Regulation of methyl farnesoate production by mandibular organs in the crayfish, Procambarus clarkii: a possible role for allatostatins

Decapod crustaceans do not appear to produce juvenile hormone, but rather its immediate precursor, methyl farnesoate (MF). Both MF and its immediate precursor, farnesoic acid (FA) are produced by the mandibular organs (MO) in crustaceans. The MO are homologous to the insect corpora allata (CA), the site of juvenile hormone biosynthesis. However, the FGLamide allatostatin (ASTs) peptides, of which there are about 60 distinct forms reported from crustaceans, have previously been found to have no effect on MO activity in crustaceans. We have identified by immunocytochemistry the presence of FGLamide-like AST immunoreactivity in neurosecretory cells throughout the CNS as well as in neurohaemal structures such as the sinus gland and pericardial organs. The ASTs are likely delivered to the MO hormonally and/or by local neurohaemal release. Using MO from adult males, we have found wide variability between animals in the in vitro rates of MF and FA biosynthesis. Treatment with Dippu-ASTs has a statistically significant stimulatory effect on MF synthesis, but only in MO that are initially producing MF at lower rates. No effect on FA production was observed, suggesting that the FGLamide ASTs exert their effect on the o-methyl transferase, the enzyme responsible for the conversion of FA to MF.     

Regulation of the Crustacean Mandibular Organ

The crustacean mandibular organ (MO) produces methyl farnesoate(MF), a juvenile hormone-related compound thought to have rolesin crustacean reproduction and development. Therefore, the controlof MF production by the MO has been of considerable interest.Current evidence indicates that the MO is negatively regulatedby peptides present in the eyestalk (MO inhibiting factor, MO-IH).Several eyestalk neuropeptides have been identified that inhibitMF synthesis by MO incubated in vitro. The amino acid sequencesof these MO-IH peptides are similar to peptides in the crustaceanhyperglycemic hormone (CHH) family of neuropeptides. In addition,there appears to be a compound in the eyestalk that lowers hemolymphlevels of MF in vivo but does not directly affect the MO invitro. The inhibition of MF synthesis by eyestalk peptides involvesthe inhibition of farnesoic acid O-methyl transferase, the lastenzyme in the MF biosynthetic pathway. The activity of thisenzyme is affected by cyclic nucleotides, suggesting that thesecompounds may be involved in the signal transduction pathwaymediating the effects of MO-IH.     

Production of farnesoic acid and methyl farnesoate by mandibular organs of the crayfish,Procambarus clarkii

The mandibular organs (MO) of crustaceans secrete methyl farnesoate (MF) and farnesoic acid (FA). To better understand the secretory activity of MO, the kinetics of production and release of both compounds were determined in vitro by following incorporation of [2-¹⁴C]acetate and l-[³H-methyl]methionine into MF and [2-¹⁴C]acetate into FA by MO of Procambarus clarkii. MO released more FA than MF but contained more MF. In medium lacking unlabeled acetate, the percentage incorporation of [¹⁴C]acetate into MF, relative to [³H]methionine, was between 21 and 40%, suggesting that there may be an alternative source of C₂ units.MO produce similar amounts of MF at concentrations of acetate from 0.08 to 10 mM. However, the addition of exogenous unlabelled FA to incubation media did not stimulate the biosynthesis of MF, raising the possibility that unlike JH biosynthesis in insects, the last step in MF production may be rate-limiting. Nonetheless, exogenous FA significantly reduced the incorporation of [¹⁴C]acetate into MF, suggesting that the glands use exogenous FA to synthesize MF. The absence of stimulation of FA production by exogenous FA indicates that there is no feedback effect of this product on the early steps in the biosynthetic pathway.     

Implications of temperature and sediment characteristics on methane formation and oxidation in lake sediments

Methane emissions from aquatic environments depend on methane formation (MF) and methane oxidation (MO) rates. One important question is to what extent increased temperatures will affect the balance between MF and MO. We measured potential MF and MO rates simultaneously at 4, 10, 20 and 30°C in sediment from eight different lakes representing typical boreal and northern temperate lake types. Potential MF rates ranged between 0.002 and 3.99 μmol CH₄ g_d.w. ^?1 day^?1, potential MO rates ranged from 0.01 to 0.39 CH₄ g_d.w. ^?1 day^?1. The potential MF rates were sensitive to temperature and increased 10 to 100 fold over the temperature interval studied. MF also differed between lakes and was correlated to sediment water content, percent of organic material and C:N ratio. Potential MO did not depend on temperature or sediment characteristics but was instead well explained by MF rates at the in situ temperature. It implies that elevated temperatures will enhance MF rates which may cause increased methane release from sediments until MO increases as well, as a response to higher methane levels.     

