Titres of juvenile hormone I, II and III in Spodoptera littoralis (Noctuidae) from the egg to the pupal moult and their modification by the egg-larval parasitoid Chelonus inanitus (Braconidae).

Physico-chemical analysis of juvenile hormones (JHs) of Spodoptera littoralis revealed highest quantities in the second half of embryonic development and in newly hatched 1st instar larvae. At these stages, mostly JH II, JH I and little JH III were found, while in later stages only JH II and JH III were found. Titres fluctuated in a similar manner in all larval instars, being lowest during the moults. In last (=6th) instar larvae, JHs disappeared in the late feeding-digging stage and again increased in the early prepupal stage. Parasitisation with Chelonus inanitus, a solitary egg-larval parasitoid which induces in its host the precocious onset of metamorphosis in the 5th instar, did not alter JH homologue composition but led to a disappearance of JHs in the 5th instar. Implantation of a parasitoid larva into early 5th instar larvae containing polydnavirus/venom caused a drop in the JH titre which indicates that the parasitoid larva plays an important role in the manipulation of the host's JH titre. In the parasitoid larva, only JH III was found; titres were highest in the 2nd larval instar, a stage when the host is in the 5th instar and contains almost no JHs. Thus, JHs of the parasitoid and the host fluctuate in an independent manner.

Behavioral response of femaleHelicoverpa (Heliothis)armigera HB. (Lepidoptera: Noctuidae) moths to synthetic pigeonpea (Cajanus cajan L.) kairomone.

FemaleH. armigera moths are highly attracted by a steam distillate from pigeonpea plants, one of their main hosts. A mixture of six compounds, all sesquiterpenes (ß-caryophyllene,α-humulene,α-guajene,α-muurolene,γ-muurolene, andα-bulnesene), mixed in the proportions as found in the steam distillate, elicited the same behavioral responses (oriented upwind flights and contacts with the odor source) as the steam distillate. Onlyα-bulnesene was attractive by itself, but still less than the whole mixture. In addition, the sesquiterpene mixture acts as an oviposition stimulant. Both behavioral responses, orientation and oviposition, are concentration dependent. Electrophysiological recordings from female and male antennae (EAG) showed the same qualitative and quantitative responses to each of the compounds of the sesquiterpene mixture. The EAG responses to the original steam distillate were higher and similar to chickpea kairomonal components, which were also tested. The pigeonpea sesquiterpene mixture and its individual components elicited weak EAG responses only. The response of the male antenna to female-produced pheromone components was in the same range as the pigeonpea steam distillate.

De novo biosynthesis of juvenile hormone III and I by the accessory glands of the male mosquito.

The role of the male accessory glands (MAG) in reproduction was investigated in the mosquito Aedes aegypti. MAG incubated with [14C]acetate synthesized radioactively labeled JH III, JH III bisepoxide and methyl farnesoate. MAG incubated with L-[methyl-3H]methionine synthesized [3H]JH III and a molecule that chromatographed on HPLC with JH I. Analysis of MAG and whole males extract by glass capillary combined gas-chromatography-selected ion monitoring mass spectrometry identified JH III and I as the main analogs that were synthesized by male mosquitoes. MAG of Culex nigripalpus, Anopheles rangeli and Anopheles trinkae also synthesized JH III from L-[methyl-3H]methionine, which indicates that the male mosquito has a complete JH III biosynthetic pathway. Unfed and unmated Culex quinquefasciatus do not develop their ovaries to the resting stage. Females injected with one MAG extract equivalent or implanted with A. aegypti MAG developed their ovaries to the resting previtellogenic stage, whereas females that were injected with saline did not. These results indicate that MAG synthesize and secrete JH III. The corpora allata (CA) of the male Aedes aegypti also synthesize JH III from L-[methyl-3H]methionine. This observation may suggest that JH synthesized by the male's CA is used for internal regulation, whereas JH synthesized by the MAG is transferred with the sperm into the female.

Polycytidylate and poly(7-deazaguanylate): a pair of complementary templates.

We have studied the template-directed oligomerization on polycytidylic acid of the 5'-phosphoro(2-methyl)imidazolides of a number of analogues of guanosine. None of the analogues reacted as efficiently as the original guanosine compound, and only the 7-deazaguanosine analogue gives a detectable yield of oligomers. Similar results are described for a reaction involving the intramolecular template-directed elongation of a short oligocytidylate primer. Oligocytidylates containing five or more cytidylate residues are extended on the single-stranded regions of poly(G). In the present study we show that these oligocytidylates are extended efficiently by reaction with cytidine-5'-phosphoro(2-methyl)imidazolide on a poly(7-deazaguanylic acid) template. The products are considerably longer than those obtained using a polyguanylic acid template. We believe that the formation of a tetrahelix inhibits the latter reaction, while poly(7-deazaguanylate) does not aggregate and, therefore, acts as a more efficient template. This work identifies for the first time a pair of homopolymers each of which facilitates the template-directed elongation of the other.

