Allatotropic and allatostatic activities in extracts of brain and the effects of Manduca sexta allatotropin and Manduca sexta allatostatin on juvenile hormone synthesis in vitro by corpora allata from the Eri silkworm, Samia cynthia ricini

Both allatotropic and allatostatic activities were found in crude extracts of brain from adult and larval Eri silkworm, Samia cynthia ricini, but it seems that allatotropic activity dominates in each stage. There was a high level of allatotropic activity in the crude extract of brain from newly emerged female adults, but allatostatic activity appeared in the bioassay when excessive amounts of crude extracts of brain were added. Crude extracts of brain from premoulting fourth‐instar larvae and from newly ecdysed fifth‐instar larvae exhibited allatotropic activities, whereas extracts of brain from the second and third day of the fifth‐instar larvae inhibited juvenile hormone (JH) release slightly. Allatotropic activity from the brains of adults and larvae stimulated both adult and larval corpora allata (CA) to synthesize JH. Manduca sexta allatotropin (AT) (Mas‐AT) and M. sexta allatostatin (AST) (Mas‐AST) also stimulated and inhibited both adult and larval S. cynthia ricini CA to synthesize JH, respectively. Higher concentrations of Mas‐AT (10^?4 or 10^?3 M) showed an inhibitory effect on adult CA. CA from newly emerged female adults were the most sensitive to inhibition by Mas‐AST, whereas CA from female pharate adults at about 6 h before adult emergence were the most sensitive to stimulation by Mas‐AT and S. cynthia ricini brain allatotropic activity. An extract of brain and Mas‐AT induced some of the non‐active female pharate adult CA at 12 h before emergence to synthesize a small amount of JH.     

Allatotropic activity in the suboesophageal ganglia and corpora cardiaca of the adult male loreyi leafworm, Mythimna loreyi

Allatotropic activity was found in the methanolic extract of the suboesophageal ganglia (SOG) and the corpora cardiaca (CC) of the Mythimna loreyi virgin males. No allatotropic activity was observed in the extract of brain or corpora allata (CA). Although CA can be activated by the SOG and CC extract, respectively, CC extract inhibited the response to the SOG extract. A significant in vitro allatotropic effect was exerted by the SOG and CC extract within 10 and 15 min, respectively, and this effect can be sustained for several hours even after transferring to fresh medium without extracts. The time course pattern of the CA activation ratio in both the SOG and CC extract-treated group is very similar to, but with significantly higher level than, that in the control group, suggesting the existence of an intrinsic pacemaker or an in vitro effect that controls the fluctuation of the CA biosynthetic activity. Synthetic Manduca sexta allatotropin had no significant effect on the M. loreyi CA. The results of treatment with the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor IBMX, and the cAMP analogue dibutyryl-cAMP did not indicate that cAMP might be involved in the allatotropic control of CA. Arch.     

Responsiveness of honey bee (Apis mellifera L.) corpora allata to allatoregulatory peptides from four insect species

Five neuropeptides with known allatotropic or allatostatic activity in other insect species were examined for their effects on honey bee corpora allata. Using an in vitro radiochemical assay, we assessed the ability of these peptides to affect the biosynthesis of juvenile hormone III and its immediate precursor methyl farnesoate, as well as their effects on the conversion of methyl farnesoate into juvenile hormone. None of the allatostatins tested affected JH biosynthesis during the last larval instar of honey bee workers. Manduca sexta allatotropin, however, stimulated JH biosynthesis in a stage-specific and dose-dependent manner. Analysis of intraglandular contents of juvenile hormone and its precursor revealed that the allatotropin significantly increased JH precursor but did not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for early fifth-instar worker larvae. Studies also indicated that the allatotropic effect was reversible at the level of methyl farnesoate production.     

Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta

Retrocerebral complexes (RCs) were isolated from adult females of the moths Heliothis virescens and Manduca sexta. Different homologs of juvenile hormone (JH) produced by the isolated RCs were identified and amounts measured by capillary gas chromatography-chemical ionization (isobutane)-mass spectroscopy. Only JH I, II and III were identified. Incubation of RCs from both species in media containing acetate, but no propionate, induced production of approximately equal amounts of JH II and JH III, but the amount of JH I present was very low in all samples. Incubation of RCs with synthetic Manduca sexta allatotropin stimulated significant increases in production of all three homologs but increases in JH I and JH II were greater than those for JH III. The effect of allatotropin was mimicked by addition of propionate to the medium, which indicated that allatotropin increased supply of acetyl- and propionyl-CoA precursors. Incubation of tissue from H. virescens females during the first 24 h after eclosion with synthetic Manduca sexta allatostatin did not reduce production of JH. However, incubation of tissue from 3-day-old females with allatostatin significantly reduced production of JH. Similarly, incubation of tissue from H. virescens females during the first 24 h after eclosion with both allatotropin and allatostatin did not increase JH over the amount present in extracts from tissue incubated without the neuropeptides, indicating that allatostatin negated the action of allatotropin. Incubation of tissue from H. virescens females with allatostatin plus farnesol or JH III acid resulted in significant production of JH III, but neither JH I nor JH II was detected. These findings indicated that allatostatin acts prior to formation of the sesquiterpene alcohol precursors of JH.     

Allatotropic activity in the brain of female Phormia regina (Diptera: Calliphoridae)

In Phormia regina, the rate of juvenile hormone (JH) synthesis rises rapidly after the ingestion of an adequate protein meal. In a previous publication we have localized the neurons containing Manduca sexta allatotropin (Mas-AT)-like substances in the brain of P. regina and demonstrated the allatotropic effect of synthetic Mas-AT in sugar-fed flies in vitro. In this current study, we examined the possible role of the brain of P. regina after the fly received a protein meal. In vitro studies showed that the brain releases, at 8 h after a protein meal, a factor(s) with a strong allatotropic effect. This factor(s) stimulates the corpus allatum (CA) to produce 6.9 times more juvenile hormones (JHs) than the control CA. Time course studies showed that the release of this allatotropic factor(s) is temporally controlled. Only the brains collected from flies at 6 and 8 h after the onset of a liver meal release allatotropic factor(s). Injection of anti-Mas-AT antiserum partially suppressed the fly follicle development in vivo. Presence of anti-Mas-AT antiserum decreased the allatotropic effect of the brain released allatotropic factor(s) in vitro. In addition to a Mas-AT-like substance, it is possible that the brains of liver-fed P. regina females may synthesize other allatotropic factors that are chemically unrelated or partially related to Mas-AT, which cannot be recognized/neutralized by our anti-Mas-AT antiserum.     

Allatotropic and nervous control of corpora allata in the adult male loreyi leafworm, Mythimna loreyi (Lepidoptera: Noctuidae)

Allatotropic activity is found in methanolic extracts of the brain–suboesophageal ganglion (SOG)–corpora cardiaca (CC) complex from virgin males of Mythimna loreyi Duponchel (Lepidoptera, Noctuidae). Corpora allata (CA) from 6‐day‐old virgin males exhibit low rates of release of Juvenile Hormone (JH) acid (JHA) in vitro. Release of JHA can be activated by the addition of an extract of brain–SOG–CC complex in a dose‐dependent manner, and this allatotropic activation can be sustained consistently in the continuous presence of such extracts. Based on its trypsin sensitivity and heat stability, the allatotropic factor is most likely a peptide. The allatotropic activity is dependent on the concentration of calcium ions in the medium, with the highest activation achieved beyond 2 m m . The results of nerve transection experiments suggest that both nervi corporis allati I (NCA I) and NCA II are involved in mediating the allatotropic control of CA in vitro. Isolated CA alone show significantly higher rates of release of JHA than the intact brain–SOG–CC–CA complex during the first 3 h of incubation, but the release of JHA reaches almost the same range in both groups by the end of the fourth hour of incubation.     

