Dietary compounds activate an insect gustatory receptor on enteroendocrine cells to elicit myosuppressin secretion.

Sensing of midgut internal contents is important for ensuring appropriate hormonal response and digestion following the ingestion of dietary components. Studies in mammals have demonstrated that taste receptors (TRs), a subgroup of G protein-coupled receptors (GPCRs), are expressed in gut enteroendocrine cells (EECs) to sense dietary compounds and regulate the production and/or secretion of peptide hormones. Although progress has been made in identifying expression patterns of gustatory receptors (GRs) in gut EECs, it is currently unknown whether these receptors, which act as ligand-gated ion channels, serve similar functions as mammalian GPCR TRs to elicit hormone production and/or secretion. A Bombyx mori Gr, BmGr6, has been demonstrated to express in cells by oral sensory organs, midgut and nervous system; and to sense isoquercitrin and chlorogenic acid, which are non-nutritional secondary metabolites of host mulberry. Here, we show that BmGr6 co-expresses with Bommo-myosuppressin (BMS) in midgut EECs, responds to dietary compounds and is involved in regulation of BMS secretion. The presence of dietary compounds in midgut lumen after food intake resulted in an increase of BMS secretions in hemolymph of both wild-type and BmGr9 knockout larvae, but BMS secretions in BmGr6 knockout larvae decreased relative to wild-type. In addition, loss of BmGr6 led to a significant decrease in weight gain, excrement, hemolymph carbohydrates levels and hemolymph lipid levels. Interestingly, although BMS is produced in both midgut EECs and brain neurosecretory cells (NSCs), BMS levels in tissue extracts suggested that the increase in hemolymph BMS during feeding conditions is primarily due to secretion from midgut EECs. Our studies indicate that BmGr6 expressed in midgut EECs responds to the presence of dietary compounds in the lumen by eliciting BMS secretion in B. mori larvae.

BmGr4 responds to sucrose and glucose and expresses in tachykinin-related peptide-secreting enteroendocrine cells.

The regulatory hormones known as tachykinin-related peptides (TRPs) are identified as brain-gut peptides in insects. Dietary components from mulberry leaves, including glucose, induce secretion of TRPs from Bombyx mori midgut. However, the sensory molecules that recognize these compounds are still unknown. Here, we identified the gustatory receptor, BmGr4, as a sucrose and glucose receptor using Ca2+ imaging. Immunostaining revealed BmGr4 expression not only in the midgut, but also in the brain. In addition, BmGr4 expression was found to co-localize with TRP-expressing cells in both midgut enteroendocrine cells (EECs) and brain neurosecretory cells (NSCs). Furthermore, dietary nutrients after food intake result in an increase of TRP-level in hemolymph of silkworm larvae. These results provide significant circumstantial evidence for the involvement of the sucrose and glucose receptor, BmGr4, in the elicitation of TRP secretion in midgut EECs and brain NSCs.

Ultrasensitive detection by maxillary palp neurons allows non-host recognition without consumption of harmful allelochemicals.

Most lepidopteran insect larvae exhibit stepwise feeding behaviors, such as palpation using the maxillary palps (MPs) followed by test biting and persistent biting. However, the purpose of palpation has been unclear. In particular, nothing is known about the neurons in the MP and their mode of recognition of undesired plants, although such neurons have been suggested to exist. In this study, we used larvae of the stenophagous insect Bombyx mori and compared the roles of palpation and test biting in the selection of feeding behavior. When the larvae were given non-host plant leaves, they did not initiate test biting, indicating that non-host plant leaves were recognized via palpation without biting, and that this behavior resulted in a lack of persistent biting, as the leaves were judged non-suitable for consumption. Surface extracts of inedible leaves significantly suppressed test biting of mulberry leaves, a host plant of B. mori, suggesting that secondary metabolites on the leaf surface of inedible leaves function as test biting suppressors, even when another conditions are suitable for test biting. The allelochemical coumarin, which is found in the inedible leaves of cherry, Cerasus speciosa, significantly suppressed test biting of mulberry leaves, suggesting that coumarin is a possible deterrent to the eating of cherry leaves. Using the electrophysiological method of tip recording and a leaf-surface extract as the test material, leaf-surface compound-responsive neurons were identified in the MP. In addition, several neurons that respond to coumarin in the attomolar range were identified, suggesting that the larvae use ultrasensitive neurons in the MP to recognize inedible leaves. In the HEK293T cell heterologous expression system, the B. mori gustatory receptors BmGr53 and BmGr19, which were previously found to be expressed in the MP and to respond to coumarin in the attomolar range, responded to a leaf-surface extract of C. speciosa, suggesting that these receptors may be present on the inedible-leaf-recognizing neurons of the MP. These findings suggest that ultrasensitive plant secondary metabolite-recognizing neurons in the MP allow for the recognition of non-host plants via palpation without risking damage caused by ingesting harmful allelochemicals.