Intensity of the microbiological processes of the methane cycle in different types of Baltic lakes

The intensity of the microbiological processes of methane formation (MF) and methane oxidation (MO) was determined in the sediments and water of different types of Baltic lakes. The emission of methane from the lake sediments and methane distribution in the water column of the lakes were studied as functions of the lake productivity and hydrologic conditions. During summers, the intensity of MF in the lake sediments and waters varied from 0.001 to 106 ml CH4/(dm3 day) and from 0 to 3.2 ml CH4/(1 day), respectively, and the intensity of MO in the sediments and water varied from 0 to 11.2 ml CH4/(dm3 day) and from 0 to 1.1 ml CH4/(1 day), respectively. The total methane production (MP) in the lakes varied from 15 to 5000 ml CH4/(m2 day). In anoxic waters, the MP comprised 9-18% of the total PM in the lakes. The consumption of organic carbon for methanogenesis varied from 0.03 to 9.7 g/(m2 day). The role of the methane cycle in the degradation of organic matter in the lakes increased with their productivity.     

Methyl farnesoate: Its site of synthesis and regulation of secretion in a juvenile crustacean

Analogy to Insecta suggests that JH(s) may play important roles in the development and reproduction of Crustacea. It has recently been shown by Laufer et al. (1987), using LC and GC/MS that the mandibular organ of adult crabs synthesized methyl farnesoate, the unepoxidated from of JH III, and that synthesis varied according to the sex, as a function of eyestalk ablation, and stage of vitellogenesis. In experiments reported here, we found that eyestalk removal from juvenile spider crabs, Libinia emarginata, resulted in a two-fold increase in the rate of MF synthesis by MOs in vitro. Furthermore, 2 h incubations of MOs from eyestalk ablated animals with eyestalk extracts inhibited the rate of synthesis of MF by about 60%. These results suggest that MF may be a gonad stimulating hormone (GSH) reported by other workers. Secretion by the MO is inhibited by an eyestalk factor (MO-IH), and MO-IH may be similar or identical to the gonad inhibiting hormone (GIH) reported by others.     

Methyl farnesoate synthesis is necessary for the environmental sex determination in the water flea Daphnia pulex

Sex-determination systems can be divided into two groups: genotypic sex determination (GSD) and environmental sex determination (ESD). ESD is an adaptive life-history strategy that allows control of sex in response to environmental cues in order to optimize fitness. However, the molecular basis of ESD remains largely unknown. The micro crustacean Daphnia pulex exhibits ESD in response to various external stimuli. Although methyl farnesoate (MF: putative juvenile hormone, JH, in daphnids) has been reported to induce male production in daphnids, the role of MF as a sex-determining factor remains elusive due to the lack of a suitable model system for its study. Here, we establish such a system for ESD studies in D. pulex. The WTN6 strain switches from producing females to producing males in response to the shortened day condition, while the MFP strain only produces females, irrespective of day-length. To clarify whether MF has a novel physiological role as a sex-determining factor in D. pulex, we demonstrate that a MF/JH biosynthesis inhibitor suppressed male production in WTN6 strain reared under the male-inducible condition, shortened day-length. Moreover, we show that juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme of MF/JH biosynthesis, displays MF-generating activity by catalyzing farnesoic acid. Expression of the JHAMT gene increased significantly just before the MF-sensitive period for male production in the WTN6 strain, but not in the MFP strain, when maintained under male-inducible conditions. These results suggest that MF synthesis regulated by JHAMT is necessary for male offspring production in D. pulex. Our findings provide novel insights into the genetic underpinnings of ESD and they begin to shed light on the physiological function of MF as a male-fate determiner in D. pulex.     

