RNA synthesis in imaginal disks of Galleria mellonella: Effects of alpha- and beta-ecdysone and fat body in vitro

The effects of alpha-ecdysone (α-E), beta-ecdysone (β-E), and larval fat body on morphogenesis and total RNA synthesis in wing imaginal disks of Galleria mellonella were studied. Both ecdysones induce morphogenesis of disks in vitro. Alpha-ecdysone and β-E (0·3–3·0 μg/ml) stimulate RNA synthesis 30 and 60 per cent above control levels, respectively. While less α-E (0·3 μg/ml) is required to increase RNA synthesis than to induce morphogenesis, the reverse is true for β-E. Morphogenesis (i.e. tracheole migration and evagination) can proceed in the presence of concentrations of β-E (0·03 μg/ml) that are subthreshold for the induction of RNA synthesis (0·3 μg/ml). We conclude, therefore, that the increase in total RNA (presumbly ribosomal) is unrelated to and not a prerequisite for tracheole migration or evagination. If morphogenically active preconditioned medium (i.e. medium in which α-E and fat body have been incubated for 48 hr and the fat body then removed) is added to disk cultures, RNA synthesis is not stimulated. Apparently, increases in total RNA caused by both ecdysones may not be necessary for early in vitro disk development. The independent nature of some ecdysone-induced events and implications of our conclusions are discussed.     

Relation between cell number and pupal development of wing disks in Bombyx mori

The effects of JH and ecdysone on pupal differentiation of the wing disk of Bombyx mori were studied in vivo and in vitro. JH prevents pupal differentiation during larval life by stopping a particular stage of the cell cycle. Immediately after allatectomy, a cell cycle sets in without ecdysone, but afterwards wing disks obtain the competence to differentiate to the pupal type in response to ecdysterone. Disks older than 2 days after allatectomy can develop to the pupal type with an abrupt increase of mitosis in a certain concentration of ecdysterone in vitro. Once the disks have gained such competence and begun pupal development, JH no longer exhibits the effect that prevents DNA synthesis, which is enhanced by ecdysterone. It is therefore suggested that there are two phases in DNA synthesis: one which is important for achieving competence and is inhibited by JH and relatively independent of ecdysone; and another which is important for morphogenetic development and depends on ecdysone but is not inhibited by JH.     

The influence of larval fat body on wing disk development in vitro

The effects are noted of various fat body treatments on betaecdysone-induced cuticle deposition in cultured wing disks of Plodia interpunctella. The percentage of disks which responded to hormone treatment in vitro depended upon the age of the disk, the amount of fat body, the time the medium was incubated with fat body, and the time the disks were exposed to beta-ecdysone in fat body-conditioned medium. Fat body from pharate pupae did not stimulate disk development.     

Cuticle deposition in imaginal disks of three species of Lepidoptera: Effects of ecdysones in vitro

We report the effects of three ecdysone analogues in the presence and absence of fat body on the wing imaginal disks of Cadra cautella, Paramyelois transitella, and Plodia interpunctella cultured in vitro in a modified Grace's medium. Alpha-ecdysone, unlike beta-ecdysone, did not stimulate cuticle deposition in Plodia disks at low doses and was only slightly effective at high doses. Also, the response of wing disks of Cadra and Paramyelois to beta-ecdysone and alpha ecdysone with and without fat body was similar to that of Plodia. Combinations of alpha-ecdysone and beta-ecdysone were more effective than beta-ecdysone alone in Plodia disks, but a synthetic analogue, 22-iso-ecdysone, had no effect on cuticle deposition when it was used alone or with beta-ecdysone in the presence or absence of fat body. Thus, the results with alpha-ecdysone and beta-ecdysone were not non-specific steroidal effects.     

Effects of fat body on α-ecdysone induced morphogenesis in cultured wing disks of the wax moth, Galleria mellonella

Fat body promoted ecdysone induced morphogenesis in Galleria wing disks cultured in vitro. Medium preincubated with fat body and α-ecdysone from any of the first 5 days of the final larval instar enhanced tracheal migration and elongation in the disks. Disks from the third, fourth, and fifth days of the final larval instar responded equally well to the fat body and α-ecdysone. We suggest a physiological rôle for Galleria fat body as an intermediary in the stimulation of wing disk development by α-ecdysone.     

Overexpression of CD44 in Neural Precursor Cells Improves Trans- Endothelial Migration and Facilitates Their Invasion of Perivascular Tissues In Vivo

Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional in vivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.     

Juvenile hormone and DNA synthesis in imaginal disks of Calliphora erythrocephala: results of a new incubation technique.

An in vitro incubation technique in which imaginal disks are exposed to juvenile hormone and some of its analogues is presented. These substances were shown to have an inhibitory effect on the incorporation of tritiated thymidine (³HTdR) during the post-feeding period of the last larval instar of Calliphora. The technique makes it possible to investigate the nature of the effects of ecdysterone and juvenile hormones on the DNA synthesis in imaginal disks of exo- and endopterygote insects.     

