Cloning and characterization of an ecdysone receptor cDNA from Locusta migratoria

It has been suggested that some mitochondrial genes are important in cellular senescence. In order to identify the mitochondrial genes that are involved in cellular senescence, we have constructed a cDNA library from senescent human vascular endothelial cells and isolated 86 senescence-specific cDNA clones by differential screening. Among the clones, we identified four distinct mitochondrial genes including NADH dehydrogenase subunit 2 (ND2), ND3, ATPase 6 and 16S ribosomal RNA. We then compared the levels of expression of these genes in young and senescent cells by using two endothelial and two fibroblast cell strains. Northern blot and slot blot hybridization confirmed that the expression levels of ND3, ATPase 6 and 16S rRNA were elevated in senescent cells of all four strains. The expression level of ND2 was also elevated during cellular senescence in three of the four strains. Because mitochondria are actively involved in oxidative phosphorylation and respiratory functions, the altered expression levels of these genes may participate in aging processes.     

Characterization of ATPases of apical membrane fractions from Locusta migratoria Malpighian tubules

Fetal magnetic resonance imaging (FMRI) has gained considerable interest during the last decade, especially in its intracranial applications. Due to its high soft-tissue contrast and presumed safety, FMRI should be accepted as a complementary technique in prenatal diagnosis, useful either to elucidate equivocal findings on routine US studies or to further delineate some pathological entities. Normal patterns of fetal brain maturation, as shown by FMRI, are described because they are of paramount importance to accurately evaluate intracranial diseases. To date, however, FMRI requires specialised facilities and should be considered as an evolving clinical research modality and performed within precise guidelines in a multidisciplinary approach to prenatal pathology.     

Primary structure of proteins from the wing cuticle of the migratory locust, Locusta migratoria

Wing cuticle from pharate adult locusts, Locusta migratoria, contains several prominent proteins which occur as minor components or are completely absent in other cuticular regions. Six of the wing-specific proteins have been purified and their amino acid sequences determined by combined use of mass spectrometry and automated Edman degradation. During the sequence determination very long sequence runs (90-121 residues) were necessary in order to establish the primary structure. All the wing-specific cuticular proteins from locusts contain the repeated short sequence motif -Ala-Ala-Pro-Ala/Val-, which is common for all hitherto sequenced cuticular proteins from pharate locusts. Several of the wing-specific proteins also possess an N-terminal region rich in glycine, tyrosine and leucine, characteristic for many locust cuticular proteins. Two of the analysed proteins have a conserved 61-residue sequence in common with a previously sequenced protein from locust wing cuticle and with two proteins from the pharate cuticle of adult Tenebrio molitor. Possible roles for the various sequence motifs are discussed.     

Immunological similarities between two cerebral substances from Schistocerca gregaria and two neurohormones from Locusta migratoria

Two neurohormones produced by two distinctive neurosecretory median cells (A1, B) of the protocerebum have been characterized in Locusta migratoria; on with a multiple functions one called neuroparsin and one stimulating the ovarian maturation called lom OMP. Using the specific immunoserum of these two neurohormones in Schistocerca gregaria, we could demonstrate the occurring of molecules related to the neuroparsin and lom OMP of Locusta. Through histology studies of the brain and corpora cardiaca complex, the immunoserum revealed the presence of the two types of these neurosecretory cells suggesting the occurring in Locusta as well as Schistocerca of the same cells releasing molecules immunologically apparented to neuroparsin and lom OMP. The results were confirmed by electrophoretic separation of corpora cardiaca extract under unreduced conditions followed by a transfer on immobilon membrane.     

First results of the antibacterial defense reactions of Locusta migratoria larva and imago

The echocardiographic features of mitral valvular motion in a patient with classic rheumatic mitral stenosis are presented. Two unusual features were noted, and the important of careful echocardiographic scanning of the mitral valve is imphasized. The theories for the classic echocardiographic abnormalities of mitral stenosis are briefly considered in light of the findings in this case.     

Role of phagocytosis and soluble antibacterial factors in experimental immunization of Locusta migratoria

Last instar larvae of Locusta migratoria can be protected ("vaccinated") against lethal doses of Bacillus thuringiensis by previous injections of low doses of this pathogen. If iron saccharate is injected in to larvae prior to the administration of the "vaccinating" dose of Bacillus thuringiensis, no antibacterial protection can be induced. Injection of iron saccharate in to "vaccinated" larvae does not interfere with the induced protection; such larvae resist lethal doses.     

The post-ecdysis development of female fat tissue Locusta migratoria and its endocrine control

Cellular events associated with degeneration of the projection of the olfactory bulb to the molecular layer of the piriform cortex of the mouse have been studied with rapid-Golgi and Fink-Heimer impregnations and with the electron microscope. Four classes of axon terminals: s-1, s-d, f-1, and f-d, are differentiated on the basis of whether the synaptic vesicles are spherical or flattened and whether the axoplasm is lightly or darkly stained. The majority of s-1 terminals, the predominant class in sublamina Ia of the molecular layer, degenerate after bulb ablation. Degeneration of axon terminals is associated with dilation and, eventually, degeneration of segments of dendrites in Ia. Both s-1 and s-d terminals contribute to a partial reconstitution of the neuropil of Ia during the weeks after bulb ablation.     

