Mg, K-containing microparticle: A possible active principle of a culture extract produced by a microbial consortium.

A synthetic microbial consortium called Effective Microorganisms (EM) consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects of EM∙XGOLD, a health drink produced by EM, on life cycle of Dictyostelium discoideum were described previously. Here, we report our attempt to identify the active principle, termed EMF, that brought about the observed effects. Throughout the purification processes, the presence of the active principle was monitored by promoted fruiting body formation. By liquid-liquid separation the activity was recovered in aqueous phase, which, after concentration, was further subjected to reverse-phase column chromatography. No activity was detected in any eluant, while almost all the activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures to the residual fraction did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, the residue revealed spherical particles of µm size range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence, on the surface of the particles, of magnesium and, to a certain extent, of potassium. In separate experiments, acid treatment and alkali neutralization of EM∙XGOLD completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticles to be an active principle of EM culture extract. How these particles exert their effect is currently under intensive investigation.

The Life Cycle of Dictyostelium discoideum Is Accelerated via MAP Kinase Cascade by a Culture Extract Produced by a Synthetic Microbial Consortium.

A cellular slime mold, Dictyostelium discoideum, is an amoeboid organism that has a unique life cycle consisting of distinctly separated vegetative and developmental phases. Thus, this organism presents a rare opportunity in which to examine the effects of bioactive substances on separate cellular activities. In this research, we investigated the effect of a culture extract, termed EMXG, produced by a synthetic microbial consortium. EMXG promoted proliferative response of amoeba cells. It further accelerated the developmental phase, leading to the preferred fruiting body formation from fewer cells. Furthermore, EMXG modulated biological rhythm of this organism, that is, interval of oscillation of cAMP level observed in suspension starvation was significantly shortened. Concomitantly, the level of ERKB, a MAP kinase, was found to oscillate in a similar fashion to that of cAMP. Additionally, ErkB-deficient mutant amoeboid cells did not respond to proliferative stimulation by EMXG. These lines of evidence point to a likelihood that MAP kinase cascade is involved and further that ErkB could be the molecular target of EMXG.

Characterization of a novel KRAS mutation identified in Noonan syndrome.

Noonan syndrome (NS) is the most common non-chromosomal syndrome seen in children and is characterized by short stature, dysmorphic facial features, chest deformity, a wide range of congenital heart defects and developmental delay of variable degree. Mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathways cause about 70% of NS cases with a KRAS mutation present in about 2%. In a cohort of 65 clinically confirmed NS patients of Japanese origin, we screened for mutations in the RAS genes by direct sequencing. We found a novel mutation in KRAS with an amino acid substitution of asparagine to serine at codon 116 (N116S). We analyzed the biological activity of this mutant by ectopic expression of wild-type or mutant KRAS. NS-associated KRAS mutation resulted in Erk activation and active Ras-GTP levels, and exhibited mild cell proliferation. In addition, kras-targeted morpholino knocked-down zebrafish embryos caused heart and craniofacial malformations, while the expression of mutated kras resulted in maldevelopment of the heart. Our findings implicate that N116S change in KRAS is a hyperactive mutation which is a causative agent of NS through maldevelopment of the heart.

Phosphorylation of the chromodomain changes the binding specificity of Cbx2 for methylated histone H3.

The chromatin organizer modifier domain (chromodomain) is present in proteins that contribute to chromatin organization and mediates their binding to methylated histone H3. Despite a high level of sequence conservation, individual chromodomains manifest substantial differences in binding preference for methylated forms of histone H3, suggesting that posttranslational modification of the chromodomain might be an important determinant of binding specificity. We now show that mouse Cbx2 (also known as M33), a homolog of Drosophila Polycomb protein, is highly phosphorylated in some cell lines. A low-mobility band of Cbx2 observed on SDS-polyacrylamide gel electrophoresis was thus converted to a higher-mobility band by treatment with alkaline phosphatase. Mass spectrometric analysis revealed serine-42, a conserved amino acid in the chromodomain, as a phosphorylation site of Cbx2. Phosphorylation of the chromodomain of Cbx2 on this residue in vitro resulted in a reduced level of binding to an H3 peptide containing trimethylated lysine-9 as well as an increase in the extent of binding to an H3 peptide containing trimethylated lysine-27, suggesting that such phosphorylation changes the binding specificity of Cbx2 for modified histone H3. Phosphorylation of the chromodomain of Cbx2 may therefore serve as a molecular switch that affects the reading of the histone modification code and thereby controls epigenetic cellular memory.

