Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo.

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo. Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions.

Two neuronal groups for NaCl with differential taste response properties and topographical distributions in the rat parabrachial nucleus.

It is crucial for animals to discriminate between palatable (safe) and aversive (toxic) tastants. The mechanisms underlying neuronal discrimination of taste stimuli remain unclear. We examined relations between taste response properties (spike counts, response duration, and coefficient of variation [CV]) and location of taste-sensitive neurons in the pontine parabrachial nucleus (PBN). Extracellular single units' activity in the PBN of Wistar rats was recorded using multibarrel glass micropipettes under urethane anesthesia. Forty taste-sensitive neurons were classified as NaCl (N)-best (n = 15), NaCl/HCl (NH)-best (n = 14), HCl (H)-best (n = 8), and sucrose (S)-best (n = 3) neurons. The net response to NaCl (15.2 ± 2.3 spikes/s) among the N-best neurons was significantly larger than that among the NH-best (4.5 ± 0.8 spikes/s) neurons. The response duration (4.5 ± 0.2 s) of the N-best neurons to NaCl was significantly longer than that of the NH-best (2.2 ± 0.3 s) neurons. These differences in the spike counts and the response durations between the two neuronal types in the PBN were similar to that previously reported in the rostral nucleus of the solitary tract (rNST). The CVs in the N-best and the NH-best neurons were significantly smaller in the PBN than those in the rNST. Histologically, most N-best neurons (12/13, 92%) were localized to the medial region, while NH-best neurons (11/13, 85%) were primarily found within the brachium conjunctivum. These results suggest that NaCl-specific taste information is transmitted by two distinct neuronal groups (N-best and NH-best), with different taste properties and locations within rNST to PBN tractography. Future studies on the higher order nuclei for taste could reveal more palatable and aversive taste pathways.

Cyanidioschyzon merolae aurora kinase phosphorylates evolutionarily conserved sites on its target to regulate mitochondrial division.

The mitochondrion is an organelle that was derived from an endosymbiosis. Although regulation of mitochondrial growth by the host cell is necessary for the maintenance of mitochondria, it is unclear how this regulatory mechanism was acquired. To address this, we studied the primitive unicellular red alga Cyanidioschyzon merolae, which has the simplest eukaryotic genome and a single mitochondrion. Here we show that the C. merolae Aurora kinase ortholog CmAUR regulates mitochondrial division through phosphorylation of mitochondrial division ring components. One of the components, the Drp1 ortholog CmDnm1, has at least four sites phosphorylated by CmAUR. Depletion of the phosphorylation site conserved among eukaryotes induced defects such as mitochondrial distribution on one side of the cell. Taken together with the observation that human Aurora kinase phosphorylates Drp1 in vitro, we suggest that the phosphoregulation is conserved from the simplest eukaryotes to mammals, and was acquired at the primitive stage of endosymbiosis.

Pyrenocine A induces monopolar spindle formation and suppresses proliferation of cancer cells.

Pyrenocine A, a phytotoxin, was found to exhibit cytotoxicity against cancer cells with an IC50 value of 2.6-12.9 µM. Live cell imaging analysis revealed that pyrenocine A arrested HeLa cells at the M phase with characteristic ring-shaped chromosomes. Furthermore, as a result of immunofluorescence staining analysis, we found that pyrenocine A resulted in the formation of monopolar spindles in HeLa cells. Monopolar spindles are known to be induced by inhibitors of the kinesin motor protein Eg5 such as monastrol and STLC. Monastrol and STLC induce monopolar spindle formation and M phase arrest via inhibition of the ATPase activity of Eg5. Interestingly, our data revealed that pyrenocine A had no effect on the ATPase activity of Eg5 in vitro, which suggested the compound induces a monopolar spindle by an unknown mechanism. Structure-activity relationship analysis indicates that the enone structure of pyrenocine A is likely to be important for its cytotoxicity. An alkyne-tagged analog of pyrenocine A was synthesized and suppressed proliferation of HeLa cells with an IC50 value of 2.3 µM. We concluded that pyrenocine A induced monopolar spindle formation by a novel mechanism other than direct inhibition of Eg5 motor activity, and the activity of pyrenocine A may suggest a new anticancer mechanism.

