[Show abstract][Hide abstract] ABSTRACT: The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography.
Philosophical Transactions of The Royal Society B Biological Sciences 07/2014; 369(1647). DOI:10.1098/rstb.2013.0497 · 7.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Farnesoic acid (FA) and methyl farnesoate (MF) are juvenile hormone-related compounds secreted by the mandibular organ (MO) of crustaceans and play an important role in stimulation of ovarian maturation. To better understand how the MO activity influences female reproduction by secretion of FA and MF, the biosynthesis and release of these two compounds were measured in vitro by the incorporation of l-[(3)H-methyl]methionine into MF and [2-(14)C]acetate into FA by the MO of Homarus americanus. The production of FA is 7.5 times that of MF, and most FA and MF synthesized remained within the gland, and was not released into the surrounding medium. Most FA and MF were synthesized in the anterior fan-fold region of the MO. The rates of biosynthesis of FA and MF were stage-related, with maximal production occurring during secondary vitellogenesis (i.e. stages 4 and 5). A potential juvenoid receptor, retinoid X receptor (RXR), HaRXR, was characterized using PCR cloning techniques. HaRXR belongs to the nuclear hormone receptor superfamily and its deduced amino acid sequence shares a high homology to other RXRs of crustaceans, insects, and vertebrates. Transcripts of HaRXR can be detected in many tissues, and significant high expression level was detected in the MO, especially in the anterior fan-fold region. Expression of HaRXR was also related to reproductive stage, and maximal level of expression was observed at stage 4, in which secondary vitellogenesis is occurring. Changes in transcript level of HaRXR and the rates of FA/MF biosynthesis in the female reproductive cycle indicate that HaRXR and FA/MF may play important roles in crustacean reproduction.
General and Comparative Endocrinology 11/2011; 175(2):259-69. DOI:10.1016/j.ygcen.2011.11.016 · 2.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two genes coding for enzymes previously reported to be involved in the final steps of juvenile hormone (JH) biosynthesis in different insect species, were characterised in the desert locust, Schistocerca gregaria. Juvenile hormone acid O-methyltransferase (JHAMT) was previously described to catalyse the conversion of farnesoic acid (FA) and JH acid to their methyl esters, methyl farnesoate (MF) and JH respectively. A second gene, CYP15A1 was reported to encode a cytochrome P450 enzyme responsible for the epoxidation of MF to JH. Additionally, a third gene, FAMeT (originally reported to encode a farnesoic acid methyltransferase) was included in this study. Using q-RT-PCR, all three genes (JHAMT, CYP15A1 and FAMeT) were found to be primarily expressed in the CA of the desert locust, the main biosynthetic tissue of JH. An RNA interference approach was used to verify the orthologous function of these genes in S. gregaria. Knockdown of the three genes in adult animals followed by the radiochemical assay (RCA) for JH biosynthesis and release showed that SgJHAMT and SgCYP15A1 are responsible for synthesis of MF and JH respectively. Our experiments did not show any involvement of SgFAMeT in JH biosynthesis in the desert locust. Effective and selective inhibitors of SgJHAMT and SgCYP15A1 would likely represent selective biorational locust control agents.
[Show abstract][Hide abstract] ABSTRACT: Immunoreactivity to cockroach Diploptera punctata allatostatin-7 (Dippu AST-7) has been demonstrated previously in axons innervating the corpora allata of the termite Reticulitermes flavipes. This peptide and Dippu AST-11 inhibited juvenile hormone (JH) synthesis by corpora allata (CA) of brachypterous neotenic reproductives (secondary reproductives) of termites. The present study shows that R. flavipes CA are also inhibited by Dippu AST-2, AST-5, AST-8, and AST-9 at approximately the same rank order of potency as demonstrated in D. punctata. Another allatostatin from Periplaneta americana (Peram AST-12) also inhibits JH synthesis by R. flavipes CA. Sensitivity to the allatostatins is higher in glands with low rates of JH synthesis than in those with relatively high JH synthetic rates as has been demonstrated in CA from male and female secondary reproductives as well as in those from non-egg-laying and egg-laying females. The identical inhibitory effects of R. flavipes brain extract on CA from both D. punctata and R. flavipes and the isolation and identification of five cockroach allatostatins (Dippu AST-1, AST-2, AST-5, AST-8, and Peram AST-12) from termite brain extract reflect the close relationship between cockroaches and termites.