Single-strand regions of poly(G) act as templates for oligo(C) synthesis.

Oligonucleotide primers consisting of a sequence of four or more deoxycytidylate residues terminated by a single ribocytidylate residue are extended by reaction with cytidine 5'-phosphoro(2-methyl)imidazolide using polyguanylic acid as a template. The efficiency of the reaction decreases as the length of the primer increases. The reaction does not seem to depend on the dissociation of poly(G) tetrahelices but uses as templates single-stranded segments that are already present in enzymatically synthesized polyguanylic acid.

Synthesis of Kdo-alpha-glycosides of lipid A derivatives.

The synthesis of the lipopolysaccharide fragment O-(4,5,7,8-tetra-O-acetyl-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide)-(2-->6)-O-(2-deoxy-2-[(3R)-3- dodecanoyloxytetradecanamido]-4-O- phosphono-3-O-tetradecanoyl-beta-D-glucopyranosyl)-(1-->6)-1-O-acetyl-2- deoxy-2 - [(3R)-3-dodecanoyloxytetradecanamido]-3-O-tetradecanoyl-alpha-D- glucopyranose (35 alpha) is performed via anomeric O-alkylation. With this objective, the 2-azido-3-O-benzyl-2-deoxy-6-O-trifluoromethanesulfonyl-beta-D-glu copyranosides 5, 7, and 19 alpha, beta were synthesized from D-glucal and employed as alkylating agents. Reaction of 5 with the O-cyclohexylidene-protected Kdo-derivative 10 afforded the desired alpha-linked disaccharide, tert-butyldimethylsilyl 4-O-allyl-2-azido-3-O-benzyl-2-deoxy-6- O-(4,5:7,8-di-O-cyclohexylidene-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide)-beta-D-glucopyranoside (11); even better yields of the structurally related disaccharide 12 were obtained with the 4-O-unprotected 7 as alkylating agent. 1-O-Desilylation of 12 furnished the lactol 20, which could be alkylated at the anomeric position with 1-O-allyl protected alkylating agents 19 alpha and 19 beta, both of which furnished exclusively the desired beta-(1-->6)-linked trisaccharides allyl O-(4,5:7,8-di-O-cyclohexylidene-3- deoxy-N-methyl-alpha-D-manno-2-octulopyranosylonamide)-(2-->6)-O-( 2-azido-3- O-benzyl-2-deoxy-beta-D-glucopyranosyl)-(1-->6)-2-azido-3, 4-di-O-benzyl-2-deoxy-alpha- (21 alpha) and -beta-D-glucopyranoside (21 beta), respectively. Phosphorylation with diphenyl phosphorochloridate, replacement of the O-cyclohexylidene protective group by O-triethylsilyl (TES) protective groups, removal of the 1-O-allyl group, azido group reduction, subsequent N-acylation, and then O-acetylation provided the key 1-O-acetyl protected intermediate 30 alpha. Removal of the O-TES groups, subsequent O-acetylation, and hydrogenolytic O-debenzylation furnished O-[4,5:7,8-tetra-O-acetyl-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide]-(2-->6)-O-(2-deoxy-4-O-diphenoxyphospho ryl-2-[(3R)- 3-dodecanoyloxytetradecanamido]-beta-D-glucopyranosyl)-(1-->6)-1-O -acetyl-2- deoxy-2[(3R)-dodecanoyloxytetradecanamido]-alpha-D-glucopyranose (33 alpha), which underwent the required selective O-tetradecanoylation at the 3-O- and 3'-O-position, thus furnishing, after hydrogenolytic O-dephenylation of the diphenoxyphosphoryl group, the target molecule 35 alpha.

Azadirachtin inhibits proliferation of Sf 9 cells in monolayer culture.

Azadirachtin A in ppm quantity inhibits proliferation and monolayer formation of Spodoptera frugiperda (Sf9) insect cells in monolayer culture. The incubated cells demonstrate reduced rates of protein synthesis which finally leads to cell death. This growth inhibiting activity is compared with other botanicals such as ohchinin, salannin and volkensin. The high and specific activity of azadirachtin A on insect cells is discussed in comparison with its effect on a mammalian cell line, based on 2 D PAGE analysis of the total protein contents.

High resolution two-dimensional mapping of tissue-specific polypeptides in the desert locust, Schistocerca gregaria.