Assay of nicotinamide deamidase activity using high-performance liquid chromatography

A rapid, simple and reproducible method has been developed for the determination of nicotinamide deamidase activity using high-performance liquid chromatography (HPLC). Nicotinic acid (NA) liberated from nicotinamide (NAA) after a 15-min enzyme reaction was determined directly by HPLC without further separation steps. Both NA, the product, and NAA, the substrate were separated by reversed-phase ion-pair isocratic chromatography and detected at 261 nm. The present method could be applied to the measurement of deamidase activity in crude cell extracts prepared from several bacterial strains. The Michaelis constant of nicotinamide deamidase in Enterobacter agglomerans was 36 μM for NAA. This method is useful for the measurement of nicotinamide deamidase from various sources.     

Biochemical and molecular characterization of allatotropin and allatostatin from the Eri silkworm, Samia cynthia ricini

In a previous study, allatotropic and allatostatic activities were observed in brain extract from the Eri silkworm, Samia cynthia ricini (Samcri) [Li, S., Jiang, R.-J., Cao, M.-X., 2002b. Allatotropic and allatostatic activities in brain extracts of the Eri silkworm, S. cynthia ricini, and the effects of Manduca sexta allatotropin and M. sexta allatostatin on juvenile hormone in vitro. Physiol. Entomol. 27, 322-329]. In the present study, the HPLC purified Samcri-allatotropin (AT) and -allatostatin (AST) factors were shown to have the same retention time as those of M. sexta (Manse)-AT and -AST, respectively. Moreover, the amino acid sequences of mature Samcri-AT and -AST deduced from their encoding cDNAs are identical to the Manse-AT and -AST amino acid sequences. Both Samcri-AT and -AST genes were expressed in brain, nerve cord, and midgut, with Samcri-AT also detected in gonads and epidermis, suggesting their pleiotropic physiological functions. The expression levels of Samcri-AT and -AST genes correlated well with the allatoregulatory activities during the period of adult emergence indicating the two peptides tightly control JH synthesis, in a contradictive and cooperative manner. Our biochemical and molecular data of Samcri-AT and -AST and other studies demonstrate that these two peptides regulate JH synthesis by corpora allata in Lepidoptera and have pleiotropic physiological effects.     

A method for determination of iododeoxyuridine substitution of thymidine using reversed-phase high-performance liquid chromatography

An assay for thymidine substitution by iododeoxyuridine (IdUrd) using reversed-phase high-performance liquid chromatography (HPLC) has been developed. Three principal steps in this procedure are: extraction of DNA from cell or tissues, hydrolysis of DNA into deoxynucleosides and separation using HPLC. Approximately 1 microgram of DNA was recovered from 10(5) cells by phenol extraction, and subjected to hydrolysis into deoxynucleosides which required a three-stage DNA digestion using enzymes DNAse I. phosphodiesterase I and alkaline phosphatase. The deoxynucleosides were separated on the Microsorb C18 column with isocratic elution; 90-100% of the DNA was recovered as deoxynucleosides on the column. The method was used to determine quantitatively the percent IdUrd substitution of thymidine in Chinese hamster lung cells in vitro and BA1112 rhabdomyosarcoma in WAG/Rij rats perfused with IdUrd. It was possible to determine the thymidine substitution by IdUrd as small as 1% using a few micrograms of DNA. The close correspondence between the percent substitutions determined by HPLC and those determined by radioactive assay using [125I]-labelled IdUrd, confirmed the accuracy of our HPLC method. The HPLC analysis is especially suitable for the determination of percent IdUrd substitution of thymidine in tissue biopsies from animals used in in vivo experiments or humans undergoing radiation treatment.     

