Steroid regulated programmed cell death during Drosophila metamorphosis

During insect metamorphosis, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) direct the destruction of obsolete larval tissues and their replacement by tissues and structures that form the adult fly. We show here that larval midgut and salivary gland histolysis are stage-specific steroid-triggered programmed cell death responses. Dying larval midgut and salivary gland cell nuclei become permeable to the vital dye acridine orange and their DNA undergoes fragmentation, indicative of apoptosis. Furthermore, the histolysis of these tissues can be inhibited by ectopic expression of the baculovirus anti-apoptotic protein p35, implicating a role for caspases in the death response. Coordinate stage-specific induction of the Drosophila death genes reaper (rpr) and head involution defective (hid) immediately precedes the destruction of the larval midgut and salivary gland. In addition, the diap2 anti-cell death gene is repressed in larval salivary glands as rpr and hid are induced, suggesting that the death of this tissue is under both positive and negative regulation. Finally, diap2 is repressed by ecdysone in cultured salivary glands under the same conditions that induce rpr expression and trigger programmed cell death. These studies indicate that ecdysone directs the death of larval tissues via the precise stage- and tissue-specific regulation of key death effector genes.     

Severe pulmonary hemorrhage in the premature newborn infant: analysis of presurfactant and surfactant eras

Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. During the molt and metamorphosis of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ecdysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and that the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high concentration (10(-5) M) of 20E equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurred at lower concentrations (effective concentrations, EC50=6.3x10(-7) M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until pupation. When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 microgram/ml JH I which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increased steadily with the same EC50 as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, each in its own time-dependent manner, similar to that seen in vivo. These initial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected.     

Primary structure and possible functions of a trypsin inhibitor of Bombyx mori

A protein with a low molecular mass of 6027 was purified from cocoon shell of silkworm, Bombyx mori. Two-dimensional polyacrylamide gel electrophoresis (2D/PAGE) resolved this protein into a single spot with pI 4.3 and Mr 6000. Amino acid sequence analysis revealed that this protein consists of 55 amino acids, six of these being cysteine residues and is highly homologous to bovine pancreatic trypsin inhibitor-type inhibitors. The 6-kDa protein is heat stable and acid stable and inhibits bovine trypsin by forming a low-dissociation complex with trypsin in a 1 : 1 molar ratio (Ki = 2.8 x 10-10), but does not alpha-chymotrypsin. This cocoon shell-associated trypsin inhibitor (CSTI) was thus concluded to belong to the bovine pancreatic trypsin inhibitor class. CSTI was developmentally regulated in the silk gland at the final stage of larval growth, and its specific distribution in the middle silk gland, an organ in which silk proteins are stored during the final larval instar, occurred before the onset of spinning. This inhibitor protects the tryptic degradation of fibroin light (L) chain in vitro. These results suggest that this trypsin inhibitor may play an important part on regulating proteolytic activity in the silk gland or protecting silk proteins from degradation during histolysis.     

Application of NMR microscopy to the morphological study of the silkworm, Bombyx mori, during its metamorphosis

Zero (ZQ) and double (DQ) quantum 2D chemical shift selective and spin-echo 3D NMR imaging at microscopy resolution, has been applied to the morphological study of silkworm, Bombyx mori, during its metamorphosis. Attention had been focused on the evolution of the internal structure of the insect during its postembryonal life occurring through the larval, pupal and adult development. A major objective of this work was the characterization of the silk glands, responsible for the synthesis and secretion of fibroin and sericin, through the changes of distribution and mobility of water, by imaging the water protons during postembryonal life stages. Moreover, alanine deriving from silk gland proteins was imaged during the last life stage of Bombyx mori.     

Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.     

