Sex-specific expression profiles of ecdysteroid biosynthesis and ecdysone response genes in extreme sexual dimorphism of the mealybug Planococcus kraunhiae (Kuwana).

Insect molting hormone (ecdysteroids) and juvenile hormone regulate molting and metamorphic events in a variety of insect species. Mealybugs undergo sexually dimorphic metamorphosis: males develop into winged adults through non-feeding, pupa-like stages called prepupa and pupa, while females emerge as neotenic wingless adults. We previously demonstrated, in the Japanese mealybug Planococcus kraunhiae (Kuwana), that the juvenile hormone titer is higher in males than in females at the end of the juvenile stage, which suggests that juvenile hormone may regulate male-specific adult morphogenesis. Here, we examined the involvement of ecdysteroids in sexually dimorphic metamorphosis. To estimate ecdysteroid titers, quantitative RT-PCR analyses of four Halloween genes encoding for cytochrome P450 monooxygenases in ecdysteroid biosynthesis, i.e., spook, disembodied, shadow and shade, were performed. Overall, their expression levels peaked before each nymphal molt. Transcript levels of spook, disembodied and shadow, genes that catalyze the steps in ecdysteroid biosynthesis in the prothoracic gland, were higher in males from the middle of the second nymphal instar to adult emergence. In contrast, the expression of shade, which was reported to be involved in the conversion of ecdysone into 20-hydroxyecdysone in peripheral tissues, was similar between males and females. These results suggest that ecdysteroid biosynthesis in the prothoracic gland is more active in males than in females, although the final conversion into 20-hydroxyecdysone occurs at similar levels in both sexes. Moreover, expression profiles of ecdysone response genes, ecdysone receptor and ecdysone-induced protein 75B, were also analyzed. Based on these expression profiles, we propose that the changes in ecdysteroid titer differ between males and females, and that high ecdysteroid titer is essential for directing male adult development.

E93 expression and links to the juvenile hormone in hemipteran mealybugs with insights on female neoteny.

Insect metamorphosis produces reproductive adults and is commonly accompanied with the direct or indirect development of wings. In some winged insects, the imago is altered by life history changes. For instance, in scale insects and mealybugs, reproductive females retain juvenile features and are wingless. The transcription factor E93 triggers metamorphosis and plays in concert with the juvenile hormone pathway to guarantee the successful transition from juvenile to adult. We previously provided evidence of an atypical down-regulation of the juvenile hormone pathway during female development in the Japanese mealybug. Here, we further investigate how E93 is involved in the production of neotenic wingless females, by identifying its isoforms, assessing their expression patterns and evaluating the effect of exogenous juvenile hormone mimic treatment on E93. This study identifies three E93 isoforms on the 5' end, based on Japanese mealybug cDNA and shows that female development occurs with the near absence of E93 transcripts, as opposed to male metamorphosis. Additionally, while male development is typically affected by exogenous juvenile hormone mimic treatments, females seem to remain insensitive to the treatment, and up-regulation of the juvenile hormone signaling is not observed. Furthermore, juvenile hormone mimic treatment on female nymphs did not have an obvious effect on E93 transcription, while treatment on male prepupae resulted in depleted E93 transcripts. In this study, we emphasize the importance in examining atypical cases of metamorphosis as complementary systems to provide a better understanding on the molecular mechanisms underlying insect metamorphosis. For instance, the factors regulating the expression of E93 are largely unclear. Investigating the regulatory mechanism of E93 transcription could provide clues towards identifying the factors that induce or suppress E93 transcription, in turn triggering male adult development or female neoteny.

Molecular Characterization of Eye Pigmentation-Related ABC Transporter Genes in the Ladybird Beetle Harmonia axyridis Reveals Striking Gene Duplication of the white Gene.

Many species of ladybird beetles (Coccinellidae) possess vivid body colors. These colors and patterns show diversity between coccinellid species, or even within species. However, the molecular underpinnings of these striking body colors are scarcely understood. One of the candidate pigmentation molecules responsible for ladybird body color is ommochrome pigment, which is well known as the red pigment molecule responsible for the red eyes of Drosophila. Various insects also use ommochrome in body coloration. It is known that ommochrome pigment precursors are imported into appropriate cells by the ATP binding cassette (ABC) transporter proteins White and Scarlet. Thus, these ABC transporter genes are potentially involved in various color and pattern expressions seen in ladybird beetle species. In this study, in order to identify the repertory of ABC transporter genes responsible for such body colors, we performed molecular characterization of pigment-related ABC transporter genes, especially white and scarlet, in the coccinellid Harmonia axyridis. By using whole genome data for H. axyridis and subsequent RACE-PCR, six white orthologs and one scarlet ortholog were successfully identified. According to the results of functional analyses via RNA interference (RNAi), only one of these genes had a major function in eye pigmentation. Specific effects on body color and pattern were not detected by our RNAi experiments of any of these genes. This is the first report of this striking duplication of white genes and their functional analyses in insects.