[Show abstract][Hide abstract] ABSTRACT: In the subterranean termite Reticulitermes flavipes, allatostatins (ASTs) with the C-terminus Phe-Gly Leu-amide were localized by immunocytochemistry with antibody against a cockroach AST, Dippu AST-7. AST-immunoreactivity occurred in the corpus cardiacum and corpus allatum and in the lateral and medial neurosecretory cells of the brain that innervate these organs as well as in many other nerve cells of the brain. This was observed in workers, nymphs, soldiers and secondary reproductives. A radioimmunoassay, using anti-Dippu AST-11, demonstrated about 40 fmole equivalents of AST in brains of soldiers and secondary reproductives. The product of the corpora allata in this species was determined to be juvenile hormone III. Its synthesis by corpora allata of secondary reproductives, determined by in vitro radiochemical assay, was inhibited in a dose-dependent fashion by two cockroach allatostatins, Dippu AST-7 and Dippu AST-11. Thus, as in cockroaches and crickets, allatostatin-containing nerves innervate the corpora allata of this termite species and their production of juvenile hormone is inhibited by these neuropeptides.
[Show abstract][Hide abstract] ABSTRACT: In the subterranean termite Reticulitermes flavipes, allatostatins (ASTs) with the C-terminus Phe-Gly Leu-amide were localized by immunocytochemistry with antibody against a cockroach AST, Dippu AST-7. AST-immunoreactivity occurred in the corpus cardiacum and corpus allatum and in the lateral and medial neurosecretory cells of the brain that innervate these organs as well as in many other nerve cells of the brain. This was observed in workers, nymphs, soldiers and secondary reproductives. A radioimmunoassay, using anti-Dippu AST-11, demonstrated about 40 fmole equivalents of AST in brains of soldiers and secondary reproductives. The product of the corpora allata in this species was determined to be juvenile hormone III. Its synthesis by corpora allata of secondary reproductives, determined by in vitro radiochemical assay, was inhibited in a dose-dependent fashion by two cockroach allatostatins, Dippu AST-7 and Dippu AST-11. Thus, as in cockroaches and crickets, allatostatin-containing nerves innervate the corpora allata of this termite species and their production of juvenile hormone is inhibited by these neuropeptides. (c) 2005 Elsevier Ltd. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.
European Journal of Biochemistry 08/2002; 269(14):3587-95. DOI:10.1046/j.1432-1033.2002.03048.x · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Measurement of juvenile hormone (JH) production using the radiochemical assay (RCA) for JH biosynthesis and release is usually a reliable and precise technique. However problems with radiolabeled precursors and misunderstanding of the data, the techniques and the calculations have contributed towards uncertainty with respect to published experimental results. Problems with the purity of [methyl-3H]-methionine or determination of its specific radioactivity have had detrimental effects on the reliability of results using the RCA. Proper control procedures and the use of 14C/3H-double-label RCA can be useful in detecting irregularities in the experimental results, and in determining contributing factors to any problems. The use of [methyl-14C]-methionine and an awareness of normally expected RCA values can also assist the researcher in checking the validity of results. The radiolabeled methyl moiety of methionine is incorporated into JH without discrimination relative to unlabeled methyl methionine, by the o-methyl transferase. However unexpected preferential incorporation of the [methyl-14C]- vs. [methyl-3H]-moiety into JH occurs, but is only evident at concentrations of radiolabeled methionine outside the normal range of the RCA. Changes in radioactive precursor formulation have no effect on the RCA.
[Show abstract][Hide abstract] ABSTRACT: Corpora allata of the ring-legged earwig, Euborellia annulipes, produce and secrete juvenile hormone III (JH III). The primary degradation product of JH III in vitro by diluted hemolymph was juvenile hormone acid, as assessed by thin-layer and high-performance liquid chromatography. This result was confirmed by derivatization of the degradation product using diazomethane. Conversion of JH III to JH acid by E. annulipes hemolymph in vitro occurred at a constant rate for at least 4 hr. The addition of 10−6 M O-ethyl-S-phenyl phosphoamidothiolate or, alternatively, 3-octyl thio-1,1,1-trifluoropropan-2-one inhibited hemolymph esterase activity; O,O-diisopropylphosphorofluoridate and ethanol alone had no effect on the conversion of JH III to JH acid. At 10−4 M, each of the selected inhibitors reduced hemolymph esterase activity relative to ethanol-treated and untreated controls. JH esterase activity of diluted hemolymph was relatively high in females with basal follicles < 0.2 mm and low in those with larger basal follicles. The change in JH esterase activity of the hemolymph was not correlated with changes in hemolymph protein content; hemolymph protein content was generally 50–100 μg/μ1 and did not change significantly with either age or basal follicle length.