Effect of dietary intake and lifestyle factors on in vivo mutant frequency at the HPRT gene locus in healthy human subjects

This paper describes the results of a study designed to assess the effects of a variety of dietary and lifestyle factors on background levels of mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus in humans. Eighty-three healthy and free-living subjects (aged 20–80 yr; 61 males and 22 females; mean age of 63.07±14.71 yr) were recruited. Background levels of MF were determined for each subject using a cloning assay. The mean MF/10⁶ clonable cells (MF) for the study subjects was 4.63±2.20. An interview-administered questionnaire was completed by each study subject in order to assess details of dietary history, physical activity, health and potential genotoxin exposure history. A 7-day estimated dietary record method with a food frequency questionnaire was used to determine average intakes of energy and macronutrients (including alcohol), and a range of micronutrients (including vitamin and mineral supplement usage). The relationships between individual dietary and lifestyle factors and HPRT MF were investigated by univariate and multivariate analysis (data was adjusted for age, lymphocyte plating efficiency [PE] and energy intake [EI]). Univariate analysis revealed a significant positive correlation between EI and MF and multivariate analysis revealed significant positive correlations between, body mass index (BMI), % energy intake from total carbohydrate, starch, fat and MF. These findings suggest that a reduction in EI may be a useful preventative measure against the onset of carcinogenesis in humans. No correlations were found between alcohol intake and MF or between estimated antioxidant intake and MF. Thus, estimated intakes of antioxidants may not reflect their bioavailability and functional capacity in vivo and it may be more useful to examine actual plasma/cell levels vs. MF to establish if any significant relationship exists.     

Effect of dietary intake and lifestyle factors on in vivo mutant frequency at the HPRT gene locus in healthy human subjects

This paper describes the results of a study designed to assess the effects of a variety of dietary and lifestyle factors on background levels of mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus in humans. Eighty-three healthy and free-living subjects (aged 20–80 yr; 61 males and 22 females; mean age of 63.07±14.71 yr) were recruited. Background levels of MF were determined for each subject using a cloning assay. The mean MF/10⁶ clonable cells (MF) for the study subjects was 4.63±2.20. An interview-administered questionnaire was completed by each study subject in order to assess details of dietary history, physical activity, health and potential genotoxin exposure history. A 7-day estimated dietary record method with a food frequency questionnaire was used to determine average intakes of energy and macronutrients (including alcohol), and a range of micronutrients (including vitamin and mineral supplement usage). The relationships between individual dietary and lifestyle factors and HPRT MF were investigated by univariate and multivariate analysis (data was adjusted for age, lymphocyte plating efficiency [PE] and energy intake [EI]). Univariate analysis revealed a significant positive correlation between EI and MF and multivariate analysis revealed significant positive correlations between, body mass index (BMI), % energy intake from total carbohydrate, starch, fat and MF. These findings suggest that a reduction in EI may be a useful preventative measure against the onset of carcinogenesis in humans. No correlations were found between alcohol intake and MF or between estimated antioxidant intake and MF. Thus, estimated intakes of antioxidants may not reflect their bioavailability and functional capacity in vivo and it may be more useful to examine actual plasma/cell levels vs. MF to establish if any significant relationship exists.     

Impact of a static magnetic field on the electricity production of Shewanella-inoculated microbial fuel cells

The electricity production of Shewanella-inoculated microbial fuel cells (MFCs) under magnetic field (MF) exposure was investigated in different reactor systems. The persistency of the MF effect and the influences of MF intensity and direction on MFC performance were also studied. Application of a 100-mT static MF to the MFCs improved electricity production considerably, with an increase in the maximum voltage by 20-27% in both single- and two-chamber MFCs, while a more conspicuous improvement in the electricity generation was observed in a three-electrode cell. The MF effects were found to be immediate and reversible, and adverse effects seemed to occur when the MF was suddenly removed. The medium components analysis demonstrated that the application of MF led to an enhanced bioelectrochemical activity of Shewanella, and no significant promotion in mediator secretion was found. The improvement in the electricity production of MFCs under MF was mainly attributed to the enhanced bioelectrochemical activity, possibly through the oxidative stress mechanism. An accelerated cell growth under MF might also contribute to the enhanced substrate degradation and power generation.     

cDNA cloning of a mandibular organ inhibiting hormone from the spider crabLibinia emarginata

Mandibular organs (MO) produce a crustacean juvenile hormone, methyl farnesoate (MF). MO activity is negatively regulated by factors, called mandibular organ inhibiting hormones (MOIHs), from the crustacean sinus gland X-organ complex in the eyestalks. Three MOIHs have been isolated previously from the spider crabLibinia emarginata and are characterized as members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. In the research reported here, a full length cDNA sequence of 972 bp of a MOIH was isolated by screening a cDNA library constructed from the eyestalks ofLibinia emarginata. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH, and a tri-peptide Gly-Lys-Lys which designates the potential amidation site at the C-terminus of the mature peptide.     