Plant hormone cytokinins control cell cycle progression and plastid replication in apicomplexan parasites.

Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites.

Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes.

Drug resistance compromises control of malaria. Here, we show that resistance to a commonly used antimalarial medication, atovaquone, is apparently unable to spread. Atovaquone pressure selects parasites with mutations in cytochrome b, a respiratory protein with low but essential activity in the mammalian blood phase of the parasite life cycle. Resistance mutations rescue parasites from the drug but later prove lethal in the mosquito phase, where parasites require full respiration. Unable to respire efficiently, resistant parasites fail to complete mosquito development, arresting their life cycle. Because cytochrome b is encoded by the maternally inherited parasite mitochondrion, even outcrossing with wild-type strains cannot facilitate spread of resistance. Lack of transmission suggests that resistance will be unable to spread in the field, greatly enhancing the utility of atovaquone in malaria control.

Lateralization, maturation, and anteroposterior topography in the lateral habenula revealed by ZIF268/EGR1 immunoreactivity and labeling history of neuronal activity.

We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response.

Axons from the medial habenular nucleus are topographically sorted in the fasciculus retroflexus.

We generated transgenic mice lines with a construct consisting of the zif268/egr1 promoter and the gene for the normal long-life yellow fluorescent protein (Venus) with a membrane localization sequence. One of the lines exhibited topographic labeling in the medial habenular nucleus (MHb) during postnatal development, which confirmed the previous findings that the medial, lateral, and dorsal areas of MHb project to the ventral, dorsal, and lateral parts of the interpeduncular nucleus, respectively. In addition, the membranous localization of the labeling allowed us to observe spacial arrangement of the labeled axons in the fasciculus retroflexus (FR) in the transgenic mice. Here, we report topographic sorting of the MHb axons in the FR. At postnatal day (P) 5 and P10, the labeled axons from the medial MHb were fasciculated and ran through the narrow path in the core of the FR. At P24, the labeled axons from the medial and dorsal MHb were fasciculated and ran through the broad path in the FR core. No labeling occurred in the lateral MHb throughout development; correspondingly, parts of the FR core remained unlabeled. The results indicated that the axons from the medial and dorsal areas of the MHb are grouped together in the FR of this transgenic line and are sorted out from the axons from the lateral MHb.

Developmental epigenetic modification regulates stochastic expression of clustered protocadherin genes, generating single neuron diversity.

In the brain, enormous numbers of neurons have functional individuality and distinct circuit specificities. Clustered Protocadherins (Pcdhs), diversified cell-surface proteins, are stochastically expressed by alternative promoter choice and affect dendritic arborization in individual neurons. Here we found that the Pcdh promoters are differentially methylated by the de novo DNA methyltransferase Dnmt3b during early embryogenesis. To determine this methylation's role in neurons, we produced chimeric mice from Dnmt3b-deficient induced pluripotent stem cells (iPSCs). Single-cell expression analysis revealed that individual Dnmt3b-deficient Purkinje cells expressed increased numbers of Pcdh isoforms; in vivo, they exhibited abnormal dendritic arborization. These results indicate that DNA methylation by Dnmt3b at early embryonic stages regulates the probability of expression for the stochastically expressed Pcdh isoforms. They also suggest a mechanism for a rare human recessive disease, the ICF (Immunodeficiency, Centromere instability, and Facial anomalies) syndrome, which is caused by Dnmt3b mutations.