Comparative Biochemistry and Physiology Part C Comparative Pharmacology and Toxicology 06/1996; 114(2). DOI:10.1016/0742-8413(96)00035-7
[Show abstract][Hide abstract] ABSTRACT: Corpora allata of adult female Euborellia annulipes, incubated in medium containing 3H-methionine, synthesized and released juvenile hormone III. Labelled material co-migrating with methyl farnesoate was also found, suggesting this as an intermediate in the pathway of juvenile hormone III production. Juvenile hormone was not appreciably stored in the glands, but was released into the medium. In normal medium, 93.6 ± 1.6% of the total juvenile hormone III synthesized was released and 96.5% ± 0.3 in medium supplemented with 60 μM farnesoic acid. The rate of juvenile hormone III biosynthesis/release in vitro remained constant for at least 8 hr for glands of different activities. The rate of juvenile hormone production was closely correlated with the gonadotrophic cycle. In females with previtellogenic ovarian follicles (0.26 ± 0.004 mm), hormone production was only 0.59 ± 0.13 fmol hr−/corpus allatum; production increased to 1.52 ± 0.25 fmol hr−1/corpus allatum when basal follicles were growing rapidly, and remained high during the period of oviposition. By 3 days following oviposition when females were brooding clutches, hormone production had declined to 0.46 ± 0.13 fmol hr−1/corpus allatum. The addition of 60 μM farnesoic acid to the medium enhanced juvenile hormone biosynthesis at each stage examined. Lastly, elevating the level of l-methionine in the medium also enhanced hormone biosynthesis. Maximal hormone production was 32.8 ± 10.9 fmol hr−1/corpus allatum, at an l-methionine concentration of 51 μM.
Comparative Biochemistry and Physiology Part C Comparative Pharmacology and Toxicology 03/1995; 110(3-110):241-251. DOI:10.1016/0742-8413(95)00016-H
[Show abstract][Hide abstract] ABSTRACT: The O-methyltransferase, which is responsible for the methylation of farnesoic acid in the corpora allata of Diploptera punctata, is a cytosolic enzyme. The activity of O-methyltransferase closely parallels JH biosynthesis in last instars and adult females. Because allatostatin 4 (AST 4) from D. punctata and callatostatin 5 (CAST 5) from Calliphora vomitoria can inhibit juvenile hormone biosynthesis, their effects on the activity of O-methyltransferase and epoxidase, the enzymes involved in the final two steps of juvenile hormone biosynthesis, were investigated in vitro. AST 4 can inhibit methyltransferase activity whereas CAST 5 stimulates it. AST 4 inhibits epoxidase activity slightly whereas CAST 5 inhibits it significantly (36%). Treatment of corpora allata with farnesoic acid (40 μM) can reverse the inhibitory effect of AST 4 and CAST 5 on JH release by corpora allata. Thus, allatostatins appear to exert their inhibitory effect on JH biosynthesis at least partially through inhibition of the activity of terminal enzymes. Two biosynthetic pathways for the conversion of farnesoic acid to JH may exist in corpora allata of D. punctata: the predominant pathway is farnesoic acid to methyl farnesoate, then to JH whereas the other, representing about 5–10% of total JH production, is farnesoic acid to JH III acid, then to JH.