1,5-Disubstituted imidazoles inhibit juvenile hormone biosynthesis by the corpora allata of the mosquito Aedes aegypti

We investigated the effect of fifteen 1,5-disubstituted imidazoles (1,5-dis) on juvenile hormone III (JH III) and methyl farnesoate (MF) biosynthesis by the corpora allata (CA) of the mosquito Aedes aegypti in vitro. Four compounds (TH-35, TH-83, TH-62 and TH-28) significantly decreased JH biosynthesis in the CA dissected from 3-day old sugar-fed females. The decrease of JH synthesis was not always associated with increased MF. TH-30 and TH-83 increased MF levels, while TH-85 and TH-61 significantly decreased MF levels. Five compounds (TH-26, TH-60, TH-83, TH-35 and TH-30) significantly inhibited JH biosynthesis in the CA dissected from females 15 h after a blood meal. Four 1,5-dis (TH-30, TH-26, TH-28 and TH-66) caused MF increases in CA from blood-fed females. 1,5-Disubstituted imidazoles had higher inhibitory activity on JH synthesis when substituted at position 5 by a 3-benzyloxyphenyl group and at position 1 by a benzyl group (such as TH-35). Inhibition of JH and MF biosynthesis by TH-35 was age-dependent and influenced by nutritional status; inhibition differed when evaluated in the CA dissected from sugar-fed females at different days after emergence and in the CA dissected from females at different hours after a blood meal. Inhibition was always higher when the CA was more active. The addition of TH-35 significantly reduced the stimulatory effect of Aedes-allatotropin and farnesoic acid on JH synthesis. This is the first report of an inhibitory effect of 1,5-disubstituted imidazoles on JH synthesis in Diptera.     

Low frequency therapeutic EMF differently influences experimental muscle pain in female and male subjects

Effects of a pulsating, half sine wave magnetic field (MF) with a frequency of 100 pps and 15 mT rms flux density, generated by the MD TEMF device (EMF Therapeutics, Inc., Chattanooga), on subjective pain rating, heart rate, and arterial blood pressure were tested in a double blind, crossover design study employing experimental muscle pain. Each of 24 healthy volunteers (12 females and 12 males, 24.7 +/- 3.2 years of age) received painful stimulation induced by the infusion of 5% hypertonic saline (HS) into the erector spinae muscle during real and sham MF exposure, in counterbalanced order. Exposure to MF differently affects subjective pain estimates in females and males. MF exposure increased averaged pain level and time integral of pain ratings in females, whereas no statistically significant difference for these characteristics was found in males. Pain related elevation of systolic and diastolic blood pressure was observed during both real and sham EMF exposure in female and male subjects.     

Effect of static magnetic field on electricity production and wastewater treatment in microbial fuel cells

The effect of a magnetic field (MF) on electricity production and wastewater treatment in two-chamber microbial fuel cells (MFCs) has been investigated. Electricity production capacity could be improved by the application of a low-intensity static MF. When a MF of 50 mT was applied to MFCs, the maximum voltage, total phosphorus (TP) removal efficiency, and chemical oxygen demand (COD) removal efficiency increased from 523?±?2 to 553?±?2 mV, ～93 to ～96 %, and ～80 to >90 %, respectively, while the start-up time and coulombic efficiency decreased from 16 to 10 days and ～50 to ～43 %, respectively. The MF effects were immediate, reversible, and not long lasting, and negative effects on electricity generation and COD removal seemed to occur after the MF was removed. The start-up and voltage output were less affected by the MF direction. Nitrogen compounds in magnetic MFCs were nitrified more thoroughly; furthermore, a higher proportion of electrochemically inactive microorganisms were found in magnetic systems. TP was effectively removed by the co-effects of microbe absorption and chemical precipitation. Chemical precipitates were analyzed by a scanning electron microscope capable of energy-dispersive spectroscopy (SEM-EDS) to be a mixture of phosphate, carbonate, and hydroxyl compounds.     