Effect of ecdysterone contact period on development of wing disks of the fleshfly, Sarcophaga peregrina, in vitro

The ecdysterone contact period required for pupal development of Sarcophaga wing disks was studied in vitro. When the disks were cultured in a medium with 1 × 10^?6 M ecdysterone for about 21 hr, evagination of wing disks occurred independent of a later transfer into a hormone-free medium. The contact period required for wing evagination was dependent on the concentration of ecdysterone.When the disks cultured in the ecdysterone-containing medium were subjected to an intervening ecdysterone-free condition, evagination of the wing occurred if the period of the hormone contact before and after the ecdysterone-free period totalled a certain length. The total period required for wing evagination was altered both by the duration of the intervening hormone-free culture and duration of the first culture with ecdysterone.The morphogenetic effect of ecdysterone is discussed in relation to RNA synthesis in vitro.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

Plexin-A3 and plexin-A4 restrict the migration of sympathetic neurons but not their neural crest precursors

During development, the semaphorin family of guidance molecules is required for proper formation of the sympathetic nervous system. Plexins are receptors that mediate semaphorin signaling, but how plexins function during sympathetic development is not fully understood. Using phenotypic analyses of mutant mice in vivo, expression pattern studies, and in vitro assays, we show that plexin-A3 and plexin-A4 are essential for normal sympathetic development. This study confirms our previous in vitro findings that the two plexins differentially regulate the guidance of sympathetic axons. In addition, we find that semaphorin signaling through plexin-A3 and plexin-A4 restricts the migration of sympathetic neurons, but these two plexins function redundantly since migration defects are only observed in plexin-A3/-A4 double mutants. Surprisingly, our analysis also indicates that plexin-A3 and plexin-A4 are not required for guiding neural crest precursors prior to reaching the sympathetic anlagen. Immunoprecipitation studies suggest that these two plexins independently mediate secreted semaphorin signaling. Thus, plexin-A3 and plexin-A4 are expressed in newly-differentiated sympathetic neurons, but not their neural crest precursors. They function cooperatively to regulate the migration of sympathetic neurons and then differentially to guide the sympathetic axons.     

Range of potential functions of the Drosophila melanogaster hdc gene

The Drosophila melanogaster hdc gene controls trachea branching, which starts during embryo development. Expression in imaginal disks and reproductive organs suggests additional functions for the hdc gene. The gene was demonstrated to have a maternal effect, which was denied previously. Analysis of cell proliferation in imaginal disks with hdc mutations showed that the gene does not possess tumor suppressor properties at the levels of mosaic cuticle clones of adults and transplanted imaginal disks. Transplanted imaginal disks homozygous, but not heterozygous, for an hdc mutation were found to affect oogenesis in the recipient females, implicating the hdc activity in exchanging signals between different organs. Amino acid sequence analysis of the HDC protein revealed a region homologous to the human HRS proteins, which directly interact with the NF2 tumor suppressor on experimental evidence.     

Changes in ribosomal activity during incubation of Daucus carota root disks

Cytoplasmic monoribosomes from freshly cut and ‘aged’ carrot root disks were characterized relative to the Mg²⁺ optima for poly U (polyuridylic acid)-directed phenylalanine incorporation, the ease of dissociation by KCl in the presence of Mg²⁺, the ability to bind ³H-poly U, and acrylamide gel fractionation of the ribosomal proteins. The differences in in vitro amino acid incorporation by ribosomes and supernatant from fresh and ‘aged’ disks were confined to the ribosome fraction. The Mg²⁺ optima for poly U-directed ¹⁴C-phenylalanine incorporation was 16 mM for ribosomes from ‘aged’ disks compared to 20 mM for ribosomes from fresh disks. Monoribosomes from the fresh disks were easily dissociated into subunits (0·2 M KCl in 5 mM Mg²⁺) while the ribosomes from ‘aged’ disks were not completely dissociated even in 0·5 M KCl. Ribosomes from ‘aged’ disks were more effective in binding ³H-poly U than ribosomes from fresh disks. When the disks were subjected to an anaerobic environment prior to ribosome extraction (to strip monoribosomes of peptidyl-t RNA) the above effects of ‘aging’ were reversed. These results suggest that increased monoribosome activity associated with ‘aging’ may be related in part to an increase in the level of peptidyl-tRNA associated with the ribosomes. Acrylamide gel electrophoresis profiles of ribosomal proteins extracted from ribosomes of fresh and ‘aged’ tissue suggest that a change in the protein complement may also be important to the observed changes in ribosomal activity. The ribosomes from ‘aged’ disks contained at least two components not associated with ribosomes from fresh disks.     