High resolution two-dimensional gel electrophoresis (2-DE) using immobilized pH gradients was used to map the tissue-specific polypeptides of the desert locust, Schistocerca gregaria. Highly specific comprehensive 2-DE reference maps ("master gels") were developed for the brain, corpus cardiacum, subesophageal ganglion, and hemolymph. The polypeptides were well resolved within the pH 4-7 range in the first dimension and within the 14-94 kDa molecular mass range in the second dimension.

Biosemiotics--a paradigm of biology. Biological signalling on the verge of deterministic chaos.

Biosemiotics is presented as an interdisciplinary approach to the diversity and irregularity of living systems. Emphasizing the interconnectedness by a multitude of signals as a hallmark of life, it goes beyond the scope of a new discipline. As an overarching concept and a new perspective it should be able to integrate a vast array of biological phenomena which till now appear unrelated or incompatible. The basic tenet is that biology, on all levels from molecular biology to ecosystems, can be viewed and investigated as communication, and that life processes can be defined as sign-mediated interaction. Biology thus is, in itself and in all its aspects, natural semiotics with a pronounced proximity to deterministic chaos. Pervading all of biology, the signal is a viable key and a practically feasible access to the understanding of life.

In vivo 19F nuclear magnetic resonance spectroscopy of trifluorinated neuroleptics in the rat.

In vivo 19F NMR measurements of trifluorinated neuroleptics in the rat brain were made using a 13 x 18 mm surface coil at 4.7 T. The signal of fluphenazine was obtained within 8-15 min from brains of living rats treated chronically with drug doses of 5-30 mg/kg. Following the intravenous injection of a single dose of trifluoperazine (30 mg/kg), brain levels could be monitored with a time resolution of 30 min. The data demonstrate the possibility of obtaining in vivo pharmacokinetics of fluorinated agents in the rat brain. 19F NMR is expected to become an important tool in neurochemical research.

O-alkylation at the anomeric centre for the stereoselective synthesis of Kdo-alpha-glycosides.

O-Alkylation at the anomeric centre of the dianion of 4,5:7,8-di-O-cyclohexylidene-3-deoxy-N-methyl-alpha-D-manno- octulopyranosonamide (1) with several triflates led diastereoselectively to the alpha-glycosides. In this way, lipopolysaccharide building-blocks containing alpha-Kdo-(2----6)-GlcN and alpha-Kdo-(2----6)-beta-GlcN-(1----6)-GlcN moieties were obtained and deployed in the synthesis of decyl 4,5,7,8-tetra-O-acetyl-3-deoxy-N-methyl-alpha-D-manno-2-octulopyranos idonamide (18), 2,3-di-O-tetradecanoyl-D-glycer-1-yl 4,5,7,8-tetra-O-acetyl-3-deoxy-N-methyl- alpha-D-manno-2-octulopyranosid-onamide (22), methyl 2,3,4-tri-O-acetyl-6-O-(methyl 4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D- manno-2-octulopyranosylonate)-alpha-D-glucopyranoside (28), 1-O-acetyl-2-deoxy-6-O-(methyl 4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2- octulopyranosylonate)-2-tetradecanoylamino-alpha-D-glucopyra nose (33), tert-butyldimethylsilyl O-(methyl 4,5:7,8-di-O-cyclohexylidene-3-deoxy-alpha-D- manno-2-octulopyranosylonate)-(2----6)-O-(3,4-di-O-benzyl-2-deoxy- 2- tetradecanoylamino-beta-D-glucopyranosyl)-(1----6)-3,4-di-O-benzyl -2-deoxy-2- tetradecanoylamino-alpha-D-glucopyranoside (36).

Short- and long-term effects of azadirachtin A on development and egg production of Rhodnius prolixus.

Azadirachtin A was given through a blood meal to 4th-instar larvae and to adult females of Rhodnius prolixus. Development (ecdysis) and egg production were inhibited in a dose-dependent manner. Long-term experiments with subsequent four feedings on azadirachtin-free blood were performed with 4th-instar larvae and with adult females. Only in the low-dose azadirachtin larval groups (0.01 and 0.1 microgram/ml of blood), development was partially restored; after a single 1.0 microgram/ml treatment about 50% of the treated larvae were still alive 120 days later without any adult emergence. Similarly fed females had a dose-dependent lower survival and egg deposition rate. The results are discussed in relation to the mode of azadirachtin A action.

Chagas' disease and its insect vector. Effect of azadirachtin A on the interaction of a triatomine host (Rhodnius prolixus) and its parasite (Trypanosoma cruzi).