Control of ecdysteroidogenesis: activation and inhibition of prothoracic gland activity

The ecdysteroid hormones, mainly 20-hydroxyecdysone (20E), play a pivotal role in insect development by controlling gene expression involved in molting and metamorphosis. In the model insect Manduca sexta the production of ecdysteroids by the prothoracic gland is acutely controlled by a brain neurohormone, prothoracicotropic hormone (PTTH). PTTH initiates a cascade of events that progresses from the influx of Ca2+ and cAMP generation through phosphorylation of the ribosomal protein S6 and S6-dependent protein synthesis, and concludes with an increase in the synthesis and export of ecdysteroids from the gland. Recent studies indicate that S6 phosphorylation probably controls the steroidogenic effect of PTTH by gating the translation of selected mRNAs whose protein products are required for increased ecdysteroid synthesis. Inhibition of S6 phosphorylation prevents an increase in PTTH-stimulated protein synthesis and subsequent ecdysteroid synthesis. Two of the proteins whose translations are specifically stimulated by PTTH have been identified, one being a beta tubulin and the other a heat shock protein 70 family member. Current data suggest that these two proteins could be involved in supporting microtubule-dependent protein synthesis and ecdysone receptor assembly and/or function. Recent data also indicate that the 20E produced by the prothoracic gland feeds back upon the gland by increasing expression and phosphorylation of a specific USP isoform that is a constituent of the functional ecdysone receptor. Changes in the concentration and composition of the ecdysone receptor complex of the prothoracic gland could modulate the gland's potential for ecdysteroid synthesis (e.g. feedback inhibition) by controlling the levels of enzymes or other proteins in the ecdysteroid biosynthetic pathway.     

Ecdysone signaling cascade and regulation of Drosophila metamorphosis

Pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) regulate diverse biological responses during the life history of insects. Studies of the fruit fly, Drosophila melanogaster, have provided significant insights into the mechanisms underlying ecdysone mediated regulation of development. During the dramatic metamorphosis of Drosophila, ecdysone induces the histolysis of nearly all of the larval tissues and differentiation and morphogenesis of the structures composing the adult fly. These changes are mediated by a genetic signaling cascade that was first recognized as puffs in the giant polytene chromosomes of the salivary gland. This genetic regulatory cascade is composed of early and late genes that are intricately coordinated by changes in hormone titer. Early genes encode regulatory proteins that are involved in the proper regulation of late genes, which are thought to play a more direct role in development. The regulation and function of these genes is discussed in the context of the cell- and tissue-specific changes required for the reorganization of a larva to form an adult fly.     

The ecdysone receptor and ultraspiracle regulate the timing and progression of ovarian morphogenesis during Drosophila metamorphosis

Ecdysteroids regulate insect metamorphosis through the edysone receptor complex, a heterodimeric nuclear receptor consisting of the ecdysone receptor (EcR) and its partner ultraspiracle (USP). Differentiation in the Drosophila ovary at metamorphosis correlates with colocalization of USP and the EcR-A isoform in all but one of eight mesoderm-derived somatic cell types. The one exception is the larval terminal filament (TF) cells, in which only USP is detectable during cell differentiation. In cells destined to form the basal stalks and anterior oviduct, USP colocalizes with what appears to be the EcR-B2 isoform. Flies heterozygous for a deletion of the EcR gene exhibit several defects in ovarian morphogenesis, including a heterochronic delay in the onset of terminal filament differentiation. Flies heterozygous for a strong usp allele exhibit accelerated TF differentiation. Flies simultaneously heterozygous for both EcR and usp have additional phenotypes, including several heterochronic shifts, delayed initiation and completion of terminal filament morphogenesis and delayed ovarian differentiation during the first day of metamorphosis. Terminal filament morphogenesis is severely disrupted in homozygous usp clones. Our results demonstrate that proper expression of the ecdysone receptor complex is required to maintain the normal progression and timing of the events of ovarian differentiation in Drosophila. These findings are discussed in the context of a developmental and evolutionary role for the ecdysone receptor complex in regulating the timing of ovarian differentiation in dipteran insects.     