[Show abstract][Hide abstract] ABSTRACT: The release of juvenile hormones (JH) by female and JH acids (JHA) by male corpora allata (CA) of Pseudaletia unipuncta was monitored in vitro using a radiochemical assay. Separation and quantification of the homologues was accomplished by HPLC and liquid scintillation counting of collected fractions. At emergence, CA of females maintained at 25°C, 16 h light:8 h dark (16L:8D) released only trace amounts of JH. On days 1–2, JH III remained virtually undetectable, but similar, low quantities of JH I and JH II were released. Between days 3 and 7, an increase in the release of each homologue was observed, the largest being for JH II. Similarly, the amount of JH released by CA of females maintained at 10°C, 12L:12D, increased as a function of age up to 25 days following emergence, with JH II again being the most abundant homologue. However, even after 25 days, the levels observed at 10°C were considerably less than those seen at 25°C. Under conditions of 25°C, 16L:8D, males exhibited a pattern of JHA release similar to that described for females, with the exception that JHA I rather than JHA II was generally the dominant homologue. CA from male moths held for 5–25 days at 10°C, 12L:12D, released little JHA III, but comparatively high and similar amounts of JHA I and II. However, contrary to the continuous age-related increase seen in females, JHA release peaked on day 10 and subsequently declined. We discuss these results in the context of ovarian development, pheromone production, calling behaviour and migration in armyworm females, and we argue that JH or JH acid regulates the maturation of the pheromonal communication system in males.
[Show abstract][Hide abstract] ABSTRACT: Five neuropeptides with C-terminal amino acid sequence homology to cockroach allatostatins have been identified in the blowfly Calliphora vomitoria. Three have the same pentapeptide C-terminal amino acid sequence as allatostatin 1 of the cockroach Diploptera punctata. A hexadecapeptide designated callatostatin 1, isolated from thoracic ganglia, brains, and heads, has the sequence Asp-Pro-Leu-Asn-Glu-Glu-Arg-Arg-Ala-Asn-Arg-Tyr-Gly-Phe-Gly-Leu-NH2. Callatostatins 2 and 3 have been isolated from heads and thoracic ganglia, respectively; they comprise the last 14 and 8 residues of callatostatin 1. Callatostatin 4, isolated from thoracic ganglia, has the sequence Xaa-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2, where Xaa is either Asp or Asn. This peptide, with a serine substitution for glycine at position 5, has a C-terminal pentapeptide sequence identical to that of allatostatins 3 and 4 of D. punctata. Callatostatin 5, with the sequence Gly-Pro-Pro-Tyr-Asp-Phe-Gly-Met-NH2, was identified from whole flies. All five peptides inhibit juvenile hormone production by the corpora allata of D. punctata in vitro. Callatostatin 5 was the most potent allatostatin so far tested in this species, with maximum inhibition occurring at 1 nM. In contrast, none of the callatostatins or the allatostatins showed allatostatic activity in mature female C. vomitoria when tested at concentrations of 100 to 0.1 microM. In accordance with these results, immunoreactivity to an antiserum directed against the common C terminus of callatostatin 1 and allatostatin 1 was observed in the corpora allata of D. punctata but not in the corpus allatum of C. vomitoria, despite its presence in neurons of the brain. Neurons in the thoracic ganglion of C. vomitoria that are immunoreactive against this antiserum project to the hindgut, rectum, rectal papillae, and oviduct, suggestive of a function different from that of a true allatostatin.
Proceedings of the National Academy of Sciences 04/1993; 90(6):2456-60. DOI:10.1073/pnas.90.6.2456 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an effort to identify the signal transduction mechanism associated with the inhibition of juvenile hormone (JH) biosynthesis by the neuropeptides allatostatins, levels of the cyclic nucleotides cAMP and cGMP were measured in corpora allata (CA) of virgin and mated Diploptera punctata females using radioimmunoassays. Treatment of isolated CA with varying concentrations of synthetic allatostatins 1, 2, 3 or 4 did not elicit significant changes in the levels of either cAMP or cGMP in any of the test glands, suggesting that these compounds do not act as second messengers for the four allatostatins tested. Simultaneous treatment of CA with allatostatin 4 and the adenylate cyclase activator forskolin did not increase the degree of inhibition of juvenile hormone biosynthesis relative to that obtained with forskolin (5 or 50 microM) alone. We interpret these results as lending further support to the suggestion that cyclic nucleotides do not play a role in the signal transduction of allatostatins 1-4 in cockroach CA.
