Steroid hormone activation of wandering in the isolated nervous system of Manduca sexta

Steroid hormones modulate motor circuits in both vertebrates and invertebrates. The insect Manduca sexta, with its well-characterized developmental and endocrinological history, is a useful model system in which to study these effects. Wandering is a stage-specific locomotor behavior triggered by the steroid hormone 20-hydroxyecdysone (20E), consisting of crawling and burrowing movements as the animal searches for a pupation site. This study was undertaken to determine whether the wandering motor pattern is activated by direct action of 20E on the CNS. 20E acts on the isolated larval nervous system to induce a fictive motor pattern showing features of crawling and burrowing. The latency of the response to 20E is long, suggestive of a genomic mechanism of action. The abdominal motoneurons or segmental pattern generating circuits are unlikely to be the primary targets of 20E action in inducing fictive wandering. Exposure of the segmental ganglia alone to hormone did not evoke fictive wandering. Therefore, as suggested by an earlier study, the likely site of 20E action is within the brain.     

MsGC-II,a receptor guanylyl cyclase isolated from the CNS of Manduca sexta that is inhibited by calcium

We describe the cloning of a receptor guanylyl cyclase, MsGC-II, from the CNS of the insect Manduca sexta. Sequence comparisons with other receptor guanylyl cyclases show that MsGC-II is most similar to a predicted guanylyl cyclase in the Drosophila genome and to vertebrate retinal guanylyl cyclases. When expressed in COS-7 cells, MsGC-II exhibited a low level of basal activity that was nearly abolished in the presence of 10 micro m calcium. Incubation with either a mammalian guanylyl cyclase-activating protein or Drosophila frequenin resulted in only mild stimulation of activity, whereas incubation of COS-7 cells expressing MsGC-II with a variety of Manduca tissue extracts failed to stimulate enzyme activity above basal levels. Analysis of the tissue distribution of MsGC-II revealed that it is nervous system specific. In the adult, MsGC-II is present in neurons in the optic lobes, antennal lobes and cellular cortex, but it is most highly expressed in subsets of intrinsic mushroom body neurons. Thus, MsGC-II appears to be a neural-specific receptor guanylyl cyclase whose activity may be regulated either directly or indirectly by calcium.     

A new peptide in the FMRFamide family isolated from the CNS of the hawkmoth, Manduca sexta

We have purified a FMRFamide-like peptide from extracts of brain-subesophageal ganglion of the moth, Manduca sexta. The purification was monitored with a new, competitive ELISA, and accomplished with ion exchange and reverse-phase HPLC. The peptide structure was determined by a combination of tandem mass spectrometry and automated Edman degradation. The amino acid sequence of the peptide is less than Glu-Asp-Val-Val-His-Ser-Phe-Leu-Arg-Phe-amide (pEDVVHSFLRF-NH2). In a separate purification, an identical peptide was isolated from extracts of brain-associated neurohemal structures. We have named this peptide ManducaFLRFamide, to indicate its homology with other members of the "FMRFamide" family. In bioassays, chemically synthesized peptide increased the force of neurally evoked contractions in the major power-producing flight muscles, the dorsal longitudinal muscles. This observation suggests that hormonally released ManducaFLRFamide may play a role in sustaining or promoting the flight behavior necessary for mate-seeking (in males) or oviposition (in females) in sphingid moths.     