Allatotropin regulation of juvenile hormone synthesis by the corpora allata from the lubber grasshopper, Romalea microptera

The in vitro synthesis of juvenile hormone (JH) by corpora allata (CA) from the lubber grasshopper (Romalea microptera) was stimulated by low concentrations of brain extract and this effect was reduced at higher concentrations, suggesting the presence of allatotropin (AT) and allatostatin (AST) factors in the brain. The AT activity of brain extracts caused a rapid and reversible stimulation and appeared to be a peptide(s). Reversed phase (C18) HPLC analysis of brain extracts disclosed two peaks of AT activity but no significant AST activity. Manse-AT, Schgr-NPF, and Locmi-FLRF had no effect on JH synthesis by lubber CA, indicating that the Rommi-AT factors are distinct from these peptides. High concentrations of Dippu-AST-7 and Grybi-AST-1 inhibited JH synthesis, implying that AST factors might be present in lubber grasshoppers. CA response to AT activity of brain extracts varied during the oviposition cycle ( approximately 35 days), with the maximum response occurring on days 16-18. AT activity of brain extracts also varied during the cycle, being highest on day 25. Our data suggest that the lubber CA is largely regulated by AT activity, and that JH synthesis reflects both CA response to AT activity and the level of AT activity in the brain.     

Determination of glutamate decarboxylase by high-performance liquid chromatography

An improved method for the determination of glutamate decarboxylase (GAD) activity is described. The enzyme was evaluated by incubation with glutamic acid (l-Glu) in the presence of pyridoxal 5 ′-phosphate (PLP): the γ-aminobutyric acid (GABA) formed was derivatized to PTC-GABA; the latter was subsequently separated and assayed by isocratic HPLC (LiChrospher RP-18 column; isocratic elution with pH 5.8 acetate buffer in acetonitrile-water) with UV absorbance detection at 254 nm. The method described is a sensitive, reproducible and specific assay useful for following variations of GAD activity in vitro; this assay was subsequently used for the evaluation of GAD activity variations after irradiation with low doses of HeNe laser radiation.     

Effect of the brain and corpus cardiacum on egg production in Rhodnius prolixus

Using total egg production corrected for size of blood meal as an index of the activity of the corpus allatum (CA), the effects of various surgical manipulations of the neuroendocrine system have been examined. Isolation of the CA from its nervous connections increases egg production well beyond that of a normal insect, thus confirming that the CA is at least partly controlled by inhibitory nerves from the brain. Removal of the corpora cardiaca (CC) reduces the level of this increased egg production, and decapitation anterior to the CC results in a level of egg production that is greater than that found in females decapitated between the CA and CC. Implanting a CC together with a CA into a decapitated female results in a higher egg production than implanting a CA alone. These results demonstrate that an allatotropic influence is exerted by the CC. Experiments designed to examine the role of the brain were inconclusive and did not eliminate the possibility that the allatotropin from the CC originated in the brain.     

Allatotropin, leucokinin and AKH in honey bees and other Hymenoptera

In the honey bee no allatotropin gene has been found, even though allatotropin stimulates the synthesis of juvenile hormone in this species. We report here that honey bees and other Hymenoptera do have a typical allatotropin gene, although the peptides predicted have a somewhat different structure from that of other insect allatotropins. Polyclonal antisera to honey bee allatotropin reacted with material in the neurohemal organs of the segmental nerves of abdominal ganglia. We were unable to find the allatotropin peptide using mass spectrometry in extracts from these tissues. Thus the expression of this gene in honey bees is less important than in other insect species. We also characterized the leucokinin gene which similarly appears to be very weakly expressed in worker honey bees. Unlike the allatotropin gene, which is conserved within Hymenoptera, the leucokinin gene is much more variable in structure and was not found in ants nor the parasitic wasp Nasonia vitripennis. The absence of significant expression of adipokinetic hormone (AKH) in the honey bee may be due to the existence of a second TATA box in the promotor region of the gene, which explains the production of an mRNA encoding a putative peptide precursor from which no AKH should be released. Such a second TATA box was not found in other Hymenoptera, and may therefore be specific for the two Apis species. It is suggested that functional disintegration of this important metabolic gene became possible in Apis because of the highly evolved social nature of the species.     