Changes in masseter muscle fibers by liquid diet rearing in rabbits and recovery by chewing of solid diet.

OBJECTIVE: To investigate the effects of liquid diet on the development of masseter muscle fibers and whether the changes in the masseter muscle can be recovered by chewing of solid diet. DESIGN: Masseter muscles from 40 rabbits (solid- and liquid-diet groups, n = 30; unweaned group, n = 5; recovery group, n = 5) were histochemically examined at 4, 12, 18, and 33 weeks after birth. Six fiber types (I, IC, IIC, IIA, IIAB, and IIB) were distinguished via mATPase staining. Muscle fiber diameter and fiber type composition were measured and compared between groups. RESULTS: In the liquid diet group, the diameter of types IIAB (solid group: 81.7 µm, liquid group: 60.9 µm) and IIB (solid group: 89.3 µm, liquid group: 68.8 µm) and the fiber type composition of type I (solid group: 18.4%, liquid group: 9.6%) decreased significantly at 33 weeks of age. In the recovery group, the fiber type composition of type I fibers recovered to 16.5%, while no recovery of type IIAB (56.6 µm) and IIB (64.6 µm) fiber diameter was observed. CONCLUSIONS: Liquid diet caused atrophy of muscle fibers and an increase in the proportion of fast-twitch fibers. Although the diameter and ratio of slow-twitch fibers were recovered by chewing of solid diet, recovery was not observed for fast-twitch fibers. Our findings are relevant for dental medicine as it explored the possibility of masticatory muscle function recovery by hard food.

Intracellular localization of histone deacetylase HDA6 in plants.

Histone modification is an important epigenetic mechanism in eukaryotes. Histone acetyltransferase and deacetylase regulate histone acetylation levels antagonistically, leading to dynamic control of chromatin structure. One of the histone deacetylases, HDA6, is involved in gene silencing in the heterochromatin regions, chromocenter formation, and metabolic adaptation under drought stress. Although HDA6 plays an important role in chromatin control and response to drought stress, its intracellular localization has not been observed in detail. In this paper, we generated transformants expressing HDA6-GFP in the model plant, Arabidopsis thaliana, and the crops, rice, and cassava. We observed the localization of the fusion protein and showed that HDA6-GFP was expressed in the whole root and localized at the nucleus in Arabidopsis, rice, and cassava. Remarkably, HDA6-GFP clearly formed speckles that were actively colocalized with chromocenters in Arabidopsis root meristem. In contrast, such speckles were unlikely to be formed in rice or cassava. Because AtHDA6 directly binds to the acetate synthesis genes, which function in drought tolerance, we performed live imaging analyses to examine the cellular dynamics of pH in roots and the subnuclear dynamics of AtHDA6 responding to acetic acid treatment. The number of HDA6 speckles increased during drought stress, suggesting a role in contributing to drought stress tolerance.

Acetic Acid Treatment Enhances Drought Avoidance in Cassava (Manihot esculenta Crantz).