Induction of metamorphosis from the larval to the polyp stage is similar in Hydrozoa and a subgroup of Scyphozoa (Cnidaria, Semaeostomeae)

Larvae of cnidarians need an external cue for metamorphosis to start. The larvae of various hydrozoa, in particular of Hydractinia echinata, respond to Cs⁺, Li⁺, NH₄ ⁺ and seawater in which the concentration of Mg²⁺ ions is reduced. They further respond to the phorbolester, tetradecanoyl-phorbol-13-acetate (TPA) and the diacylglycerol (DAG) diC8, which both are argued to stimulate a protein kinase C. The only well-studied scyphozoa, Cassiopea spp., respond differently, i.e. to TPA and diC8 only. We found that larvae of the scyphozoa Aurelia aurita, Chrysaora hysoscella and Cyanea lamarckii respond to all the compounds mentioned. Trigonelline (N-methylnicotinic acid), a metamorphosis inhibitor found in Hydractinia larvae, is assumed to act by delivering a methyl group for transmethylation processes antagonising metamorphosis induction in Chrysaora hysoscella and Cyanea lamarckii. The three species tested are scyphozoa belonging to the subgroup of semaeostomeae, while Cassiopea spp. belong to the rhizostomeae. The results obtained may contribute to the discussion concerning the evolution of cnidarians and may help to clarify whether the way metamorphosis can be induced in rhizostomeae as a whole is different from that in hydrozoa and those scyphozoa belonging to the subgroup semaeostomeae. Electronic Publication     

Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori

Synthesis and secretion of bactericidal protein (cecropin) and lysozyme were induced by soluble peptidoglycan fragments (SPG) from Escherichia coli in a culture of fat body from Bombyx mori larvae. The rate of the secretion by fat body increased as a function of SPG concentration added to the culture medium. The induction of bactericidal activity was specific for peptidoglycan of a particular structure. Thus, SPG from Micrococcus luteus was 500-times less potent than E. coli SPG, and various glucans and peptides structurally related to peptidoglycan were all ineffective as elicitor. These results support the hypothesis that bacteria invading the haemocoel have to be partially degraded to generate peptidoglycan fragments as a signal molecule, which subsequently acts on a receptor on fat body cells and induces antibacterial protein synthesis.     

Diuretic activity of Mas-DP II,an identified neuropeptide from Manduca sexta: An in vivo and in vitro examination in the adult moth

Mas-DP II, a recently identified 30 amino acid diuretic peptide isolated from the tobacco hornworm moth, Manduca sexta, was tested for its ability to increase fluid excretion in adult M. sexta, and for the ability to elevate the rate of fluid secretion from isolated Malpighian tubules cultured in vitro. Mas-DP II was found to increase fluid weight loss from decapitated adult moths in a dose-dependent manner; weight loss increased significantly at doses as low as 5 ng for female moths and 25 ng for male moths. Male moths injected with large doses of Mas-DP II continued to exhibit increased rates of fluid loss up to 4 h post-injection. In vitro, Mas-DP II stimulated fluid secretion from isolated Malpighian tubules at concentrations as low as 4 nM for tubules from both male and female moths. © 1994 Wiley-Liss, Inc.     

The nuclease activity of the yeast DNA2 protein, which is related to the RecB-like nucleases, is essential in vivo

Saccharomyces cerevisiae Dna2 protein is required for DNA replication and repair and is associated with multiple biochemical activities: DNA-dependent ATPase, DNA helicase, and DNA nuclease. To investigate which of these activities is important for the cellular functions of Dna2, we have identified separation of function mutations that selectively inactivate the helicase or nuclease. We describe the effect of six such mutations on ATPase, helicase, and nuclease after purification of the mutant proteins from yeast or baculovirus-infected insect cells. A mutation in the Walker A box in the C-terminal third of the protein affects helicase and ATPase but not nuclease; a mutation in the N-terminal domain (amino acid 504) affects ATPase, helicase, and nuclease. Two mutations in the N-terminal domain abolish nuclease but do not reduce helicase activity (amino acids 657 and 675) and identify the putative nuclease active site. Two mutations immediately adjacent to the proposed nuclease active site (amino acids 640 and 693) impair nuclease activity in the absence of ATP but completely abolish nuclease activity in the presence of ATP. These results suggest that, although the Dna2 helicase and nuclease activities can be independently affected by some mutations, the two activities appear to interact, and the nuclease activity is regulated in a complex manner by ATP. Physiological analysis shows that both ATPase and nuclease are important for the essential function of DNA2 in DNA replication and for its role in double-strand break repair. Four of the nuclease mutants are not only loss of function mutations but also exhibit a dominant negative phenotype.     

