Breakdown of the silk glands of Galleria mellonella—Acid phosphatase in involuting glands

The silk glands of larvae of Galleria mellonella and Malacosoma americana contain sedimentable enzymes digesting paranitrophenyl phosphate, sodium-β-glycerophosphate, and naphthol phosphates at pH 5·0. These enzymes increase significantly in amount, but do not alter in distribution, as the larva pupates.     

An in vitro system for studying juvenile hormone induction of juvenile hormone esterase from the fat body of Trichoplusia ni (Hübner)

An in vitro fat body culture was used to study juvenile hormone esterase (JHE) regulation. The present study shows juvenile hormone can directly induce JHE activity to appear in the culture medium in a dose-dependent manner at physiological concentrations of JH. This induced appearance of JHE can be blocked with actinomycin D. Biochemical characterization of the in vitro produced JHE demonstrated that it had the same isoelectric points as that of the in vivo JHE activity. The JHE inhibitor 1-1-1 trifluoro-tetradecan-2-one gave the same inhibition profile and I₅₀ toward both in vivo and in vitro produced JHE activities. Finally, the JHE activity induced in vitro was immunologically similar to that occurring in vivo. The system should be useful for high resolution studies on the regulation of JHE.     

Pleiotropic effects of juvenile hormone in ant queens and the escape from the reproduction–immunocompetence trade-off

The ubiquitous trade-off between survival and costly reproduction is one of the most fundamental constraints governing life-history evolution. In numerous animals, gonadotropic hormones antagonistically suppressing immunocompetence cause this trade-off. The queens of many social insects defy the reproduction–survival trade-off, achieving both an extraordinarily long life and high reproductive output, but how they achieve this is unknown. Here we show experimentally, by integrating quantification of gene expression, physiology and behaviour, that the long-lived queens of the ant Lasius niger have escaped the reproduction–immunocompetence trade-off by decoupling the effects of a key endocrine regulator of fertility and immunocompetence in solitary insects, juvenile hormone (JH). This modification of the regulatory architecture enables queens to sustain a high reproductive output without elevated JH titres and suppressed immunocompetence, providing an escape from the reproduction–immunocompetence trade-off that may contribute to the extraordinary lifespan of many social insect queens.     

Proteins binding to the 5‘—flanking regualtory elements of the human β—globin gene

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5‘-flanking regulatory elements of human β-globin gene,two negative control regions(NCR1,-610to-490 bp;NCR2,-338,to-233bp),was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5‘-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in art for the silence of β-glolbin gene in the embryonic and fetal stages.     

In silico and bio assay of juvenile hormone analogs as an insect growth regulator against Galleria mellonella (wax moth) – Part I

Juvenile hormone (JH) analogs are nowadays in use to control harmful pests. In order to develop new bioactive molecules as potential pesticides, we have incorporated different active structural features like sulfonamide, aromatic rings, amide group, and amino acid moiety to the base structure. We have screened a series of designed novel JH analogs against JH receptor protein (jhbpGm-2RCK) of Galleria mellonella in comparison to commercial insect growth regulators (IGRs) – Pyriproxyfen (T₁) and Fenoxycarb (T₂). All analogs exhibit the binding energy profile comparable to commercial IGRs. Based upon these results, a series of sulfonamide-based JHAs (T₃–T₈) as IGRs have been synthesized and characterized. Further, the efficacy of synthesized analogs (T₃–T₈) and commercial IGRs (Pyriproxyfen and Fenoxycarb) has been assessed against fourth instars larvae of G. mellonella under the laboratory conditions. LC₅₀ values of all the analogs (T₁–T₈) against the fourth instars larvae were 9.99, 10.12, 24.76, 30.73, 38.45, 34.15, 34.14, 19.48 ppm and the LC₉₀ 153.27, 131.69, 112.15, 191.46, 427.02, 167.13, 217.10, 172.00 ppm, respectively. Among these analogs, N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl)-p-toluene sulfonamide (T₈) and N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl) benzene sulfonamide (T₇) exhibited the good pest larval mortality at different exposure periods (in hours) and different concentrations (in ppm) in comparison to in use IGRs- T₁ and T₂. Bio assay results are supported by docking at higher concentration. The present investigation clearly exhibits that analog T₈ could serve as a potential IGR in comparison to in use IGRs (T₁ and T₂). The results are promising and provide new array of synthetic chemicals that may be utilized as IGRs.     