Gibberellin biosynthetic inhibitors make human malaria parasite Plasmodium falciparum cells swell and rupture to death.

Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites.

Concatenated mitochondrial DNA of the coccidian parasite Eimeria tenella.

Apicomplexan parasites of the genus Plasmodium, pathogens causing malaria, and the genera Babesia and Theileria, aetiological agents of piroplasmosis, are closely related. However, their mitochondrial (mt) genome structures are highly divergent: Plasmodium has a concatemer of 6-kb unit and Babesia/Theileria a monomer of 6.6- to 8.2-kb with terminal inverted repeats. Fragmentation of ribosomal RNA (rRNA) genes and gene arrangements are remarkably distinctive. To elucidate the evolutionary origin of this structural divergence, we determined the mt genome of Eimeria tenella, pathogens of coccidiosis in domestic fowls. Analysis revealed that E. tenella mt genome was concatemeric with similar protein-coding genes and rRNA gene fragments to Plasmodium. Copy number was 50-fold of the nuclear genome. Evolution of structural divergence in the apicomplexan mt genomes is discussed.

Identification of Plasmodium malariae, a human malaria parasite, in imported chimpanzees.

It is widely believed that human malaria parasites infect only man as a natural host. However, earlier morphological observations suggest that great apes are likely to be natural reservoirs as well. To identify malaria parasites in great apes, we screened 60 chimpanzees imported into Japan. Using the sequences of small subunit rRNA and the mitochondrial genome, we identified infection of Plasmodium malariae, a human malaria parasite, in two chimpanzees that were imported about thirty years ago. The chimpanzees have been asymptomatic to the present. In Japan, indigenous malaria disappeared more than fifty years ago; and thus, it is most likely inferred that the chimpanzees were infected in Africa, and P. malariae isolates were brought into Japan from Africa with their hosts, suggesting persistence of parasites at low level for thirty years. Such a long term latent infection is a unique feature of P. malariae infection in humans. To our knowledge, this is the first to report P. malariae infection in chimpanzees and a human malaria parasite from nonhuman primates imported to a nonendemic country.

Two cases of childhood bullous pemphigoid.

This report describes two cases of childhood bullous pemphigoid (BP). These cases showed vesiculobullous lesions on the face, trunk, extremities, hands and feet. Histopathological analysis of skin lesions showed infiltration of numerous lymphocytes and eosinophils in the superficial dermis. Immunofluorescent analysis showed a linear IgG deposit along the basement membrane zone. In both cases ELISA showed circulating IgG autoantibodies against the NC16A domain of 180-KDa BP antigen (BP180). Both IgG and IgA (faint deposit) autoantibodies against the NC16A domain of BP180 were detected by an immunoblot analysis in both cases. Both patients showed a similar clinical course with a rapid remission after treatment with topical corticosteroids. Both patients received vaccinations within two weeks before the appearance of the eruption. These cases were considered to be childhood BP presenting both IgG and IgA autoantibodies against the NC16A domains of BP180.

Expression levels of Protocadherin-alpha transcripts are decreased by nonsense-mediated mRNA decay with frameshift mutations and by high DNA methylation in their promoter regions.

The mouse protocadherin (Pcdh) clusters, Pcdh-alpha, -beta, and -gamma, are located on chromosome 18. Many polymorphic variations are found in the Pcdh-alpha genes in wild-derived and laboratory mouse strains. In comparing the expression levels of Pcdh-alpha isoforms among several strains, we observed lower expression levels of Pcdh-alpha9 in BLG2 and BFM/2, and of Pcdh-alpha8 in C57BL/6 (B6) than in the other strains. For Pcdh-alpha8, high DNA methylation (72.7%) in the promoter region was found only in B6, whereas 36.4-44.3% methylation was seen in the other strains. On the other hand, the Pcdh-alpha9 DNA-methylation levels were similar (23.6-36.3%) among the strains regardless of the difference in expression levels. Interestingly, however, the Pcdh-alpha9 variable exon in both BLG2 and BFM/2 included a premature termination codon (PTC) generated by a nucleotide deletion or insertion. Treatment with emetine, a potent inhibitor of nonsense-mediated mRNA decay (NMD), increased the expression level of Pcdh-alpha9 from the BLG2-Pcdh-alpha locus. These data indicate that the transcription levels of mature Pcdh-alpha mRNAs are decreased by the DNA-methylation state of the Pcdh-alpha promoter regions and by the NMD pathway during RNA maturation. And we correct some previous data on Sugino, H., Toyama, T., Taguchi, Y., Esumi, S., Miyazaki, M., Yagi, T., (2004) Negative and positive effects of an IAP-LTR on nearby Pcdaalpha gene expression in the central nervous system and neuroblastoma cell lines, Gene 337 91-103.