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phos-phorylated form of the protein allow counter-ciockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephos-phorylation rates of these mutants, CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of CheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephospborylation rates, interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of inter-action with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.     

Changes in the functional activity of mitochondria isolated from the liver of rat that passed the preadaptation to ultra-low deuterium concentration

The incubation in deuterium-depleted medium of mitochondria isolated from the liver of rats that consumed drinking diet with depleted deuterium (46 ppm) revealed a higher (by 35%) generation of hydrogen peroxide in comparison with the mitochondria (isolated from the liver of rats that consumed drinking diet with 152 ppm deuterium) incubated in medium that contained 152 ppm deuterium. Succinate addition to the reaction system led to an increase in the production of hydrogen peroxide in isolated mitochondria by 44–81%, whereas the difference in the generation of H₂O₂ between the organelles incubated in mediums 46 and 152 ppm was reduced by 14%. The revealed change in the functional activity of mitochondria suggests the ability of the organism to adapt to the deuterium-depleted drinking diet, which is probably due to the formation of the D/H isotope transmembrane gradient.     

Arabidopsis TARGET OF RAPAMYCIN interacts with RAPTOR, which regulates the activity of S6 kinase in response to osmotic stress signals

TARGET OF RAPAMYCIN (TOR) kinase controls many cellular functions in eukaryotic cells in response to stress and nutrient availability and was shown to be essential for embryonic development in Arabidopsis thaliana. We demonstrated that Arabidopsis RAPTOR1 (a TOR regulatory protein) interacts with the HEAT repeats of TOR and that RAPTOR1 regulates the activity of S6 kinase (S6K) in response to osmotic stress. RAPTOR1 also interacts in vivo with Arabidopsis S6K1, a putative substrate for TOR. S6K1 fused to green fluorescent protein and immunoprecipitated from tobacco (Nicotiana tabacum) leaves after transient expression was active in phosphorylating the Arabidopsis ribosomal S6 protein. The catalytic domain of S6K1 could be phosphorylated by Arabidopsis 3-phosphoinositide-dependent protein kinase-1 (PDK1), indicating the involvement of PDK1 in the regulation of S6K. The S6K1 activity was sensitive to osmotic stress, while PDK1 activity was not affected. However, S6K1 sensitivity to osmotic stress was relieved by co-overexpression of RAPTOR1. Overall, these observations demonstrated the existence of a functional TOR kinase pathway in plants. However, Arabidopsis seedlings do not respond to normal physiological levels of rapamycin, which appears to be due its inability to bind to the Arabidopsis homolog of FKBP12, a protein that is essential for the binding of rapamycin with TOR. Replacement of the Arabidopsis FKBP12 with the human FKBP12 allowed rapamycin-dependent interaction with TOR. Since homozygous mutation in TOR is lethal, it suggests that this pathway is essential for integrating the stress signals into the growth regulation.     

Lipopolysaccharide promotes the survival of osteoclasts via Toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages

Lipopolysaccharide is a pathogen that causes inflammatory bone loss. Monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNF-alpha, and IL-6 in response to LPS. We examined the effects of LPS on the function of osteoclasts formed in vitro in comparison with its effect on bone marrow macrophages, osteoclast precursors. Both osteoclasts and bone marrow macrophages expressed mRNA of Toll-like receptor 4 (TLR4) and CD14, components of the LPS receptor system. LPS induced rapid degradation of I-kappaB in osteoclasts, and stimulated the survival of osteoclasts. LPS failed to support the survival of osteoclasts derived from C3H/HeJ mice, which possess a missense mutation in the TLR4 gene. The LPS-promoted survival of osteoclasts was not mediated by any of the cytokines known to prolong the survival of osteoclasts, such as IL-1beta, TNF-alpha, and receptor activator of NF-kappaB ligand. LPS stimulated the production of proinflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in bone marrow macrophages and peritoneal macrophages, but not in osteoclasts. These results indicate that osteoclasts respond to LPS through TLR4, but the characteristics of osteoclasts are quite different from those of their precursors, macrophages, in terms of proinflammatory cytokine production in response to LPS.     

Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.     

Molecular cloning, nucleotide sequencing, and expression of the Bacillus subtilis (natto) IAM1212 alpha-amylase gene, which encodes an alpha-amylase structurally similar to but enzymatically distinct from that of B. subtilis 2633.

An alpha-amylase gene of Bacillus subtilis (natto) IAM1212 was cloned in a lambda EMBL3 bacteriophage vector, and the nucleotide sequence was determined. An open reading frame encoding the alpha-amylase (AMY1212) consists of 1,431 base pairs and contains 477 amino acid residues, which is the same in size as the alpha-amylase (AMY2633) of B. subtilis 2633, an alpha-amylase-hyperproducing strain, and smaller than that of B. subtilis 168, Marburg strain. The amino acid sequence of AMY1212 is different from that of AMY2633 at five residues. Enzymatic properties of these two alpha-amylases were examined by introducing the cloned genes into an alpha-amylase-deficient strain, B. subtilis M15. It was revealed that products of soluble starch hydrolyzed by AMY1212 are maltose and maltotriose, while those of AMY2633 are glucose and maltose. From the detailed analyses with oligosaccharides as substrates, it was concluded that the difference in hydrolysis products of the two similar alpha-amylases should be ascribed to the different activity hydrolyzing low-molecular-weight substrates, especially maltotriose; AMY1212 slowly hydrolyzes maltotetraose and cannot hydrolyze maltotriose, while AMY2633 efficiently hydrolyzes maltotetraose and maltotriose. Further analyses with chimeric alpha-amylase molecules constructed from the cloned genes revealed that only one amino acid substitution is responsible for the differences in hydrolysis products.     

Evidence that the hydrophobicity of isolated, in situ, and de novo-synthesized native human placental folate receptors is a function of glycosyl-phosphatidylinositol anchoring to membranes.

Although normal human chorionic villi-associated hydrophobic placental folate receptors (PFR) are converted to hydrophilic forms by an endogenous, EDTA-sensitive, Mg(2+)-dependent protease under serum-free conditions (Verma, R. S., and Antony, A. C. (1991) J. Biol. Chem. 266, 12522-12535), it is not known whether hydrophobic PFR are also susceptible to conversion by endogenous phospholipases. We isolated and characterized hydrophobic PFR, and tested the hypothesis that purified, in situ, and de novo-synthesized native PFR were covalently linked to glycosyl-phosphatidylinositol (GPI) anchors. 125I-hydrophobic PFR, but not 125I-hydrophilic PFR, (i) separated into the Triton X-114 micellar phase at 30 degrees C, (ii) efficiently incorporated into phosphatidylcholine-cholesterol liposomes, and (iii) were covalently labeled by the hydrophobic probe 3-(trifluoromethyl)-3-(meta[125I]iodophenyl)diazirine, [125I]TID. (iv) [125I]TID-labeled- and [phenyl-3H]Triton X-100-bound hydrophobic PFR, as well as native PFR in situ, were released as hydrophilic forms by recombinant (r) GPI-specific phospholipase(PL) C (GPI-PLC), and GPI-PLD (but not by PLC), in the absence and presence of a concentration of EDTA known to inhibit endogenous Mg(2+)-dependent protease. (v) Nitrous acid deamination of [125I]TID-labeled hydrophobic PFR as well as (r)GPI-PLC cleavage of [phenyl-3H]Triton-X-100- and [125I] TID-labeled hydrophobic PFR, released hydrophobic radiolabeled moieties which comigrated on thin layer chromatography distinct from free radiolabel. Finally, (vi) biosynthetic studies on chorionic villi cultured in vitro revealed incorporation of radiolabeled precursors into the GPI anchor of hydrophobic PFR. We conclude that native hydrophobic PFR are linked to GPI anchors and are therefore potential substrates for three distinct endogenous enzymes (GPI-PLC, GPI-PLD, and specific Mg(2+)-dependent metalloprotease) in maternal serum and placenta in vivo.     