Influence of a haemolymph protein fraction on the binding of juvenile hormone in homogenates of insect epidermis (Plodia interpunctella (Hübner))

Summary A carrier protein fraction (CPF) from larval haemolymph was found to influence binding and catabolism of tritiated juvenile hormone (JH) in homogenates of larval epidermis. The CPF reduced binding of tritiated JH in all of the particulate fractions but did not alter the relative binding pattern when compared with JH alone. The CPF also protected the hormone from degradative enzymes in the membrane vesicle and microsomal + cytosol fractions but not in the nuclear and mitochondrial fractions. Preliminary evidence exists for high-affinity binding sites for JH in the nuclear and mitochondrial fractions. We conclude that the CPF influences catabolism of the tritiated JH but does not participate in subcellular recognition of JH in homogenized target tissue.Mention of a proprietary product in this paper does not constitute an endorsement of that product by the U.S. Department of Agriculture     

Dissecting water binding sites at protein–protein interfaces: a lesson from the atomic structures in the Protein Data Bank

We dissect the protein–protein interfaces into water preservation (WP), water hydration (WH) and water dehydration (WD) sites by comparing the water-mediated hydrogen bonds (H-bond) in the bound and unbound states of the interacting subunits. Upon subunit complexation, if a H-bond between an interface water and a protein polar group is retained, we assign it as WP site; if it is lost, we assign it as WD site and if a new H-bond is created, we assign it as WH site. We find that the density of WD sites is highest followed by WH and WP sites except in antigen and (or) antibody complexes, where the density of WH sites is highest followed by WD and WP sites. Furthermore, we find that WP sites are the most conserved followed by WD and WH sites in all class of complexes except in antigen and (or) antibody complexes, where WD sites are the most conserved followed by WH and WP sites. A significant number of WP and WH sites are involved in water bridges that stabilize the subunit interactions. At WH sites, the residues involved in water bridges are significantly better conserved than the other residues. However, no such difference is observed at WP sites. Interestingly, WD sites are generally replaced with direct H-bonds upon subunit complexation. Significantly, we observe many water-mediated H-bonds remain preserved in spite of large conformational changes upon subunit complexation. These findings have implications in predicting and engineering water binding sites at protein–protein interfaces.     

Importance of spores,crystals, and δ-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae

Physical methods were used to produce spores containing impurities of 0.02–0.05% crystals and crystals containing impurities of 0.001–0.01% spores from cultures of Bacillus thuringiensis. In Galleria mellonella larvae, these preparations from varieties galleriae, aizawai, and wuhanensis were only moderately active compared to 1:1 mixtures of spores and crystals. Spores of an acrystalliferous aizawai mutant were inactive and did not contain a polypeptide of the same size as the potent M_r 138000 δ-endotoxin present in spores and crystals of all three wild-type strains. Thus, this polypeptide probably contributed to the moderate activity of wild-type spores. Spore impurities in the crystal preparation were killed by γ irradiation without harming the crystals. The crystals without live spores were virtually inactive (LC₅₀s, ca. 10¹⁰ crystals/g insect food). Addition of 10³ spores to 10⁸ crystals/g food (0.001% spores) increased the mortality of larvae from 0 to 36%, and addition of 10⁴ spores (0.01% spores) killed 64% larvae. Thus, the addition of low levels of spores increased the potency of crystals in G. mellonella from virtually zero to moderate levels, suggesting that the live spore impurities in the crystal preparations were responsible for the observed moderate potency of crystals before γ irradiation, a view supported by a reduction of potency of crystal preparations following admixture of streptomycin to the insect food. In contrast to the results with G. mellonella, crystals were ca. 30 times as active as spores in Pieris brassicae larvae. Many authors have found crystals purified by physical methods to be highly active in a range of lepidopterous hosts. The present work indicates that the role of the spore impurities in these species may need further investigation. Absence of live spores of B. thuringiensis may impair the control of some insect species feeding on spore-free products and on microorganisms or plants into which endotoxins have been introduced by genetic manipulation.     