Effects of the ecdysteroid agonists RH 5849 an RH 5992, alone and in combination with a juvenile hormone analogue, pyriproxyfen, on larvae ofSpodoptera exigua

Biological activity assays with RH 5849 and RH 5992 indicated that both compounds affected growth and development of last-instar larvae ofSpodoptera exigua (Hübner) (Lepidoptera: Noctuidae) in a dose-dependent manner. Within the first 24 h after treatment by continuously offering leaves dipped in a water solution of ≥50 mg/l RH 5849 and ≥0.5 mg/l RH 5992, symptoms of a prematurely induced larval moult and head capsule apolysis were visible. Intoxicated larvae died shortly afterwards, showing signs of unsuccessful ecdysis. LC₅₀-values of RH 5849 and RH 5992 for fifth-instarS. exigua larvae were 110 and 2.5 mg/l, respectively. Pyriproxyfen alone affected the larval stage and disturbed normal metamorphosis. One supernumerary larval instar occurred occasionally. LC₅₀-value for pyriproxyfen was 1.7 mg/l. Larvae simultaneously treated with RH 5849 or RH 5992 and pyriproxyfen, continued to grow until they attained a size and weight about 2–3 times that of the controls. This growth was accompanied by at least one and sometimes two supernumerary moults. Concerning thein vivo imaginal wing disc growth and development, only in larvae treated with 10 and 50 mg/l RH 5849 or 0.5 mg/l RH 5992, tracheole migration was observed earlier than in the controls. When applying 300 mg/l RH 5849 or 3–7 mg/l RH 5992, the discs remained small and no signs of tracheole migration were observed. In larvae simultaneously treated with RH 5849 or RH 5992 and pyriproxyfen, tracheole migration was not prematurely induced and a pupal cuticle was produced in the discs of larvae, undergoing a supernumerary moult. No clear signs of evagination were observed.     

Effects of ecdysone and juvenile hormone on DNA metabolism of imaginal disks of Drosophila melanogaster

Imaginal disks of Drosophila melanogaster isolated en masse and incubated in Robb's tissue culture medium incorporate ³H-thymidine into nuclear DNA. Both α- and β-ecdysone stimulate the rate of ³H-thymidine incorporation into disk DNA. Concentrations of ecdysone that induce complete evagination of disks in vitro cause the initiation of DNA synthesis in some disk cells. Juvenile hormone has no effect on DNA synthesis in control disks. However, juvenile hormone blocks the ecdysone stimulation of DNA synthesis. It is proposed that juvenile hormone and ecdysone act in a balanced fashion to regulate DNA synthesis in imaginal disks.     

Quantitative ultrastructural autoradiographic study of RNA transport in rat ventral prostate

The nucleocytoplasmic RNA transport in rat ventral prostate was studied by electron microscope autoradiography. Isolated prostate acini from normal, castrated, and DHT-treated animals were labeled in vitro with [³H]uridine for 5 min and chased for 0, 15, 30 min and 4 hr. The results show that (a) DHT induces a significant nucleolar enlargement but intranuclear migration of rRNA is not apparently affected by androgens; (b) migration of RNA through euchromatin is delayed by castration and stimulated by DHT; (c) migration through the nuclear envelope is androgen-dependent. In addition prostate acini were maintained for 24 hr in suspension culture in order to study the in vitro effects of DHT. The result show that (a) DHT stimulates uridine uptake and/or incorporation but induces no nucleolar enlargement; (b) DHT has no clear effects on RNA migration kinetics; (c) cytoplasmic transport of RNA in cells cultured in medium with or without DHT is severely impaired but is restored after supplementation of medium with insulin and dexamethasone.     

CXCR4 and matrix metalloproteinase-2 are involved in mesenchymal stromal cell homing and engraftment to tumors

Background aimsBone marrow-derived mesenchymal stromal cells (BMSC) have been shown to migrate to injury, ischemia and tumor microenvironments. The mechanisms by which mesenchymal stromal cells (MSC) migrate across endothelium and home to the target tissues are not yet fully understood.MethodsWe used rat BMSC to investigate the molecular mechanisms involved in their tropism to tumors in vitro and in vivo.ResultsBMSC were shown to migrate toward four different tumor cells in vitro, and home to both subcutaneous and lung metastatic prostate tumor models in vivo. Gene expression profiles of MSC exposed to conditioned medium (CM) of various tumor cells were compared and revealed that matrix metalloproteinase-2 (MMP-2) expression in BMSC was downregulated after 24 h exposure to tumor CM. Chemokine (C–X–C motif) Receptor 4 (CXCR4) upregulation was also found in BMSC after 24 h exposure to tumor CM. Exposure to tumor cell CM enhanced migration of BMSC toward tumor cells. Stromal Cell-Derived Factor (SDF-1) inhibitor AMD3100 and MMP-2 inhibitor partly abolished the BMSC migration toward tumor cells in vitro.ConclusionsThese results suggest that the CXCR4 and MMP-2 are involved in the multistep migration processes of BMSC tropism to tumors.     

Interactions between beta-ecdysone and fat body during wing disk development in vitro

We have investigated the actions of beta-ecdysone and fat body on wing disks of Plodia interpunctella in a series of sequential incubations in vitro. These experiments revealed that extended treatment times of beta-ecdysone at concentrations of 0·5 μg/ml or greater inhibited development of disks, and confirm that the presence of a fat body factor in the culture medium prevents this inhibition.     

Angiogenic activity of sesamin through the activation of multiple signal pathways

The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125^FAK-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.