Differential expression of SNAP-25 isoforms and SNAP-23 in the adrenal gland

In the rat adrenal gland, we previously observed that SNAP-25 is not restricted to the plasmalemma in noradrenergic cells as it is in adrenergic cells, and hypothesized that SNAP-25 isoform expression is different in the two phenotypes. Expression of SNAP-25 isoforms and SNAP-23 was examined by immunoblotting, immunofluorescence, and RT-PCR. Amplifications of SNAP-25 mRNAs were combined with Southern hybridization, restriction enzyme analysis, and sequencing of cloned PCR products to compare SNAP-25 isoform expression in rat and bovine adrenal glands. SNAP-25 and SNAP-23 mRNA and protein are expressed in the glands; SNAP-23 is enriched in the adrenal cortex, whereas SNAP-25 is restricted to the adrenal medulla. Furthermore, high levels of SNAP-25 and low levels of SNAP-23 are observed in the PC12 cells, whereas both SNAP-25 and SNAP-23 are expressed in adrenal medullary cultures. In all extracts, the SNAP-23 mRNA corresponded to SNAP-23a. SNAP-25a is the major form expressed in rat adrenal glands (75%), as it is in PC12 cells (80%), but both SNAP-25a and SNAP-25b (40% vs. 60%) are expressed in bovine adrenal medulla in situ and in culture. In addition, an enriched population of adrenergic cells (93%) expressed a higher level of SNAP-25b (70%), suggesting that this isoform may not be restricted to fast neurotransmission.     

Ultrastructural changes of Drosophila larval and prepupal salivary glands cultured in vitro with ecdysone

Alterations in the ultrastructure of in vitro cultured larval salivary glands of Drosophila melanogaster in response to the steroid hormone ecdysone were studied in relation to complex changes in puffing patterns. We found that the changes in the fine structure of cultured glands reflected progression of the puffing pattern, and they paralleled those seen in vivo. We observed that glue secretion by exocytosis, the main function of salivary glands, took place between puff stage 5 (PS5) and PS7. Glue could not be expectorated under culture conditions but was slowly released from the lumen through a duct into the medium. After the cultured glands reached PS13/PS14, further progress of puffing and fine structural alterations required that the ecdysteroid titer be transiently extremely low or absent. Under in vitro conditions we did not observe the putative new secretory program(s) described for glands in vivo after PS12. However, ultrastructural changes which unambiguously indicated that an autohistolytic process had begun in vitro started to appear after PS17. Many salivary gland cells developed numerous features of progressive self-degradation between PS18 and PS21. Actual degradation of salivary glands in vivo seemed to be rapid, but in vitro degradation was never completed, probably due to a lack of exogenous factors from the hemolymph. Manipulations of ecdysone titer in vitro in the culture medium, known during the larval puffing cycle to cause premature induction of developmentally specific puffing patterns, did not affect the normal development of ultrastructural features of the cytoplasm and nucleus.     

Structure and function of prothoracic glands and oenocytes in embryos and last larval instars of Oncopeltus fasciatus Dallas (Insecta, Heteroptera)

1. Active prothoracic glands and oenocytes of last larval stage are both characteristized by well-developed smooth and rough endoplasmic reticulum (ER). Prothoracic glands also show plasma membrane infoldings, but not oenocytes which contain a large number of pleomorphic vesicles. 2. The fine structure of embryonic oenocytes corresponds after blastokinesis with that of active larval and adult cells. Thus, an activity in the late embryo can be assumed. Embryonic prothoracic glands reveal no signs of activity: smooth and rough ER are absent. The subcellular structure resembles that of organ anlagen, i.e. not yet fully differentiated tissue. Hormone synthesis is not likely. 3. Ecdysone titer was determined throughout embryonic development and in mature adults. Although prothoracic glands break down during adult ecdysis, imagines contain in the Calliphora-bioassay active factors: females 0.9 CU/g and males 0.5 CU/g. As sites of synthesis the oenocytes are suggested. 4. A relatively high ecdysone titer of 7 CU/g is measured in newly deposited eggs. The hormone is presumably of maternal origin. Subsequent to blastokinesis the hormone content increases dramatically up to about 180 CU/g, apparently due to endocrine function of the embryo. Oenocytes are proposed as the source of ecdysone during late embryonic development. 5. The function of ecdysone during early and advanced embryogenesis, especially in view of "embryonic molts", is discussed.     