The adipokinetic cells in the corpus cardiacum of Locusta migratoria preferentially release young secretory granules

The influence of flight activity on the release of secretory granules from the adipokinetic cells in the corpus cardiacum of Locusta migratoria was studied. Two labeling methods, an enzymatical and a radioactive one, were used to label young, newly synthesized secretory granules and so distinguish them from older, preexisting granules. Both methods demonstrated that the ratio between the numbers of labeled and unlabeled secretory granules was lower in flight-stimulated adipokinetic cells than in unstimulated cells. This ratio was lower in both the cell bodies and the cell processes of flight-stimulated cells. After flight there was no detectable change in the total number of secretory granules, which indicates that the synthesis of new secretory granules is not inhibited by flight activity. Rather, the tendency of flight-stimulated cells to have more trans-Golgi networks labeled with wheat-germ agglutinin-conjugated horseradish peroxidase suggests that the synthesis of new secretory granules was enhanced by flight. The results led to the conclusion that young secretory granules were preferentially released over older secretory granules.     

Neural parameters contributing to temperature compensation in the flight CPG of the locust, Locusta migratoria

Elevated thoracic temperature increases the wingbeat frequency of flying locusts. We investigated the extent to which temperature-induced changes in resting membrane potential and postsynaptic potential amplitude contribute to the effects of increased temperature on the frequency of the central flight rhythm. Flight neurons were hyperpolarized by changing the K+ concentration of the superfusing saline from 10 mM to 2 mM. 5 min of low-K+ superfusion hyperpolarized flight motoneurons from -42.8 mV to -50.1 mV with a concomitant decrease of the frequency of the central flight rhythm from 11.6 Hz to 10.5 Hz. The amplitude of postsynaptic potentials was halved after 10 min of zero Ca2+/high Mg2+ superfusion, but the frequency of the central rhythm did not change significantly. GABAergic inhibitory connections were reduced in amplitude using picrotoxin. This treatment increased the frequency of the central rhythm from 11.6 Hz to 12.9 Hz, and increased the thermosensitivity of the rhythm frequency. We conclude that the excitatory effect of increased temperature on rhythm frequency is not mediated by temperature effects on membrane potential and/or synaptic potential amplitude. We propose that the inhibitory effect of temperature-induced hyperpolarization of the membrane potential compensates for the excitatory effect of temperature on rhythm frequency (e.g. via increased conduction velocity). We further suggest that some measure of temperature compensation is afforded by equal effects on the amplitudes of excitatory and inhibitory postsynaptic potentials, such that the net effect on the level of excitation is zero.     

The metabolism of 3-deoxy-3-fluoro-D-glucose by Locusta migratoria and Schistocerca gregaria

3-Deoxy-3-fluoro-D-glucose (3FG) administered by injection is toxic to adult Locusta migratoria or Schistocerca gregaria (LD50, 4.8 mg/g). temperature-programmed and isothermal gas chromatographic analysis of poisoned locust haemolymph reveals the presence of a fluorinated metabolite identified as 3-deoxy-3-fluoro-D-glucitol (3FGL). The enzymes responsible for the accumulation of this metabolite are located in the fat body of the insect and partially purified as aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) and sorbitol dehydrogenase (L-iditol: NAD+ 5-oxidoreductase EC 1.1.1.14) 3FGL is shown to be both a competitive inhibitor of the NAD-linked sorbitol dehydrogenase with Ki 8?x 10(-2) M as well as a substrate with Km 0.5 M. A kinetic rate equation is derived and verified to account for the kinetic duality of 3FGL. These results partially explain the toxic effects of 3FG and are consistent with the presence of a hitherto undetected sorbitol metabolism in locusts.     

Gloverin, an antibacterial protein from the immune hemolymph of Hyalophora pupae

Gloverin is an inducible antibacterial insect protein isolated from pupae of the giant silk moth Hyalophora. It is a basic (pI 8.5) protein with a molecular mass of 13.8 kDa, containing a large number of glycine residues (18.5%) but no cysteine, and has an amino acid sequence that reveals no strong degree of identity with any known proteins. Gloverin inhibits the growth of Escherichia coli at a minimal concentration of 1-3 microM, i.e. less than 5% of the concentration of gloverin in the hemolymph of infected pupae. The prime effect of gloverin, following its interaction with lipopolysaccharide (LPS) in the bacterial envelope, is a specific inhibition of the synthesis of vital outer membrane proteins, leading to an increased permeability of the outer membrane. The activity of gloverin is not affected by heating (100 degrees C, 10 min) but is inhibited by Mg2+ and by free LPS. The gloverin molecule will undergo conformational transitions from a monomeric random coil to an alpha-helix upon transfer from an aqueous to a hydrophobic environment, a property likely to be of importance for its interaction with cell-bound LPS. The activity of gloverin is in many respects similar to that of attacin, another antibacterial protein, originally found in Hyalophora [for a review see Boman, H. G., Faye, I., Gudmundsson, G. H., Lee, J.-Y. & Lindholm, D. A. (1991) Eur J. Biochem. 201, 23-31].     