Polycomb group protein Ezh1 represses Nodal and maintains the left-right axis.

The left-right (LR) axis is essential for the proper function of internal organs. In mammals and fish, left-sided Nodal expression governs LR patterning. Here, we show that the Polycomb group protein Ezh1, which is highly conserved from fish to human, participates in LR patterning. Knockdown of olezh1, a medaka homologue of Ezh1, led to LR reversal of internal organs. It was shown that OLEZH1 acts in silencing the expression of Spaw (a medaka homolog of Nodal) via a previously unknown pathway. Furthermore, coimmunoprecipitation showed physical interaction of Ezh1 with FoxH1, a Nodal regulator. This represents a novel mechanism for LR patterning and implies that Ezh1 has developmental importance.

Zebrafish gene knockdowns imply roles for human YWHAG in infantile spasms and cardiomegaly.

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder presenting with an elfin-like face, supravalvular aortic stenosis, a specific cognitive-behavioral profile, and infantile hypercalcemia. We encountered two WBS patients presenting with infantile spasms, which is extremely rare in WBS. Array comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) analyses revealed atypical 5.7-Mb and 4.1-Mb deletions at 7q11.23 in the two patients, including the WBS critical region and expanding into the proximal side and the telomeric side, respectively. On the proximal side, AUTS2 and CALN1 may contribute to the phenotype. On the telomeric side, there are two candidate genes HIP1 and YWHAG. Because detailed information of them was unavailable, we investigated their functions using gene knockdowns of zebrafish. When zebrafish ywhag1 was knocked down, reduced brain size and increased diameter of the heart tube were observed, indicating that the infantile spasms and cardiomegaly seen in the patient with the telomeric deletion may be derived from haploinsufficiency of YWHAG.

A functional analysis of GABARAP on 17p13.1 by knockdown zebrafish.

Array-based comparative genomic hybridization identified a 2.3-Mb microdeletion of 17p13.2p13.1 in a boy presenting with moderate mental retardation, intractable epilepsy and dysmorphic features. This deletion region was overlapped with the previously proposed shortest region overlapped for microdeletion of 17p13.1 in patients with mental retardation, microcephaly, microretrognathia and abnormal magnetic resonance imaging (MRI) findings of cerebral white matter, in which at least 17 known genes are included. Among them, DLG4/PSD95, GPS2, GABARAP and KCTD11 have a function in neuronal development. Because of the functional importance, we paid attention to DLG4/PSD95 and GABARAP, and analyzed zebrafish in which the zebrafish homolog of human DLG4/PSD95 and GABARAP was knocked down and found that gabarap knockdown resulted in small head and hypoplastic mandible. This finding would be similar to the common findings of the patients with 17p13.1 deletions. Although there were no pathogenic mutations in DLG4/PSD95 or GABARAP in a cohort study with 142 patients with idiopathic developmental delay with/without epilepsy, further studies would be required for genes included in this region.

oleed, a medaka Polycomb group gene, regulates ciliogenesis and left-right patterning.

Left-right (LR) patterning is an essential part of the animal body plan. Primary cilia are known to play a pivotal role in this process. In humans, genetic disorders of ciliogenesis cause serious congenital diseases. A comprehensive mechanism that regulates ciliogenesis has not been proposed so far. Here, we show that EED, a core member of the Polycomb group (PcG) genes and a presumed player in many epigenetic processes, is required for ciliogenesis and subsequent LR patterning in the medaka fish, Oryzias latipes. Moderate knockdown of oleed, a medaka homolog of EED, preferentially caused situs inversus. In the affected embryo, the cilia in Kupffer's vesicle showed various defects in their structure, position and motility. Furthermore, we demonstrated that oleed maintains the expression of Noto, which, in mice, regulates ciliogenesis and LR patterning. This study provides the first evidence for the involvement of epigenetic plasticity in LR patterning through ciliogenesis.