Relationship between DNA methylation states and transcription of individual isoforms encoded by the protocadherin-alpha gene cluster.

The protocadherin-alpha (Pcdh-alpha) gene encodes diverse transmembrane proteins that are differentially expressed in individual neurons in the vertebrate central nervous system. The Pcdh-alpha genomic structure contains variable first exons, each regulated by its own promoter. Here, we investigated the effect of DNA methylation on gene regulation in the Pcdh-alpha gene cluster. We studied two mouse cell lines, C1300 and M3, that expressed different combinations of Pcdh-alpha isoforms and found that 1) the transcription of specific Pcdh-alpha isoforms correlated significantly with the methylation state of the promoter and the 5' (but not the 3') region of the first exon and 2) mosaic or mixed methylation states of the promoters were associated with both active and inactive transcription. Demethylation of C1300 cells up-regulated all of the Pcdh-alpha isoforms, and, in a promoter assay, hypermethylation of the promoters repressed their transcriptional activity. Cell lines subcloned from the demethylated C1300 cells transcribed different combinations of Pcdh-alpha isoforms than the parental, nondemethylated cells, and the promoters showed differential mosaic or mixed methylation patterns. In vivo, the promoter and 5'-regions of the Pcdh-alphaC1 and alphaC2 exons, which are transcribed in all neurons, were extensively hypomethylated. In contrast, the promoters of the Pcdh-alpha1 to -alpha12 isoforms, which are transcribed differentially by individual Purkinje cells, exhibited mosaic methylation patterns. Overall, our results demonstrated that mosaic or mixed DNA methylation states in the promoter and 5'-region of the first exon may help regulate differential Pcdh-alpha transcription and that hypermethylation is sufficient to repress transcription.

Negative and positive effects of an IAP-LTR on nearby Pcdaalpha gene expression in the central nervous system and neuroblastoma cell lines.

Intracisternal A-particles (IAPs) are defective retrovirions encoded by members of a large family of endogenous proviral elements in the murine genome. An intact IAP element was found in the protocadherin alpha (Pcdhalpha) gene cluster of five laboratory mouse strains. However, IAP insertion was not detected in three wild mouse strains we investigated. This IAP insertion caused the disruption of one variable exon of laboratory mouse and down-regulated expression of the Pcdhalpha v8 exon, which is located just downstream of the IAP in the brain following the methylation of 5' regulatory region of Pcdhalpha v8. In contrast, the Pcdhalpha v8 exon was highly expressed in mouse neuroblastoma cell lines. This suggested that the IAP insertion activates the expression of the nearby Pcdhalpha v8 exon in these cell lines. In fact, the Pcdhalpha v8 exon expression was driven by the IAP-long terminal repeat (LTR) following the de-methylation of 5' regulatory region of Pcdhalpha v8. To investigate the promoter activity of the IAP, we constructed an IAP-LTR-ECFP reporter gene and introduced it into neuroblastoma, melanoma, lymphoma, and plasmacytoma cell lines. Interestingly, ECFP-positive cells were observed only in the neuroblastoma cell lines. Moreover, there were no differences in the promoter activities of the IAP-LTR whether it was in the sense or complimentary orientation. Thus, this IAP-LTR has negative and positive regulation on near by gene expression in the brain and neuroblastoma cell lines.