Search

ABSTRACTEnokipodins are antimicrobial sesquiterpenes produced by Flammulina velutipesin a mycelial culture medium. To date, enokipodin production has not been reported in other members of the genus Flammulina. Hence, in this study, the production of enokipodins A, B, C, and D by F. velutipesand F. rossicawas investigated. Some strains of F. rossicawere confirmed to produce at least one of the four enokipodins in the culture medium. However, some strains of F. velutipesdid not produce any of the enokipodins. In an antibacterial assay using liquid medium, enokipodin B showed the strongest growth inhibitory activity against Bacillus subtilisamong the four types of enokipodins. Enokipodin B inhibited the spore germination of some plant pathogenic fungi. Enokipodins B and D exerted moderate anti-proliferative activity against some cancer cell lines, and enokipodins A and C inhibited the proliferation of the malarial parasite, Plasmodium falciparum.

Enokipodins are antimicrobial sesquiterpenes produced by Flammulina velutipesin a mycelial culture medium. To date, enokipodin production has not been reported in other members of the genus Flammulina. Hence, in this study, the production of enokipodins A, B, C, and D by F. velutipesand F. rossicawas investigated. Some strains of F. rossicawere confirmed to produce at least one of the four enokipodins in the culture medium. However, some strains of F. velutipesdid not produce any of the enokipodins. In an antibacterial assay using liquid medium, enokipodin B showed the strongest growth inhibitory activity against Bacillus subtilisamong the four types of enokipodins. Enokipodin B inhibited the spore germination of some plant pathogenic fungi. Enokipodins B and D exerted moderate anti-proliferative activity against some cancer cell lines, and enokipodins A and C inhibited the proliferation of the malarial parasite, Plasmodium falciparum.

The ability of megakaryoblastic leukemia 1 (MKL1) to function as a serum response factor (SRF) coactivator is regulated through its association with G-actin. In the cytoplasm, MKL1 binds to G-actin through RPXXXEL (RPEL) motifs. However, dissociation of MKL1 from G-actin triggers its translocation into the nucleus where it stimulates SRF-mediated gene expression. Previous characterization of rat MKL1 gene products has identified several isoforms: full-length MKL1, basic, SAP, and coiled-coil domain (BSAC), MKL1-elongated derivative of yield (MELODY), and MKL1met. In this study, we have investigated whether these MKL1 isoforms, which contain different numbers of RPEL motifs, differ in their subcellular localization, transcriptional activity, and effect on dendritic number and axonal length. Immunofluorescent staining of cultured cortical neurons expressing individual FLAG-tagged MKL1 isoforms indicated that all MKL1 isoforms are present in both the cytoplasm and the nucleus. However, MKL1met, which contains two RPEL motifs, shows enhanced nuclear staining compared with the other three isoforms, full-length MKL1, basic, SAP, and coiled-coil domain, and MKL1-elongated derivative of yield, which contain three RPEL motifs. Consistent with its preferential nuclear localization, overexpression of MKL1met, but not other isoforms, increases SRF-mediated transcriptional responses and reduces the number of dendrites. In contrast to the inhibitory effect of MKL1met on dendritic number, axonal length is not affected by overexpression of any of the MKL1 isoforms. These findings suggest that the subcellular localization of MKL1 isoforms, which is mediated by the number of actin-binding RPEL motifs, regulates their effect on SRF-mediated gene expression and dendritic morphology.

We investigated the regulation of l-type amino acid transporter 1 (LAT1) in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) in response to glucose deprivation. The amounts of LAT1 and 4F2 heavy chain (4F2hc) mRNA in TR-iBRB2 cells exposed to glucose-free culture medium for 8 to 24 h were significantly elevated compared with those in control medium. Concomitantly, [3H]l-leucine uptake activity was increased, suggesting that LAT1 transport activity is induced under glucose-deprivation. To determine the transcriptional activity of the LAT1gene under glucose-free conditions, the promoter activity of the LAT1gene of approximately 2 kbp (–1958 bp to + 70 bp) in TR-iBRB2 cells was assayed using a dual-luciferase reporter assay system. The transcriptional activity of the 2 kbp LAT1promoter under the glucose-free conditions was 1.7-fold greater than that under normal glucose conditions. The presence of an activator site(s) between –162 bp and –155 bp was indicated by the low activities exhibited by the construct spanning this region and mutagenesis. These results suggest that the glucose deprivation sensitivity of LAT1 expression is transcriptionally regulated, and cis-elements within the LAT1promoter region from –162 bp to –155 bp mediate this regulation.

