Quantitative prediction of the growth inhibition of various harmful chemicals by statistical analysis of delayed fluorescence decay curves obtained from the green alga Raphidocelis subcapitata.

The toxicity of chemical substances to algal growth is generally measured by the 72-96 h algal growth inhibition test. We have developed a method to assess the toxicity of chemicals in aquatic environments more quickly and simply than conventional testing methods by delayed fluorescence (DF), which reflects the photosynthetic capacity of algae. The DF method is based on a technique for evaluating the amount of change in the decay curve due to the effects of chemicals ([Formula: see text], DF inhibition). Various studies on DF have been reported; however, few reports have evaluated the decay curve of DF by approach using inductive modeling based on measurement data such as principal component analysis (PCA) and partial least squares regression analysis (PLS). Therefore, the purpose of this study is to examine methods for estimating the magnitude and type of toxicity of chemicals by means of a principal component model (PC model) and multiple regression model (MR model) derived from changes in the decay curves of DF of algae exposed to a wide range of 37 toxic substances that have an effect of clear magnitude on algal growth. The changes in the DF decay curves due to exposure the 37 toxic substances to algae were summarized in the PC model composed of eigenvectors and scores of four principal components. For validation of usefulness, a hierarchical cluster analysis (HCA) of the amount of change in four PC scores revealed that the growth inhibition rate was more influential than the chemical type. We also found the possibility of quantitatively predicting the growth inhibition of chemicals by MR model by the amount of change in the PC scores.

Expression of SOLOIST/MRTFB i4, a novel neuronal isoform of the mouse serum response factor coactivator myocardin-related transcription factor-B, negatively regulates dendritic complexity in cortical neurons.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB), a serum response factor (SRF) coactivator, is an important regulator of gene expression and neuronal morphology. Here, we show that different mouse MRTFB splice isoforms, including a novel fourth MRTFB isoform named spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4), play distinct roles in this process. SOLOIST/MRTFB i4 has a short exon that encodes 21 amino acid residues ahead of the first RPXXXEL (RPEL) motif in MRTFB isoform 3. Quantitative PCR revealed that SOLOIST/MRTFB i4 and isoform 1 were enriched in the forebrain and neurons, and up-regulated during brain development. Conversely, isoform 3 was detected in various tissues, including both neurons and astrocytes, and was down-regulated in the developing brain. Reporter assays supported the SRF-coactivator function of SOLOIST/MRTFB i4 as well as isoform 1. Acute expression of MRTFB isoform 1, but not isoform 3 or SOLOIST/MRTFB i4, in neuronal cells within 24 hr drastically increased endogenous immediate early gene [c-fos, egr1, and activity-regulated cytoskeleton-associated protein] expression, but not endogenous actinin α1, ß-actin, gelsolin, or srf gene expression measured by qPCR. Over-expression of SOLOIST/MRTFB i4 reduced the dendritic complexity of cortical neurons, whereas over-expression of isoform 1 increased this complexity. Co-expression of isoform 1 and SOLOIST/MRTFB i4 in cortical neurons revealed that isoform 1 competitively counteracted down-regulation by SOLOIST/MRTFB i4. Our findings indicate that MRTFB isoforms have unique expression patterns and differential effects on gene expression and dendritic complexity, which contribute to shaping neuronal circuits, at least in part.

Neuron-enriched phosphatase and actin regulator 3 (Phactr3)/ nuclear scaffold-associated PP1-inhibiting protein (Scapinin) regulates dendritic morphology via its protein phosphatase 1-binding domain.

Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively.

Validation of rapid algal bioassay using delayed fluorescence in an interlaboratory ring study.

Algal growth inhibition tests are generally used to determine the toxic effects of chemical substances on algae growth. In this report, we describe a rapid and simple test procedure using delayed fluorescence (DF) to determine chemical toxicities more rapidly than the conventional 72h or 96h growth inhibition tests. We assess the suitability of DF to serve as an alternative endpoint for biomass production and determine the variability by an interlaboratory ring study using a typical reference toxicant 3,5-dichlorophenol (DCP). The results suggest that DF has the potential to be used as a surrogate measure of photosynthetically-active biomass in the algal growth inhibition tests. The half maximal effective concentration (EC50) values of DCP determined from the DF inhibition test in 6h and 24h (1.2±0.3mg/L and 2.7±0.5mg/L respectively) are in reasonable agreement with the EC50 value of DCP determined by the 72h conventional method (1.8mg/L). In the interlaboratory ring study, the intralaboratory and interlaboratory variabilities of the EC50 of the DF inhibition test for a 24h exposure period are 12% and 28% respectively. DF intensity can be considered as a surrogate of living biomass with active photosynthesis, and we conclude that a 24h exposure duration better estimates the toxic effects measured using conventional surrogate measures for dry weight such as cell counts, volume, optical density or fluorescence.

Cellular localization and dendritic function of rat isoforms of the SRF coactivator MKL1 in cortical neurons.

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.

Identification, expression and characterization of rat isoforms of the serum response factor (SRF) coactivator MKL1.

Megakaryoblastic leukemia 1 (MKL1) is a member of the MKL family of serum response factor (SRF) coactivators. Here we have identified three rat MKL1 transcripts: two are homologues of mouse MKL1 transcripts, full-length MKL1 (FLMKL1) and basic, SAP, and coiled-coil domains (BSAC), the third is a novel transcript, MKL1-elongated derivative of yield (MELODY). These rat MKL1 transcripts are differentially expressed in a wide variety of tissues with highest levels in testis and brain. During brain development, these transcripts display differential patterns of expression. The FLMKL1 transcript encodes two isoforms that utilize distinct translation start sites. The longer form possesses three actin-binding RPXXXEL (RPEL) motifs and the shorter form, MKL1met only has two RPEL motifs. All four rat MKL1 isoforms, FLMKL1, BSAC, MKL1met and MELODY increased SRF-mediated transcription, but not CREB-mediated transcription. Accordingly, the differential expression of MKL1 isoforms may help fine-tune gene expression during brain development.