Theoretical prediction of antigenic sites in the Β-subunits of human choriogonadotropin and luteinizing hormone

Using hydrophilicity and recognition values of amino acids, the antigenic sites of theΒ-subunits of human choriogonadotropin and luteinizing hormone were computed from their amino acid sequences. Six antigenic sites were calculated for human choriogonadotropinΒ-subunits: residues 3–8, 17–22, 59–65,100–106,110–116 and 134–139. For luteinizing hormoneΒ-chain three antigenic sites were calculated: residues 17–22,59–65, and 100–106; all these three sites of luteinizing hormoneΒ being identical to the corresponding sites in human choriogonadotropinΒ. There was no antigenic site in luteinizing hormone that was also not found in human choriogonadotropin. On the other hand, there were unique determinants in human choriogonadotropin that were not found in luteinizing hormone; these determinants were residues 3–8, 110–116 and 134–139     

Evolution of the scrambled germline gene encoding α-telomere binding protein in three hypotrichous ciliates

The micronuclear genes encoding α-telomere-binding protein (αTP) in Oxytricha trifallax and Stylonychia mytilus contain multiple internal eliminated segments, or IESs, that divide the gene into multiple parts called macronuclear destined segments, or MDSs. The MDSs have become disordered, or scrambled, during evolution. The scrambled structures of the αTP genes in Oxytricha trifallax and S. mytilus have been compared with the previously published scrambled structure of the αTP gene in O. nova. The scrambled patterns of the αTP gene in the three species are similar but show significant differences. The micronuclear genes in O. nova and S. mytilus consist of 13 IESs and 14 MDSs, but the gene in O. trifallax is divided into three additional MDSs by the presence of three additional IESs, believed to have been inserted into the O. trifallaxαTP gene after divergence of O. trifallax from the other two species. Corresponding IESs among the three species have shifted along the DNA during evolution, presumably by a mutational mechanism that changes the short repeat sequences that flank IESs. The IESs also have changed markedly in length by insertion and/or deletion of nucleotides. Comparison of the putative αTP amino acid sequences in the three species reveals three conserved and three nonconserved domains. The 5′ nontranslated regions of the gene-sized molecules encoding αTP contain several conserved segments, and the 3′ nontranscribed trailer contains one conserved segment. Received: 29 May 1998; in revised form: 3 August 1998 / Accepted: 18 August 1998     

The juvenile hormone—I. Methods of purification and isolation

Methods for the purification and isolation of juvenile hormone from Hyalophora cecropia are described. Ether extraction, low temperature precipitation, thin layer chromatography, and gas liquid chromatography were utilized. JH activity was followed by means of the Tenebrio bioassay. An apparent gain in activity of approximately five-fold was obtained during the course of purification. This was interpreted to be largely the result of a loss of inhibitors during the low temperature precipitation procedure. The final active material, indicated to be a single compound by preliminary gas liquid chromatographic analysis, represented 0·0045–0·0051 per cent of the crude oil used as starting material. The juvenile hormone activity was increased from 25 Tenebrio Units per μl for the crude oil to ⋍2·6×10⁶Tenebrio Units per μl for the final active substance. This represents an approximate 1·05×10⁵-fold purification.     

Metal-binding sites of concanavalin A and their role in the binding of α-methyl d-glucopyranoside

Binding of a transition metal ion to specific sites in concanavalin A induces the formation of specific Ca(2+) ion-binding sites. Sites for binding alpha-methyl d-glucopyranoside exist only when a transition metal ion and Ca(2+) ion are bound.