Cooperation of dopachrome conversion factor with phenoloxidase in the eumelanin pathway in haemolymph of Locusta migratoria (Insecta)

Dopachrome Conversion Factor (DCF) was found in the plasma of the locust Locusta migratoria. It has an apparent molecular mass of 85,000. Its K(m) was 0.2 mM at 22 degrees C and pH 7 with L-dopachrome as substrate. It had a high substrate specificity for L-dopachrome, methyl-L-dopachrome and L-dopachrome methyl ester but no activity on the corresponding D-isomers or on dopaminechrome. DCF was devoid of any phenoloxidase activity. Under action of DCF, L-dopachrome was converted into dihydroxyindole, which showed that a decarboxylation occured in the course of reaction. Locust DCF was inhibited by indole-3-propionic acid but not by indole-3- or indole-2-carboxylic acid. It was also inhibited by L-tryptophan in a competitive manner. Inhibition and substrate specificity suggest that a carboxyl group, either free or as a methyl ester, is necessary but not sufficient for enzyme recognition. When purified prophenoloxidase was activated and then added to dihydroxyindole either prepared by chemical synthesis or obtained by action of purified DCF on dopachrome, black pigments with a maximum absorption at 540 nm were generated. Therefore in the eumelanin pathway of locust plasma, phenoloxidase can catalyze the reaction that converts the product generated by DCF.     

Sequence and functional relationships between androgen-binding protein/sex hormone-binding globulin and its homologs protein S, Gas6, laminin, and agrin

Androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) is an extracellular binding protein that regulates the bioavailability of sex steroids. ABP/SHBG is closely related to the globular (G) domain of vitamin K-dependent protein S family of proteins and more distantly related to the G domains of several extracellular matrix proteins. ABP/SHBG appears to have evolved from the fusion of two ancestral G domains. Expanding evidence suggests that ABP/SHBG has other functions that are mediated through membrane binding, including signal transduction; however, the types of binding proteins (receptors) have not been identified. Sequence comparisons of ABP/SHBG with G domains of its homologs protein S, Gas6, laminin, and agrin have identified regions of ABP/SHBG that may bind receptors related to homolog receptors. These membrane receptors include beta-integrins, alpha-dystroglycan, and receptor tyrosine kinases. The G domains of laminin and related proteins have clearly evolved from a common ancestor to interact with specific receptors and binding proteins. It remains to be determined if ABP/SHBG followed this evolutionary pathway.     

Isolation and identification of three ecdysteroid conjugates with a C-20 hydroxy group in eggs of Locusta migratoria

Three ecdysteroids conjugates with a hydroxy group at C-20 were isolated from developing eggs of locusta migratoria and identified as 22-phosphate conjugates of 2-deoxy-20-hydroxy-ecdysone, 20-hydroxyecdysone and 20-hydroxyecdysone acetate.     

Barley oxalate oxidase is a hexameric protein related to seed storage proteins: evidence from X-ray crystallography

The oxalate oxidase enzyme expressed in barley roots is a thermostable, protease-resistant enzyme that generates H2O2. It has great medical importance because of its use to assay plasma and urinary oxalate, and it has also been used to generate transgenic, pathogen-resistant crops. This protein has now been purified and three types of crystals grown. X-ray analysis shows that the symmetry present in these crystals is consistent with a hexameric arrangement of subunits, probably a trimer of dimers. This structure may be similar to that found in the related seed storage proteins.     

Excess corticotropin releasing hormone-binding protein in the hypothalamic-pituitary-adrenal axis in transgenic mice

Corticotropin-releasing hormone (CRH) is the primary hypothalamic releasing factor that mediates the mammalian stress response. The CRH-binding protein (CRH-BP) is secreted from corticotropes, the pituitary CRH target cells, suggesting that the CRH-BP may modulate hypothalamic-pituitary-adrenal (HPA) axis activity by preventing CRH receptor stimulation. Transgenic mice were generated that constitutively express elevated levels of CRH-BP in the anterior pituitary gland. RNA and protein analyses confirmed the elevation of pituitary CRH-BP. Basal plasma concentrations of corticosterone and adrenocorticotropin hormone (ACTH) are unchanged, and a normal pattern of increased corticosterone and ACTH was observed after restraint stress. However, CRH and vasopressin (AVP) mRNA levels in the transgenic mice are increased by 82 and 35%, respectively, to compensate for the excess CRH-BP, consistent with the idea that CRH-BP levels are important for homeostasis. The transgenic mice exhibit increased activity in standard behavioral tests, and an altered circadian pattern of food intake which may be due to transgene expression in the brain. Alterations in CRH and AVP in response to elevated pituitary CRH-BP clearly demonstrate that regulation of CRH-BP is important in the function of the HPA axis.