The ED50 for moulting inhibition by injected azadirachtin A is for fourth instar larvae of all the triatomines, Triatoma vitticepes, T. pseudomaculata, T. maculata, T. brasiliensis, T. lecticularis, T. matogrossensis, T. infestans, Rhodnius prolixus. R. neglectus, R. robustus, Panstrongylus megistus, and P. herrera in the range of 10-25 ng/larva. In Rhodnius prolixus, the survival of T. cruzi was studied after treatment with the drug. If the trypomastigotes were fed in presence of 1.0 microgram azadirachtin A/ml blood, the number of parasites decreased near to the limit of detection within 30 days. If the drug was applied 20 days after T. cruzi infection, it still completely abolished the parasite in the host's gut within the subsequent 20 days. The same holds true if the insect larvae were pretreated with azadirachtin A 20 days before the subsequent infection with T. cruzi. Azadirachtin A. if applied at the dose of 1 microgram/ml, did not affect the hemolytic activity of the crop contents or the proteinase content of the intestine. A parallel between azadirachtin effects on the hormone balance of the host and growth inhibition of the parasite is discussed on the basis of the present results.

The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster.

Using combined gas chromatography/selected-ion mass spectroscopy, the titer of juvenile hormone was determined for whole-body extracts at various morphologically defined stages of the life cycle of Drosophila melanogaster. Only juvenile hormone III (JH-III) was detected. JH-III is present in early metamorphosis but by mid-metamorphosis it is below the level of detection (0.05 pmol/g). It then increases as the pharate adult matures and rises dramatically, beginning just prior to eclosion and reaching 5-7 pmol/g shortly after eclosion. The titer then begins to fall again as the adults mature in both males and females, though the decrease is more rapid in females. Preliminary studies show that low levels of JH-III are present during all the larval instars but are absent from eggs.

Elimination of the allatotropic activity in locusts by microsurgical and immunological methods: evidence for humoral control of the corpora allata, hemolymph proteins, and ovary development.

Transection of the nervous connections (NCC I) between the brain and the corpus cardiacum (group 1) and injection of anti-brain antibodies inhibited vitellogenin synthesis and ovary development in virgin Locusta migratoria females. Removal of the neurosecretory material by these methods changed the titer and the electrophoretic pattern of hemolymph proteins, including vitellogenin. Transection of NCC I at some distance from the brain resulted in bulbs with 30-200 micron diameter which were filled with stainable neurosecretory material (group 2). Oocyte maturation was delayed in group 2 but normal eggs were laid. The same happened if group 1 animals received daily injections of JH-III. It is concluded that a humoral factor is involved in the activation of JH-III biosynthesis. The factor is transported through the NCC I and is released into the hemolymph as allatotropin.

Caste-specific maturation of the endocrine system in the female honey bee larva.

The endocrine system of female honey bee larvae has been studied through postembryonic development with histological and autoradiographic techniques. During larval development, brain and retrocerebral complex proceed from immature cells to an active endocrine system. Caste-specific retardation occurs in the worker during this process. In the developing queen, the differentiation of the neurosecretory cells (NSC) and the outgrowth of their axons occurs from the second instar onward and is nearly completed in the fourth, whereas in the worker larva these processes are delayed by more than one instar. In the queen, RNA synthesis starts in the NSC at the end of the third instar and in the worker at the fifth instar. Stainable neurosecretory material is present only in fifth instar queen larvae. The queen's corpora cardiaca become active at the end of the fourth, those of the worker in the fifth instar. In the corpora allata (CA), nuclei undergo several phases of endomitosis. These phases of polyploidization end at the beginning (queen) or at the end (worker) of the fifth instar respectively. CA volume in the queen is twice that of a worker at its height at the end of larval development. these data demonstrate a caste-specific maturation of the endocrine organs which results in differences in hormone titres.

Regional and subcellular distribution of biopterin in the rat.

Autoradiography: Intravenous (2--14C)-Tetrahydrobiopterin (BH4; 5 muCi, spec. act. 50 Ci/mol) is incorporated into liver and kidney of the rat. The gastrointestinal tract is labelled possibly due to biliary excretion of BH4 and its metabolites. BH4 and its metabolites obviously do not pass the blood brain barrier. Estimation of Crithidia active material shows a regional and subcellular distribution of the coenzyme. The highest levels were found in the liver (4.6 mumol/kg), and in spleen (2.3 mumol/kg). In the brain, highest coenzyme concentration is in the hypothalamic region with 0.49 mumol/kg as compared with 0.25 mumol/kg for total brain value. After subcellular fractionation, biopterins (expressed as Crithidia activity) are located mainly in the soluble fraction of liver (61.3 +/- 6.7) and kidney (89.1 +/- 14.0), whereas subcellular fractions of brain show additional higher percentage distribution in lysosomal (23.4 +/- 3.3) and microsomal (24.9 +/- 5.2) fractions due to their synaptosomal content as shown by density gradient centrifugation (values in brackets: percentage +/- SD).