Relationships of diverse apoptotic death process patterns to mitochondrial membrane potential (Δψ(m)) evaluated by three-parameter flow cytometric analysis.

Recently, it has been proposed that novel methodologies are needed to re-evaluate apoptotic cell death, as studies of apoptosis have shown it to be a complex process. Since mitochondria are key regulators in cell death pathways, we developed a simultaneous 3-parameter flow cytometric analysis that incorporates the change in mitochondrial membrane potential (Δψ(m)) in an Annexin-V [for phosphatidyl-serine (PS)] and propidium iodide (PI) assay system (3 parameters with 4 colours), and evaluated the apoptotic process using various haematological malignant cell lines and death triggers. The present method enabled visualization of cell composition during apoptosis and captured complicated molecular events. For example, apoptotic cells that lost Δψ(m) did not always externalize PS, while some late apoptotic cells had polarized Δψ(m). The findings of unchanged PS-externalization and aberrant cell death suggest that there is no relationship of PS externalization and apoptosis with an unknown apoptotic mechanism. Based on PS-externalization, sensitivity to staurosporine, and the combination of cell lines and triggers, the apoptotic process was classified into 2 types. Importantly, most of our findings could not be observed by PS-PI and Δψ(m) assays when independently performed. Our method may be useful for examining mitochondrial-related apoptosis and death signalling pathways, as well as screening novel apoptosis-inducing cancer drugs.

Fabrication of a tubular FeCo bimetallic nanostructure using a cellulose-cobalt hexacyanoferrate composite as a precursor.

A tubular fibre structure with a ca. 9-11 microm outer diameter comprised of FeCo nanoparticle assemblies with an amorphous carbon core was fabricated via reductive thermal decomposition of a cellulose-cobalt hexacyanoferrate (Fe-CN-Co) composite material.

Clinical and oncologic implications in epigenetic down-regulation of CD26/dipeptidyl peptidase IV in adult T-cell leukemia cells.

CD26/dipeptidyl peptidase IV (DPPIV), a T-cell-activation antigen, is a 110-kD type II surface glycoprotein expressed on various types of normal cells. CD26/DPPIV is considered a multifunction housekeeping protein. Malignant cells often show altered CD26/DPPIV expression or no CD26/DPPIV expression, thus suggesting a useful marker for assessing some T-cell malignancies. In this study, cell surface protein and messenger RNA expression profiles for CD26/DPPIV were examined in 49 patients with adult T-cell leukemia (ATL), 10 carriers of human T-lymphotropic virus I (HTLV-I), and 4 HTLV-I-infected cell lines to assess the utility of CD26/DPPIV expression as a useful molecular marker for ATL pathology. In contrast to normal lymphocytes, ATL cells and HTLV-I-infected cell lines apparently down-regulated or completely lost the CD26/DPPIV antigen. Furthermore, the positive rate and antigen density for CD26/DPPIV in ATL cells gradually declined along with the advancement of ATL stage. Analysis of genomic DNA and the CD26/DPPIV transcript showed that CD26- ATL cells possessed faintly detected transcripts of the gene that were aberrantly methylated at the CpG islands within the promoter region in parallel with the advancement of ATL, a finding supported by a rescue experiment for transcript reexpression using 5-azacytidine as demethylation agent. Moreover, there was no relationship between loss of CD26/DPPIV and HTLV-I tax expression. These results indicate that ATL cells down-regulate CD26 antigens by means of epigenetic machinery and that this antigen abnormality is a useful molecular marker for the pathology of ATL.

Restricted expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 4 in human peripheral blood lymphocytes.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor but not normal cells, thus providing therapeutic possibilities for human cancers. However, it is not fully clear how widespread TRAIL receptors are, or how TRAIL signaling is modulated in normal cells. We characterized cell surface expression of TRAIL receptors in normal healthy donor peripheral blood and report that each of the TRAIL receptors are characteristically expressed on restricted cell populations. TRAIL-R1 is distinctively expressed on B-lymphocytes, TRAIL-R2 on monocytes, TRAIL-R3 on neutrophils and most impressively, CD8+ lymphocytes and NKT lymphocytes but not CD4+ lymphocytes express TRAIL-R4.