[Show abstract][Hide abstract] ABSTRACT: Use of enkephalinase inhibitor phosphoramidon in the in vitro radiochemical assay for juvenile hormone biosynthesis enhanced allatostatin-mediated inhibition of hormone production by corpora allata of the cockroach, Diploptera punctata. Significant increases in inhibition in day 2 virgin female CA by AST 1 (at 10(-7) M) and AST 4 (10(-8)-10(-7) M) were observed in the presence of phosphoramidon (10(-5) M or greater). No significant increase in inhibition were seen in CA from day 6 mated females with AST 4 (10(-9)-10(-7) M) and phosphoramidon combined. Phosphoramidon alone had no effect on JH biosynthesis. Analysis of allatostatin content of the CA, as determined by ELISA, revealed that addition of phosphoramidon to the medium increased the endogenous allatostatin content in CA of virgin and mated females. The similarity in primary structure between allatostatins and enkephalin-like peptides and their similar distribution makes it probable that phosphoramidon acts by preventing breakdown of allatostatins within the CA.
[Show abstract][Hide abstract] ABSTRACT: The principal juvenile hormone produced by the corpus allatum of the adult blowfly Calliphora vomitoria has been identified as juvenile hormone bisepoxide. The precursor compounds, methyl farnesoate and juvenile hormone III are also synthesized and released from the isolated gland in vitro, but in small amounts (approx. 1% of the total radiolabelled products of the gland). In females given unrestricted access to sugar, water and meat immediately after eclosion, the rate of juvenile hormone bisepoxide production rises with the initiation of oöcyte development, reaches high levels early in the first gonadotropic cycle and remains consistently high through successive cycles. Mature males on the same diet also produce juvenile hormone bisepoxide at high rates. Flies restricted to a sugar-and-water-only diet synthesize much lower levels of juvenile hormone bisepoxide (less than 50% of meat-fed flies). Partially-purified brain extracts of C. vomitoria contain material that is immunoreactive in an ELISA against the inhibitory neuropeptide, allatostatin 1, of the cockroach Diploptera punctata. This material is able to inhibit significantly the biosynthesis of juvenile hormone bisepoxide in flies, and also has strong inhibitory effects on juvenile hormone III biosynthesis by cockroach corpora allata in vitro. Cockroach allatostatins 1–4 at concentrations of 10−4–10−7 M have no significant effect on juvenile hormone bisepoxide release in the blowfly. Also tested at the same concentrations, and shown to have no significant effects on the synthesis and release of juvenile hormone-related compounds in either the blowfly or the cockroach were the vertebrate peptide Met5-enkephalin-Arg6-Gly7-Leu8 (YGGFMRGL) and its carboxyamidated analogue (YGGFMRGL-NH2). Similarly, the FMRFamide-related peptides, calliFMRFamides 1 and 5 and their non-amidated analogues, at concentrations of 10−4–10−6 M, had no effect on juvenile hormone bisepoxide biosynthesis and release. Forskolin (5 × 10−5 M) and 8-bromo-cAMP (10−4 M) appeared not to have an inhibitory influence on juvenile hormone bisepoxide release in the blowfly, suggesting that cAMP may not be the intracellular second messenger in this species. A presumed precursor of juvenile hormone bisepoxide, farnesoic acid, did not stimulate and increased production of juvenile hormone bisepoxide, but resulted in an increase in juvenile hormone III production.
[Show abstract][Hide abstract] ABSTRACT: Use of the enkephalinase inhibitor phosphoramidon in the in vitro radiochemical assay for juvenile hormone biosynthesis enhanced allatostatin-mediated inhibition of hormone production by corpora allata of the cockroach,Diploptera punctata. Significant increases in inhibition in day 2 virgin female CA by AST 1 (at 10–7 M) and AST 4 (10–8–10–7 M) were observed in the presence of phosphoramidon (10–5M or greater). No significant increases in inhibition were seen in CA from day 6 mated females with AST 4 (10–9–10–7M) and phosphoramidon combined. Phosphoramidon alone had no effect on JH biosynthesis. Analysis of allatostatin content of the CA, as determined by ELISA, revealed that addition of phosphoramidon to the medium increased the endogenous allatostatin conten in CA of virgin and mated females. The similarity in primary structure between allatostatins and enkephalin-like peptides and their similar distribution makes it probable that phosphoramidon acts by preventing breakdown of allatostatins within the CA.
Cellular and Molecular Life Sciences CMLS 07/1992; 48(8):758-761. DOI:10.1007/BF02124297 · 5.81 Impact Factor