Immunolocalization and possible effect of a moth allatotropin-like substance in a fly, Phormia regina (Diptera: Calliphoridae)

Insect allatotropin upregulates the biosynthesis of juvenile hormones by the corpus allatum. We raised two rabbit antisera against the allatotropin of Manduca sexta (Mas AT) using a synthetic, multiple-antigenic-peptide that contains a branching heptalysine core and eight Mas AT molecules. Both antisera recognized specifically the same neurons in the larval brain, frontal ganglion and terminal abdominal ganglion of M. sexta as previously reported by others. Immunoassay showed reactivity specific to the Mas AT. Very low or nearly no cross-reactivity was found for two Mas AT-like peptides, a myotropin from Locusta migratoria and a Mas AT-like peptide deduced from the DNA sequence of Aedes aegypti, respectively. Immunopositive neurons also were identified in adult Phormia regina, Dacus dorsalis, Oncopeltus fasciatus, and Mythimna loreyi, and in larval M. loreyi, Bombyx mori, and Andraca bipunctata. At 20 pmol per 25 μl incubation medium (i.e. 8x10(-7) M), synthetic Mas AT significantly stimulated in vitro juvenile hormone biosynthesis by the corpus allatum of adult, sugar-fed females of P. regina to 2.64-fold that of controls. Thus, this study provides the first demonstration that at the higher end of the physiological concentration range, the Mas AT has allatotropic effect in vitro to CA of non-lepidopterans. However, in vivo functions of Mas AT and/or Mas AT-like peptide in P. regina remain to be defined.     

Assay of soluble guanylate cyclase activity by isocratic high-performance liquid chromatography

A HPLC method alternative to labelled or unlabelled procedures was developed for the assay of guanylate cyclase (GC) activity. The substrate (GTP) and the product (cGMP) of the enzymatic reaction were separated in the isocratic mode on a μBondapak C₁₈ column. The activity of GC was linearly dependent on the amount of cGMP produced in the presence of sodium nitroprusside. This approach was applied to follow the purification of GC from bovine lung and to evaluate its stability in different storage conditions.     

The role of allatostatic and allatotropic neuropeptides in the regulation of juvenile hormone biosynthesis in Lacanobia oleracea (Lepidoptera: Noctuidae)

In the sphinghid moth Manduca sexta, two allatoactive neuropeptides appear to be responsible for regulating juvenile hormone (JH) production by the corpora allata (CA). These peptides (M. sexta allatostatin, Mas-AS, and M. sexta allatotropin, Mas-AT) respectively inhibit and stimulate in vitro JH biosynthesis by CA in this insect. However, although Mas-AS inhibits CA in both larval and adult insects, Mas-AT is active only in adult M. sexta. The situation in other lepidopteran species is less clear-cut and, although both peptides have been detected (usually by immunologic and/or molecular techniques) in several other moths (including noctuids), their function as regulators of JH production remains uncertain. In the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae), we have previously demonstrated the occurrence of Mas-AS and/or Mas-AT in extracts of CA, brain and other organs, and have shown that both peptides are present in larval and adult forms. However, in L. oleracea, although Mas-AS inhibits larval and adult CA in vitro, it does so only at relatively high concentrations, and to a maximum of only approximately 70%. By contrast, Mas-AT (which is also present in larval and adult L. oleracea) stimulates larval and adult CA, but is substantially more potent ( approximately 100 fold) than the allatostatin. In this paper we present the results of paired, concurrent measurements (using ELISA) of levels of Mas-AS and Mas-AT in brains, CA and hemolymph (plasma and hemocytes) of L. oleracea at times when there are marked changes in JH titers. We also present data on the in vitro rates of JH biosynthesis by isolated CA, and on hemolymph JH esterase activity measured at the same critical developmental times, and discuss all of these data in relation to the putative allatoregulatory roles of the M. sexta allatotropic and allatostatic neuropeptides in L. oleracea.     