The external application of acetic acid has recently been reported to enhance survival of drought in plants such as Arabidopsis, rapeseed, maize, rice, and wheat, but the effects of acetic acid application on increased drought tolerance in woody plants such as a tropical crop "cassava" remain elusive. A molecular understanding of acetic acid-induced drought avoidance in cassava will contribute to the development of technology that can be used to enhance drought tolerance, without resorting to transgenic technology or advancements in cassava cultivation. In the present study, morphological, physiological, and molecular responses to drought were analyzed in cassava after treatment with acetic acid. Results indicated that the acetic acid-treated cassava plants had a higher level of drought avoidance than water-treated, control plants. Specifically, higher leaf relative water content, and chlorophyll and carotenoid levels were observed as soils dried out during the drought treatment. Leaf temperatures in acetic acid-treated cassava plants were higher relative to leaves on plants pretreated with water and an increase of ABA content was observed in leaves of acetic acid-treated plants, suggesting that stomatal conductance and the transpiration rate in leaves of acetic acid-treated plants decreased to maintain relative water contents and to avoid drought. Transcriptome analysis revealed that acetic acid treatment increased the expression of ABA signaling-related genes, such as OPEN STOMATA 1 (OST1) and protein phosphatase 2C; as well as the drought response and tolerance-related genes, such as the outer membrane tryptophan-rich sensory protein (TSPO), and the heat shock proteins. Collectively, the external application of acetic acid enhances drought avoidance in cassava through the upregulation of ABA signaling pathway genes and several stress responses- and tolerance-related genes. These data support the idea that adjustments of the acetic acid application to plants is useful to enhance drought tolerance, to minimize the growth inhibition in the agricultural field.

Proteasomal degradation of BRAHMA promotes Boron tolerance in Arabidopsis.

High levels of boron (B) induce DNA double-strand breaks (DSBs) in eukaryotes, including plants. Here we show a molecular pathway of high B-induced DSBs by characterizing Arabidopsis thaliana hypersensitive to excess boron mutants. Molecular analysis of the mutants revealed that degradation of a SWItch/Sucrose Non-Fermentable subunit, BRAHMA (BRM), by a 26S proteasome (26SP) with specific subunits is a key process for ameliorating high-B-induced DSBs. We also found that high-B treatment induces histone hyperacetylation, which increases susceptibility to DSBs. BRM binds to acetylated histone residues and opens chromatin. Accordingly, we propose that the 26SP limits chromatin opening by BRM in conjunction with histone hyperacetylation to maintain chromatin stability and avoid DSB formation under high-B conditions. Interestingly, a positive correlation between the extent of histone acetylation and DSB formation is evident in human cultured cells, suggesting that the mechanism of DSB induction is also valid in animals.

Direct quantitative evaluation of disease symptoms on living plant leaves growing under natural light.

Leaf color is an important indicator when evaluating plant growth and responses to biotic/abiotic stress. Acquisition of images by digital cameras allows analysis and long-term storage of the acquired images. However, under field conditions, where light intensity can fluctuate and other factors (shade, reflection, and background, etc.) vary, stable and reproducible measurement and quantification of leaf color are hard to achieve. Digital scanners provide fixed conditions for obtaining image data, allowing stable and reliable comparison among samples, but require detached plant materials to capture images, and the destructive processes involved often induce deformation of plant materials (curled leaves and faded colors, etc.). In this study, by using a lightweight digital scanner connected to a mobile computer, we obtained digital image data from intact plant leaves grown in natural-light greenhouses without detaching the targets. We took images of soybean leaves infected by Xanthomonas campestris pv. glycines, and distinctively quantified two disease symptoms (brown lesions and yellow halos) using freely available image processing software. The image data were amenable to quantitative and statistical analyses, allowing precise and objective evaluation of disease resistance.

Plant Aurora kinases interact with and phosphorylate transcription factors.