Active suppression of host-vs graft reaction in pregnant mice. IX. Soluble suppressor activity obtained from allopregnant mouse decidua that blocks the cytolytic effector response to IL-2 is related to transforming growth factor-beta

Non-Ts cells in murine allopregnancy decidua release potent immunosuppressor factors in vitro that block the action of IL-2. Previous studies have shown that both primary and secondary CTL responses are inhibited as well as the generation of Il-2 activated killer cells. In this paper we show that the suppressor factor(s) can arrest ongoing IL-2 dependent CTL responses but does not block binding of anti-IL-2R antibody or radiolabeled IL-2 to the IL-2R. The suppressive activity is associated with molecules that adhere to hydroxylapatite and con A-agarose but do not bind to activated charcoal or partition as lipids. HPLC TSK 3000 separation showed a major peak of suppressive activity at 60 to 100 kDa, with additional activity at 300 kDa, and at less than 1000. Under acid conditions, suppressive activity resolved as a major peak at 13 kDa with some residual activity at 65 kDa and at less than or equal to 1000. A specific rabbit IgG antibody to transforming growth factor-beta neutralized suppressor activity in unseparated supernatant and in the 13-kDa fraction whereas neutralizing antibodies to progesterone or PGE-2 did not affect suppression but could neutralize their respective ligands. Inasmuch as transforming growth factor-beta has a 25 kDa Mr, the 13-kDa decidua-associated suppressor factor would appear to represent a related but distinct regulatory molecule that associates with a variety of carrier molecules.     

Conversion of pregnenolone to DHEA by human 17alpha-hydroxylase/17, 20-lyase (P450c17). Evidence that DHEA is produced from the released intermediate, 17alpha-hydroxypregnenolone.

Most previous studies using reconstituted systems and fast kinetics suggest that the conversion of pregnenolone to dehydroepiandrosterone (DHEA; the precursor of androgen and estrogen biosynthesis) by P450c17 does not require the release of the intermediate 17alpha-OHPreg (a precursor of cortisol biosynthesis). With such a mechanism, it is difficult to conceive how high amounts of DHEA may be produced in some cells or tissues, such as the testis and cells from the adrenal reticularis, while in other tissues such as the fasciculata zone, high levels of 17alpha-OHPreg are synthesized. In this report, we address this matter using intact transfected cells, which better reflect the actual cellular conditions. Furthermore, by using transfected cells, we can conveniently analyze human enzymes, as we are not restricted by the availability of human tissues as in the case of methods using purified or partially purified enzymes. Using intact HEK-293 cells transfected with human P450c17 in culture, we showed, in a time course study of the transformation of pregnenolone, that there is an accumulation of 17alpha-OHPreg, and that, subsequently, the accumulated 17alpha-OHPreg decreases with a concomitant increase in DHEA production. The DHEA/17alpha-OHPreg ratio changes from 0.1 :1 after 1 h incubation to 50 : 1 after 20 h. This result strongly suggests that the transformation of Preg to DHEA proceeds through two steps in which DHEA is produced from the released intermediate 17alpha-OHPreg. We also show that high levels of substrate vs. enzyme concentration will lead to high hydroxylase activity whereas the reverse will increase the lyase activity. The result is in good agreement with recent observations suggesting that surrounding enzymes and steroids could modulate the lyase activity. Cotransfection of vectors expressing cytochrome b5 and NADPH cytochrome P450 reductase indicates that both are required for an optimum production of DHEA.