Altered expressions of cardiac Na/K-ATPase isoforms in copper deficient rats

OBJECTIVE: The aim was to determine if copper deficiency affects the expression of Na/K-ATPase alpha isoforms in the rat heart. METHODS: Copper deficiency was induced by placing weanling rats on a copper deficient diet for 4-5 weeks. Adult ventricular tissue, isolated ventricular myocytes, and brain stems of the control and deficient rats were compared for Cu, Zn-superoxide dismutase (CuZn-SOD) activity and for protein and mRNA contents of Na/K-ATPase alpha isoforms. RESULTS: In brain stem, where copper deficiency did not alter CuZn-SOD activity, mRNA and protein levels of alpha isoforms also remained unchanged. In ventricular tissue and ventricular myocytes, copper deficiency reduced CuZn-SOD activity, mRNAs of alpha 1 and alpha 2 isoforms, and the alpha 2 isoform protein. The alpha 1 isoform protein of ventricular tissue and its myocytes was marginally reduced by copper deficiency. CONCLUSIONS: In the rat ventricular tissue, oxidative stress resulting from copper deficiency (1) enhances the turnover of the more oxidant sensitive alpha 2 isoform to a greater extent than the turnover of the alpha 1 isoform; (2) regulates mRNA levels of alpha 1 and alpha 2 isoforms; and (3) contributes to the cardiomyopathy of copper deficiency.     

Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family

We report the cloning and expression of the fifth member of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family. Degenerate polymerase chain reaction amplification and hybridization screening of a rat sublingual gland (RSLG) cDNA library were used to identify a novel isoform termed ppGaNTase-T5. Conceptual translation of the cDNA reveals a uniquely long stem region not observed for other members of this enzyme family. Recombinant proteins expressed transiently in COS7 cells displayed transferase activity in vitro. Relative activity and substrate preferences of ppGaNTase-T5 were compared with previously identified isoforms (ppGaNTase-T1, -T3, and -T4); ppGaNTase-T5 and -T4 glycosylated a restricted subset of peptides whereas ppGaNTase-T1 and -T3 glycosylated a broader range of substrates. Northern blot analysis revealed that ppGaNTase-T5 is expressed in a highly tissue-specific manner; abundant expression was seen in the RSLG, with lesser amounts of message in the stomach, small intestine, and colon. Therefore, the pattern of expression of ppGaNTase-T5 is the most restricted of all isoforms examined thus far. The identification of this novel isoform underscores the diversity and complexity of the family of genes controlling O-linked glycosylation.     

Retrovirus-mediated transduction of porcine keratinocytes in organ culture

Fasting is associated with a decrease in the maximal binding capacity of the T3-receptor in different tissues and the responsiveness of some genes to exogenous T3 is not as pronounced as normal. One possible explanation for these observations is a change in the expression of one or all of the isoform mRNAs. We set out to measure the levels of the alpha 1, alpha 2, and beta 1 isoform mRNAs in liver and heart of fasted rats using a competitive PCR method. We constructed competitors based on the isoforms themselves so the amplification efficiencies of the target and the competitor are similar. Using this method we found that during fasting levels of alpha 2 mRNA in the liver of rats increased threefold (P < 0.002) over a 48-h period, whereas alpha 1 and beta 1 did not show any change. In the heart, no change in any of the TR isoform mRNAs was found.