Aurora kinase (AUR) is a well-known mitotic serine/threonine kinase that regulates centromere formation, chromosome segregation, and cytokinesis in eukaryotes. In addition to regulating mitotic events, AUR has been shown to regulate protein dynamics during interphase in animal cells. In contrast, there has been no identification and characterization of substrates and/or interacting proteins during interphase in plants. The Arabidopsis thaliana genome encodes three AUR paralogues, AtAUR1, AtAUR2, and AtAUR3. Among them, AtAUR1 and AtAUR2 are considered to function redundantly. Here, we confirmed that both AtAUR1 and AtAUR3 are localized in the nucleus and cytoplasm during interphase, suggesting that they have functions during interphase. To identify novel interacting proteins, we used AlphaScreen to target 580 transcription factors (TFs) that are mainly functional during interphase, using recombinant A. thaliana TFs and AtAUR1 or AtAUR3. We found 133 and 32 TFs had high potential for interaction with AtAUR1 and AtAUR3, respectively. The highly AtAUR-interacting TFs were involved in various biological processes, suggesting the functions of the AtAURs during interphase. We found that AtAUR1 and AtAUR3 showed similar interaction affinity to almost all TFs. However, in some cases, the interaction affinity differed substantially between the two AtAUR homologues. These results suggest that AtAUR1 and AtAUR3 have both redundant and distinct functions through interactions with TFs. In addition, database analysis revealed that most of the highly AtAUR-interacting TFs contained a detectable phosphopeptide that was consistent with the consensus motifs for human AURs, suggesting that these TFs are substrates of the AtAURs. The AtAURs phosphorylated several highly interacting TFs in the AlphaScreen in vitro. Overall, in line with the regulation of TFs through interaction, our results indicate the possibility of phosphoregulation of several TFs by the AtAURs (280/300).

Postero-inferior condylar movement induced by artificial occlusal interference on the balancing side during fictive mastication in rabbits.

OBJECTIVE: Tooth contact does not occur on the balancing side during mastication. Hence, it is possible that the presence of occlusal interference on the balancing side causes mandibular rotation followed by atypical condylar movement because the jaw-closing muscle activity on the working side is greater than on the balancing side. The aim of the present study was to investigate the relationship between occlusal contact on the balancing side and condylar movement during mastication. METHODS: EMG activity of the masseter (MS), lateral pterygoid (LP) and digastric (DG) muscles and jaw movements were recorded. Condylar movements in the sagittal plane were recorded using a high speed charge-coupled device (CCD) camera. Incisal point movements were recorded using a magnet on the mentum and a magnetometric sensor on the nasal bone. A removable biting plate was used to introduce an artificial occlusal interference on the balancing side. RESULTS: Nine of the 10 rabbits showed large postero-inferior condylar movement (Pi-Cm) when a biting plate was applied on the balancing side. Five rabbits showed very small Pi-Cm when a biting plate was applied on the balancing side. In the small Pi-Cm group, MS activity decreased markedly and LP and DG transient activity appeared in the early occlusal phase in the presence of the biting plate. CONCLUSION: Interference on the balancing side always caused Pi-Cm on the ipsilateral side during mastication. However, the degree of Pi-Cm was often decreased by a jaw opening reflex response.

Unusual postero-inferior condylar movements that depend on the position of occlusal contact during fictive mastication in rabbits.

OBJECTIVE: The mandible can be modelled as a triangular plate supported at two joints and the point of occlusion. The mandible is stable if the vector of the jaw-closing muscle forces lies within the triangle of support. If this vector lies outside of the triangle of support, one of the three contact points will tend to separate as the mandible rotates around a line connecting the other two points. Here, we examined whether postero-inferior condylar movements (Pi-Cm) due to mandibular rotation may occur during fictive mastication in anaesthetized rabbits. METHODS: EMG activities of the masseter (MS) and lateral pterygoid (LP) muscles and movements of the condyle and incisal points were recorded. Condylar movements in the sagittal plane were recorded using a high speed CCD camera. RESULTS: Pi-Cm were observed on the working side during occlusal phase in half of the rabbits (altered-movement group), if the biting point was restricted at the posterior most tooth (M3) on the working side using a metal biting plate. Pi-Cm appeared in the period between the estimated maximum force of the MS and the LP during late occlusal phase. The MS EMG ratio between the working and balancing sides in the altered-movement group was significantly less than that in the unaltered-movement group. CONCLUSION: Since the space lying between the condyle and the articular eminence expanded during the Pi-Cm, it is likely that the posterior band of the articular disc tended to slip anteriorly. The clinical significance of the Pi-Cm is discussed concerning the anterior dislocation of the disc in patients with TMJ disorder.

RBMX: a regulator for maintenance and centromeric protection of sister chromatid cohesion.

Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids.

Association of serum interleukin-18 and other biomarkers with disease severity in adults with atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory disease of the skin for which there are no reliable biomarkers to assess clinical severity. Serum interleukin-18 (IL-18) levels may be associated with AD severity. To identify putative biomarkers associated with clinical severity in adult AD patients, we enrolled 121 adult AD patients (mean age 35.7 years) and 50 healthy controls (mean age 31.7 years). We compared these groups for blood eosinophils and serum levels of IL-18, thymus and activation-regulated chemokine (TARC), total IgE, and lactate dehydrogenase (LDH). We also determined S. aureus enterotoxin B (SEB) specific IgE levels and the SCORingAD (SCORAD) scores for AD patients. For AD patients, stepwise logistic regression was used to estimate odds ratios (OR) for each biomarker for the likelihood of having AD, and multiple linear regression was used to identify biomarkers associated with SCORAD scores. Compared with healthy controls, adult AD patients had higher levels of IL-18, TARC, total IgE, eosinophils, and LDH. TARC levels had the highest OR for AD occurrence, while the OR for IL-18 was insignificant. Also, IL-18 was not related to the presence of SEB-IgE. Notably, IL-18 levels were significantly associated with SCORAD scores, as were TARC, total IgE, and LDH levels. A panel of biomarkers (IL-18, TARC, total IgE, and LDH) may be more useful to accurately assess clinical severity in adult AD patients.

Positional cloning of a Bombyx wingless locus flugellos (fl) reveals a crucial role for fringe that is specific for wing morphogenesis.

Mutations at the flügellos (fl) locus in Bombyx mori produce wingless pupae and moths because of the repressed response of wing discs to ecdysteroid. Four recessive fl alleles occurred spontaneously and were mapped at 13.0 of the silkworm genetic linkage group 10. By positional cloning, we confirmed that the gene responsible for fl is fringe (fng) encoding Fng glycosyltransferase, which is involved in regulating the Notch signaling pathway. In four different fl alleles, we detected a large deletion of the fng gene in fl(k) and nonsense mutations in fl, fl(o), and fl(n). In the wild-type (WT) silkworm, fng is expressed actively in the wing discs, brain, and reproductive organs from the fourth to final instars but barely in the other tissues tested. In situ hybridization showed that fng mRNA is expressed in the dorsal layer of the WT wing discs. The wingless (wg) mRNA, a downstream marker of Fng-mediated Notch signaling, is localized at the dorsoventral boundary in the WT wing discs but repressed markedly in the fl wing discs. Although null mutants of Drosophila fng result in postembryonic lethality, loss of fng function in Bombyx affects only wing morphogenesis, suggesting different essential roles for fng in tissue differentiation among insects.

Evaluation of a medetomidine-midazolam combination for immobilizing and sedating Japanese monkeys (Macaca fuscata).

We clinically and clinicopathologically investigated the immobilizing and sedative effects of a medetomidine-midazolam (MM) combination in Japanese monkeys (Macaca fuscata) and its antagonism with atipamezole. MM (medetomidine, 60 microg/kg; midazolam, 0.3 mg/kg) was administered intramuscularly to each monkey (n = 11). All animals were laterally recumbent within 13 +/-6 min after administration of MM. This combination induced deep sedation accompanied by analgesia, muscle relaxation, and markedly depressed arousal reactions to external stimuli. After administration of atipamezole (240 microg/kg intramuscularly), the animals recovered rapidly and smoothly to their normal postures within 10 +/-2 min. In this study, the hematologic and serum biochemical parameters of Japanese monkeys given MM did not differ significantly from those of Japanese monkeys under general anesthesia via ketamine. Salivary a-amylase activities (stress indexes) ranged from 4 to 99 kU/l in Japanese monkeys, similar to levels measured in humans. An important advantage of MM was that its effects were reversible with atipamezole. We have confirmed that MM is valuable as a chemical restraint agent in Japanese monkeys for various experimental procedures.

Ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.