The mRNA expression of brain-derived neurotrophic factor (BDNF) is controlled in an activity-dependent manner through Ca(2+) influx into neurons. Pyrethroids are widely used insecticides of low acute toxicity in mammals, but their effects on sodium channels are known to lead to hyperexcitation in neuronal cells of insects. In this study, we found that deltamethrin, a type II pyrethroid insecticide, was highly effective in inducing BDNF expression in culture and in the rat brain. Addition of deltamethrin to rat cortical cells in culture markedly increased the expression of BDNF exon III-V mRNA and protein, dependent upon the neuronal activity accompanying the influx of Ca(2+) into neurons and the Ca(2+) influx-dependent phosphorylation of extracellular signal-regulated kinases 1/2. The elevated expression was maintained for at least 48 h, even after deltamethrin was withdrawn from the culture medium. Comparison of the effects of selected pyrethroids on the expression revealed that type II but not type I pyrethroids effectively induced BDNF mRNA expression. In addition, administration of deltamethrin to rats increased the level of BDNF protein in the cerebral cortex and hippocampus. These results indicate that deltamethrin is a potent inducer of BDNF expression in neurons and that it may induce neuronal hyperexcitation if it reaches the brain.

Although it is widely accepted that an activity-dependent gene transcription is induced by the calcium (Ca2+) signals in neurons, it is still unclear how the particular mRNA moieties are transiently accumulated in response to synaptic transmission that evokes multiple intracellular signals including Ca2+and cAMP ones. Promoters of the brain-derived neurotrophic factor (BDNF) and the pituitary adenylate cyclase-ativating polypeptide (PACAP) can commonly be activated through the cAMP-responsive element (CRE), to which the CRE-binding protein (CREB) predominantly bound. The activation of BDNF gene promoter I and III (BDNF-PI and -PIII, respectively) was mediated not only by the CREB but also by the upstream stimulatory factor, whereas that of PACAP gene promoter (PACAP-P) was mediated by only one CRE located at around −200. The PACAP-P was synergistically enhanced by Ca2+and cAMP signals through the CRE, whereas the BDNF-PI did not show such a synergistic activation upon the stimulation with both signals. In addition, we found that the half-lives of PACAP and BDNF mRNA were prolonged by the Ca2+influx into neurons but not that of Arc mRNA, indicating an activity-dependent stabilization of particular mRNA species in neurons. Thus, the activity-dependent gene expression is co-ordinately controlled by Ca2+and cAMP signals not only at the transcriptional level but also at the post-transcriptional level for the cumulative mRNA expression in neurons.

Although -amyloid precursor protein (APP) has been suggested to play a role in neuronal survival and plasticity, the mRNA expression of APP has not been studied in terms of neuronal activity. In cultures of mouse cerebellar granule cells, we found that the levels of APP mRNA increased when a high concentration of potassium was present in the medium. A deprivation of membrane depolarization caused by lowering the Kconcentration decreased both mRNA expression and protein synthesis of APP. Increasing the concentration, however, restored mRNA expression, which was driven by the influx of Ca2through L-type voltage-dependent calcium channels and mediated by de novoprotein synthesis. Thus, APP mRNA expression is controlled in an activity-dependent manner in neurons.