TULIP1 (RALGAPA1) haploinsufficiency with brain development delay.

A novel microdeletion of 14q13.1q13.3 was identified in a patient with developmental delay and intractable epilepsy. The 2.2-Mb deletion included 15 genes, of which TULIP1 (approved gene symbol: RALGAPA1)was the only gene highly expressed in the brain. Western blotting revealed reduced amount of TULIP1 in cell lysates derived from immortalized lymphocytes of the patient, suggesting the association between TULIP1 haploinsufficiency and the patient's phenotype, then 140 patients were screened for TULIP1 mutations and four missense mutations were identified. Although all four missense mutations were common with parents, reduced TULIP1 was observed in the cell lysates with a P297T mutation identified in a conserved region among species. A full-length homolog of human TULIP1 was identified in zebrafish with 72% identity to human. Tulip1 was highly expressed in zebrafish brain, and knockdown of which resulted in brain developmental delay. Therefore, we suggest that TULIP1 is a candidate gene for developmental delay.

A histone H1 variant is required for erythrocyte maturation in medaka.

Three histone H1 variants were identified in medaka fish and their sequence characteristics were analyzed. This paper reports one of these variants, termed H1-2, because of its possible implication in erythrocyte maturation. The amino acid sequence of H1-2 was phylogenetically similar to that of other replication-dependent histones. The mRNA transcribed from the h1-2 gene, however, possessed a poly(A) tail without a stem-loop structure, indicating that H1-2 may represent a replication-independent (RI) histone. Transcripts from the h1-2 gene were largely localized in erythrocytes, and knock-down of the h1-2 gene with morpholino antisense oligos resulted in failure to develop mature erythrocytes. In the morphants, residual erythrocytes showed severely impaired nuclear compaction. Although not structurally related to chicken RI histone H5, which is required for erythrocyte maturation, H1-2 may constitute its functional counterpart. Our findings may offer evolutionary insights into the function of H1 variants.

Germline gain-of-function mutations in RAF1 cause Noonan syndrome.

Noonan syndrome is characterized by short stature, facial dysmorphia and a wide spectrum of congenital heart defects. Mutations of PTPN11, KRAS and SOS1 in the RAS-MAPK pathway cause approximately 60% of cases of Noonan syndrome. However, the gene(s) responsible for the remainder are unknown. We have identified five different mutations in RAF1 in ten individuals with Noonan syndrome; those with any of four mutations causing changes in the CR2 domain of RAF1 had hypertrophic cardiomyopathy (HCM), whereas affected individuals with mutations leading to changes in the CR3 domain did not. Cells transfected with constructs containing Noonan syndrome-associated RAF1 mutations showed increased in vitro kinase and ERK activation, and zebrafish embryos with morpholino knockdown of raf1 demonstrated the need for raf1 for the development of normal myocardial structure and function. Thus, our findings implicate RAF1 gain-of-function mutations as a causative agent of a human developmental disorder, representing a new genetic mechanism for the activation of the MAPK pathway.

Adaptive changes in TEF-1 gene expression during cold acclimation in the medaka.

How animals adaptively respond to a cold or hot environment has been questioned for a long time. Recently, with the aid of microarray analysis, various temperature-sensitive genes have been identified in several species. However, a definitive hypothesis regarding the mechanism of adaptation has not been proposed. In the present study, we surveyed, in medaka (Oryzias latipes), genes for which the level of expression changes depending on the surrounding temperature. A messenger RNA differential display of medaka muscle total RNA revealed one such gene encoding transcription enhancer factor-1 (TEF-1). In medaka muscle, the TEF-1 gene produces two splicing variants, TEF-1A and TEF-1B mRNAs. During cold acclimation, the mRNA level of TEF-1A decreased, whereas that of TEF-1B increased. We also found that three putative downstream genes of TEF-1, two for myosin heavy chain (MyHC) and one for troponin T (TnT), a specific group of muscle proteins, were transcribed in a temperature-dependent manner. These results suggest that the transcription of MyHC and/or TnT is regulated by TEF-1 and that these molecules participate in muscle reconstruction during temperature adaptation in fish.