Purification of 125I-labeled succinyl cyclic nucleotide tyrosine methyl esters by high-performance liquid chromatography

Carrier free 125I-labeled succinyl cyclic adenosine monophosphate (ScAMP) and succinyl cyclic guanosine monophosphate (ScGMP) tyrosine methyl esters (TME) were purified by reversed phase high-performance liquid chromatography (HPLC) or descending paper chromatography. Using an isocratic buffer for HPLC, mono-ScAMP-125I-TME and mono-ScGMP-125I-TME were eluted from a C18 column at 8.9 and 6.9 min, respectively. Both of the mono-iodinated radioligands were completely separated from their noniodinated precursors and other iodinated products. The radioligands purified by HPLC or paper chromatography were used for the radioimmunoassay (RIA) of cAMP and cGMP. Cyclic AMP or cGMP inhibited binding of the HPLC purified radioligands at three- to fivefold lower concentrations than the paper chromatography purified radioligands. The sensitivity of the RIA decreased with time if paper chromatography purified radioligands were used, but remained stable for 4 months if the HPLC purified compounds were used, even with storage at 4 degrees C. We attribute these results to better purification of radioligands by the HPLC than by the paper chromatography. Using optimal conditions the HPLC method takes only 10 min and results in a high yield (greater than 95%) of added 125I into the monoiodinated products.     

A new high-performance liquid chromatography assay for glutamine synthetase and glutamate synthase in plant tissues

Glutamic acid and glutamine, formed in plant tissue extracts by glutamate synthase and glutamine synthetase, respectively, were separated by derivatization with o-phthaldialdehyde followed by reverse-phase high-performance liquid chromatography on a μBondapak C₁₈ column. The derivatives were eluted by isocratic elution, and the mobile phase was a 20 mm sodium phosphate buffer (pH 6.8) with 36% methanol. The procedure is rapid, sensitive, and requires a minimum sample.     

The biological and immunological properties of fractionated atrial extracts from young and old rats

We have previously reported that the biological activity of rat atrial extract declines with age. The present study was undertaken to further evaluate the natriuretic, hypotensive and immunological properties of fractionated and HPLC purified atrial extracts prepared from young and old rats. Acetic acid extracts were prepared and subsequently fractionated by gel permeation chromatography. The high (greater than 10,000 daltons) and low (less than or equal to 10,000 daltons) molecular weight fractions were collected, lyophilized and assayed. Radioimmunoassay competitive binding curves of the initial and fractionated extracts were parallel (p greater than 0.05) to the synthetic ANP standard. No differences in parallelism (p greater than 0.05) were observed in the natriuretic activity of the initial extracts, the low molecular weight (LMW) fractions from both age groups, the 290 day high molecular weight (HMW) fraction or the synthetic ANP standard. However, the natriuretic activity of the 15 day HMW fraction was significantly attenuated compared to the other treatment groups (p less than 0.05). The initial 15 day extract was also significantly more hypotensive than the 290 day extract (p less than 0.05). HMW extracts were subjected to HPLC and the resulting immunoreactive ANP peak was reassayed. Based on SDS-PAGE and immuno blot analysis, the HPLC purified fraction was found to contain only immunoreactive proANP. Subsequent bioassay revealed greater hypotension and reduced natriuretic activity in the 15 day proANP fraction in comparison to a similarly prepared extract from older animals. Thus, we conclude that qualitative differences in the biological properties of atrial extracts may be ascribable to age-related changes in the composition of proANP or to other undefined biologically active atrial substance(s).     

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C₁₈ column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ³-trans-hexadecenoic acid.