by cerebral ischemia is complex and not completely understood. One important factor seems to be the disturbed calcium homeostasis. Measurement of cytosolic-free Ca2+ concentration [Ca2+]i is possible with the Ca(2+)-sensitive dye fura-2.

Effects of emopamil on postischemic blood flow and neuronal damage in rat brain.

The effects of the calcium entry blocker emopamil on physiological variables, local cerebral blood flow (LCBF) and on hippocampal cell damage were evaluated after 10 min of forebrain ischemia in the rat. LCBF was determined with the 14C-iodoantipyrine technique after 2, 10, and 60 min of postischemic recirculation. Histological evaluation was performed 7 days after ischemia in cortical and hippocampal tissue by determination of the percentage of necrotic neurons.

Uncoupling of cerebral blood flow and glucose utilization by dihydroergocristine in the conscious rat.

Dose-dependent effects of the dihydrogenated ergot alkaloid dihydroergocristine on physiological variables, local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCGU) were evaluated in the conscious rat after intravenous injection. Heart rate was reduced with 2.5 mg/kg and 20 mg/kg dihydroergocristine.

[Absorption, distribution and metabolism of [14C]-levomenol in the skin].

The purpose of the present investigations was to study the cutaneous absorption of sesquiterpenic alcohol, the major active principle of chamomile. For these investigations 14C-labelled levomenol ((-)-6-methyl-2-(4-methyl-3-cyclohexen-1-yl)-5-hepten-2-ol; (-)-alpha-bisabolol) was prepared by biochemical incorporation of [14C]-acetate into the molecule. 5 h after topical application of the radiolabelled substance onto nude mice half of the radioactivity was found in the skin.