The use of different brain-derived neurotrophic factor (BDNF) gene promoters results in the differential production of 5′-alternative transcripts, suggesting versatile functions of BDNF in neurons. Among four BDNF promoters I, II, III, and IV (BDNF-PI, -PII, -PIII, and -PIV), BDNF-PI was markedly activated, as well as BDNF-PIII, by Ca2+signals evoked via neuronal activity. However, little is known about the mechanisms for the transcriptional activation of BDNF-PI. Using rat cortical neurons in culture, we assigned the promoter sequences responsible for the Ca2+signal-mediated activation of BDNF-PI and found that the Ca2+-responsive elements were located in two separate (distal and proximal) regions and that the DNA sequences in the proximal region containing cAMP-responsive element (CRE), which is overlapped by the upstream stimulatory factor (USF)-binding element, were largely responsible for the activation of BDNF-PI. CRE-binding protein (CREB) family transcription factors and USF1/USF2 bind to this overlapping site, depending upon their preferred sequences which also control the magnitude of the activation. Overexpression of dominant negative CREB or USF reduced the BDNF-PI activation. These findings support that not only CREB but also USF1/USF2 contributes to Ca2+signal-mediated activation of BDNF-PI through the recognition of an overlapping CRE and USF-binding element.

The mouse pituitary adenylate cyclase activating polypeptide (PACAP) gene consists of two alternative exons (1A and 1B) and four exons including the coding region. By the usage of alternative exons, three transcripts can be produced. In this report, however, we found that at least five preproPACAP transcripts, including two novel types, were produced in mouse cerebellar granule cells (CGCs) and their expression was induced by the Ca2influx through L-type voltage-dependent Ca2channels. The splicing occurred between the 3′-donor site of exon 1A and the two novel internal 5′-acceptor sites of exon 1B. These findings suggest that preproPACAP transcripts are produced by the complicated usage of the 5′-untranslated region in an activity-dependent manner.

Although the brain-derived neurotrophic factor (BDNF) gene is activated by the intracellular Ca2+signals evoked via Ca2+influx into neurons, little is known about how the activation of alternative BDNF gene promoters is controlled by the Ca2+signals evoked viaN-methyl-d-aspartate receptors (NMDA-R) and L-type voltage-dependent Ca2+channels (L-VDCC). There is a critical range in the membrane depolarization caused by high K+concentrations (25–50 mmKCl) for effective BDNF mRNA expression and transcriptional activation of BDNF gene promoters I and III (BDNF-PI and -PIII, respectively) in rat cortical culture. The increase in BDNF mRNA expression induced at high K+was repressed not only by nicardipine, an antagonist for L-VDCC, but also by dl-amino-5-phosphonovalerate, an antagonist for NMDA-R, which was supported by the effects of antagonists on the Ca2+influx. Although the promoter activations at 25 and 50 mmKCl were different, BDNF-PIII was activated by either the Ca2+influx through NMDA-R or L-VDCC, whereas BDNF-PI was predominantly by the Ca2+influx through L-VDCC. Direct stimulation of NMDA-R supported the activation of BDNF-PIII but not that of BDNF-PI. Thus, the alternative BDNF gene promoters responded differently to the intracellular Ca2+signals evoked via NMDA-R and L-VDCC.

Although the neuron-restrictive silencer element (NRSE/Regard) has been shown to function as a negative-acting DNA regulatory element to prevent the expression of neuron-specific genes in non-neuronal cells, little is known about its silencing effect on transcription in primary glial cells nor its effect on transcriptional activation in primary neurons. By DNA transfection in primary cultures of rat cortical neuronal or glial cells, we investigated the effect of NRSE on transcription mediated by the BDNF promoter I or c-fospromoter to which NRSE sequences derived from the SCG10 gene were linked. Transfection of plasmid DNAs to NIH3T3 fibroblasts resulted in a marked repressive effect of NRSE on BDNF promoter I- or c-fospromoter-mediated transcription. In primary neuronal cells, however, NRSE did not repress the basal promoter activities of BDNF and c-fosgenes and allowed the transcriptional activation of these genes induced by membrane depolarization although NRSE slightly reduced the magnitude of BDNF promoter I activation. In contrast to neuronal cells, a marked repression of basal promoter activities of both genes was detected in primary glial culture and a two base pair-mutation of NRSE partially recovered the repression. These results indicate that NRSE negatively acts on its linked promoters in primary glial cells and does not interfere an activation of linked promoters in neuronal cells.