Cellular responses of the ciliate, Tetrahymena thermophila, to far infrared irradiation.

Infrared rays from sunlight permeate the earth's atmosphere, yet little is known about their interactions with living organisms. To learn whether they affect cell structure and function, we tested the ciliated protozoan, Tetrahymena thermophila. These unicellular eukaryotes aggregate in swarms near the surface of freshwater habitats, where direct and diffuse solar radiation impinge upon the water-air interface. We report that populations irradiated in laboratory cultures grew and mated normally, but major changes occurred in cell physiology during the stationary phase. Early on, there were significant reductions in chromatin body size and the antibody reactivity of methyl groups on lysine residues 4 and 9 in histone H3. Later, when cells began to starve, messenger RNAs for key proteins related to chromatin structure, intermediary metabolism and cellular motility increased from two- to nearly nine-fold. Metabolic activity, swimming speed and linearity of motion also increased, and spindle shaped cells with a caudal cilium appeared. Our findings suggest that infrared radiation enhances differentiation towards a dispersal cell-like phenotype in saturated populations of Tetrahymena thermophila.

Identification of a nuclear localization signal in mouse polycomb protein, M33.

The mouse Polycomb group (PcG) protein M33 forms nuclear complexes with the products of other PcG members and maintains repressed states of developmentally important genes, including homeotic genes. In this context, nuclear localization is a prerequisite for M33 to exert its function. However, we previously found that M33 in mouse liver shuttles dynamically between the nucleus and the cytoplasm, depending on the proliferative states of cells, coupled with phosphorylation and dephosphorylation of M33 protein. To understand the mechanism and significance of this phenomenon, we identified the functional nuclear localization signal (NLS) of M33 protein. Deletion mutants that lack a particular one of three putative NLS motifs failed to localize in the nucleus. Green fluorescent protein (GFP) fused to this motif specifically localized in the nucleus. We conclude that this amino-acid stretch in M33 acts as the functional NLS for this protein.

Reduced pain sensitivity in mice lacking latexin, an inhibitor of metallocarboxypeptidases.

Latexin, the endogenous protein inhibitor of the A/B subfamily of metallocarboxypeptidases, is expressed in small nociceptive neurons in sensory ganglia and in a subset of neurons in the telencephalon. In this study, we generated latexin-deficient mice that exhibited increased tail-flick latency compared to wild-type animals upon noxious heat stimulation. The reduced pain sensitivity in the mutants was rescued by the systemic administration of a plant carboxypeptidase inhibitor that inhibits the A/B subfamily of metallocarboxypeptidases. These findings suggest that latexin is involved in the transmission of pain.

Zebrafish Polycomb group gene ph2alpha is required for epiboly and tailbud formation acting downstream of FGF signaling.

We analyzed Polycomb group gene ph2alpha functionally in zebrafish embryos by a gene knock-down procedure using morpholino antisense oligos. Inhibition of ph2alpha message translation resulted in abnormal epibolic movements as well as a thick tailbud or incomplete covering of the yolk plug. At the 24hpf stage, morphants had short trunks and tails, phenotypes similar to those with disturbances in FGF signaling. Accordingly, we looked at the effects of ph2alpha expression upstream and downstream of the FGF pathway. Treatment with SU5402, an inhibitor of Fgfrs, or injection of dominant-negative Fgfr1 DNA markedly reduced ph2alpha expression in the tailbud. In addition, cells expressing mRNAs for no tail, spadetail, myoD, and papc, which are involved in FGF-related development of posterior mesoderm, were distributed abnormally. Collectively, the data argue that ph2alpha is required for epiboly and tailbud formation, acting downstream of the FGF signaling pathway.