Although DNA transfection with calcium-phosphate/DNA precipitates for promoter analysis of genes has been previously applied to primary cultures of neuronal cells, it remains uncertain whether the expression of the introduced genes in glial cells, which are also in primary culture, affects the transcriptional signals obtained. Using plasmid DNA containing the brain-derived neurotrophic factor (BDNF) gene promoter I or c-fospromoter, we investigated the optimum conditions for calcium-phosphate-mediated DNA transfection in primary culture of rat cortical neuronal cells. Normalizing the firefly luciferase activity of the experimental reporter gene to theRenillaluciferase activity of the internal control increased experimental reliability. Maximum expression of firefly luciferase activity in the cells stimulated by membrane depolarization required 6–12 hrs of incubation after stimulation. Differences in the ratio of the experimental reporter gene to the internal control did not affect experimental gene expression. Under our optimal conditions, the activation of the BDNF gene promoter I was detected in neuronal but not in glial cells. Calcium-phosphate-mediated DNA transfection should be widely applicable for promoter analysis of inducible genes in neurons.

To understand the role of nitric oxide (NO) in controlling the specific DNA-binding activities of transcriptional factors, we investigated the in vitro effect of the NO-donor sodium nitroprusside (SNP) on the AP-1 activity of cultured mouse cerebellar granule cells. A gel-mobility assay showed that SNP inhibited AP-1 activity in the presence, but not the absence of dithiothreitol (DTT). This DTT-dependent inhibition of AP-1 activity by SNP corresponded with the activation of the chemical reactivity of SNP with DTT, which can be monitored by the production of nitrite (NO −2). In contrast, diamide, a typical sulfhydryl oxidizing agent, inhibited AP-1 activity in the absence of DTT and its inhibitory effect was reversed competitively by DTT. Studies using structurally or functionally related analogues of SNP demonstrated that S-nitrosylation of the AP-1 moiety mediated by some NO-carriers but not by free NO, which can be produced by the chemical reaction of SNP with DTT, was responsible for the inhibition of AP-1 activity, suggesting NO-mediated regulation of the AP-1 transcriptional factor.

To understand the role of nitric oxide (NO) in controlling the specific DNA‐binding activities of transcriptional factors, we investigated the in vitro effect of the NO‐donor sodium nitroprusside (SNP) on the AP‐1 activity of cultured mouse cerebellar granule cells. A gel‐mobility assay showed that SNP inhibited AP‐1 activity in the presence, but not the absence of dithiothreitol (DTT). This DTT‐dependent inhibition of AP‐1 activity by SNP corresponded with the activation of the chemical reactivity of SNP with DTT, which can be monitored by the production of nitrite (NO−2). In contrast, diamide, a typical sulfhydryl oxidizing agent, inhibited AP‐1 activity in the absence of DTT and its inhibitory effect was reversed competitively by DTT. Studies using structurally or functionally related analogues of SNP demonstrated that S‐nitrosylation of the AP‐1 moiety mediated by some NO‐carriers but not by free NO, which can be produced by the chemical reaction of SNP with DTT, was responsible for the inhibition of AP‐1 activity, suggesting NO‐mediated regulation of the AP‐1 transcriptional factor.

Although stimulation of N-methyl-D-aspartate receptors or voltage-dependent calcium channels induces both the activation of c-fosand brain-derived neurotrophic factor (BDNF) genes, it is not certain how the activation of these genes is related. Using primary cultures of rat hippocampal neurons, we found that exposing the cells to cycloheximide allowed subsequent activation of BDNF mRNA expression, although activation of AP-1 DNA-binding activity resulting from the c-fosinduction was abolished. Super-induction of BDNF gene was also caused by cycloheximide. The estimated half-life of BDNF mRNA was approximately 2.5 hrs, which was almost identical to that of c-fosmRNA. These results indicate that nascent AP-1 is not required for the activation of BDNF gene, leading to the notion that the BDNF gene can be activated by Ca2+signals as an immediate early gene.