The ESC-E(Z) complex participates in the hedgehog signaling pathway.

Polycomb group (PcG) genes are required for stable inheritance of epigenetic states throughout development, a phenomenon termed cellular memory. In Drosophila and mice, the product of the E(z) gene, one of the PcG genes, constitutes the ESC-E(Z) complex and specifically methylates histone H3. It has been argued that this methylation sets the stage for appropriate repression of certain genes. Here, we report the isolation of a well-conserved homolog of E(z), olezh2, in medaka. Hypomorphic knock-down of olezh2 resulted in a cyclopia phenotype and markedly perturbed hedgehog signaling, consistent with our previous report on oleed, a medaka esc. We also found cyclopia in embryos treated with trichostatin A, an inhibitor of histone deacetylase, which is a transient component of the ESC-E(Z) complex. The level of tri-methylation at lysine 27 of histone H3 was substantially decreased in both olezh2 and oleed knock-down embryos, and in embryos with hedgehog signaling perturbed by forskolin. We conclude that the ESC-E(Z) complex per se participates in hedgehog signaling.

Participation of Polycomb group gene extra sex combs in hedgehog signaling pathway.

Polycomb group (PcG) genes are required for stable inheritance of epigenetic states across cell divisions, a phenomenon termed cellular memory. PcG proteins form multimeric nuclear complex which modifies the chromatin structure of target site. Drosophila PcG gene extra sex combs (esc) and its vertebrate orthologs constitute a member of ESC-E(Z) complex, which possesses histone methyltransferase activity. Here we report isolation and characterization of medaka esc homolog, termed oleed. Hypomorphic knock-down of oleed using morpholino antisense oligonucleotides resulted in the fusion of eyes, termed cyclopia. Prechordal plate formation was not substantially impaired, but expression of hedgehog target genes was dependent on oleed, suggesting some link with hedgehog signaling. In support of this implication, histone methylation, which requires the activity of esc gene product, is increased in hedgehog stimulated mouse NIH-3T3 cells. Our data argue for the novel role of esc in hedgehog signaling and provide fundamental insight into the epigenetic mechanisms in general.

DNA display of biologically active proteins for in vitro protein selection.

In vitro display technologies are powerful tools for screening peptides with desired functions. We previously proposed a DNA display system in which streptavidin-fused peptides are linked with their encoding DNAs via biotin labels in emulsion compartments and successfully applied it to the screening of random peptide libraries. Here we describe its application to functional and folded proteins. By introducing peptide linkers between streptavidin and fused proteins, we achieved highly efficient (>95%) formation of DNA-protein conjugates. Furthermore, we successfully enriched a glutathione-S-transferase gene by a factor of 20-30-fold per round on glutathione-coupled beads. Thus, DNA display should be useful for rapidly screening or evolving proteins based on affinity selection.

DNA display for in vitro selection of diverse peptide libraries.

We describe the use of a DNA display system for in vitro selection of peptide ligands from a large library of peptides displayed on their encoding DNAs. The method permits completely in vitro construction of a DNA-tagged peptide library by using a wheat germ in vitro transcription/translation system compartmentalized in water-in-oil emulsions. Starting with a library of 10(9)-10(10) random decapeptides, 21 different peptide ligands were isolated for monoclonal antibody anti-FLAG M2. DNA display selected more diverse peptides with a DYKXXD consensus motif than previously reported phage display systems. Binding and recovery rates of three peptides were significantly higher than those of the original FLAG peptide, implying that these peptides would be superior to the FLAG peptide for purification of tagged proteins. The simplicity of DNA display enables two selection rounds per day to be conducted. Further, DNA display can overcome the limitations of previous display technologies by avoiding the use of bacterial cells and RNA tags. Thus, DNA display is expected to be useful for rapid screening of a wide variety of peptide ligands for corresponding receptors.