Exposure of rats to a 50-Hz, 100 muTesla magnetic field does not affect the ex vivo production of interleukins by activated T or B lymphocytes.

Two separate, independent experiments were conducted to evaluate the effects of exposure of rats to a 50-Hz linearly polarized, 100 microT magnetic field (MF) on the ex vivo production of interleukins (ILs) by mitogen-stimulated splenic lymphocytes. IL-1 and IL-2 were determined by proliferation assays, using IL-dependent murine T cell lines. In the first experiment, female Sprague-Dawley rats were treated with 7,12-dimethylbenz[a]anthracene (DMBA] at a dose of 20 mg per rat (four weekly gavage doses of 5 mg), and were either MF-exposed or sham-exposed for 14 weeks. This experimental protocol has previously been shown to result in a significant increase in breast cancer growth in response to MF exposure. Furthermore, MF exposure at 50-100 microT for 3 months was recently found to induce a suppressed ex vivo proliferation of splenic T cells in response to mitogen stimulation, which could be a result of reduced IL production of spleen lymphocytes. However, the present experiments failed to demonstrate any significant difference between MF- and sham-exposed groups in production of IL-1 by mitogen-activated splenic B cells. In a second experiment, shorter MF exposure periods were studied with respect to IL production from mitogen-stimulated B and T cells. Groups of rats were MF- or sham-exposed for 1 day, 1 week, or 2 weeks, followed by preparation and activation of spleen lymphocytes. No significant difference in IL-1 or IL-2 production from stimulated B or T cells was seen. The data indicate that in vivo MF exposure of rats does not affect the ex vivo IL production of B or T lymphocytes, suggesting that the recently reported changes in T cell proliferation in response to MF exposure may not be mediated via alterations in B or T cell IL production.     

Hormonal induction of undescribed males resolves cryptic species of cladocerans

Cyclic parthenogens have a mixed breeding system with both meiotic and ameiotic eggs. Although investment in sexual stages is often synchronized with seasonal cycles, the degree of investment is a quantitative trait associated with habitat instability. Populations of cyclic parthenogens from stable environments, such as large lakes and oceans, generally show reduced or undetectable investment in males. Indeed, males of many species of lacustrine cyclic parthenogens are unknown to science. Methyl farnesoate (MF), a crustacean juvenile hormone, has been implicated as an inducer of male formation in Daphnia magna (Crustacea: Cladocera), a denizen of unstable habitats with marked sexual recruitment. Here, we show experimentally that MF induces male production in four distantly related lacustrine species of cladocerans under growth conditions unfavourable for male production. The males of three species are new to science. Unlike females, the anatomy of the previously unknown males of Bosmina (Lunobosmina) oriens permitted ready morphological diagnosis of sibling species and subfossils. The results suggest that the role for MF in the sex determination of cladocerans is general.     

Intensity of the Microbiological Processes of the Methane Cycle in Different Types of Baltic Lakes

The intensity of the microbiological processes of methane formation (MF) and methane oxidation (MO) was determined in the sediments and water of different types of Baltic lakes. The emission of methane from the lake sediments and methane distribution in the water column of the lakes were studied as functions of the lake productivity and hydrologic conditions. During summers, the intensity of MF in the lake sediments and waters varied from 0.001 to 106 ml CH₄/(dm³ day) and from 0 to 3.2 ml CH₄/(l day), respectively, and the intensity of MO in the sediments and water varied from 0 to 11.2 ml CH₄/(dm³ day) and from 0 to 1.1 ml CH₄/(l day), respectively. The total methane production (MP) in the lakes varied from 15 to 5000 ml CH₄/(m² day). In anoxic waters, the MP comprised 9–18% of the total PM in the lakes. The consumption of organic carbon for methanogenesis varied from 0.03 to 9.7 g/(m² day). The role of the methane cycle in the degradation of organic matter in the lakes increased with their productivity.     

Characterization of a human mitogenic factor (MF) released by phytohemagglutinin-activated lymphocytes.

Phytohemagglutinin (PHA)-stimulated human lymphocytes release soluble products upon subsequent incubation in fresh medium, which are strongly mitogenic for other lymphocytes. In the present investigation, some of the biochemical properties of such a factor (MF) were investigated. It was found that serum is not required in the production of MF. The mitogenic factor was stable at 56 °C for 30 min and at 80 °C for 10 min but was destroyed by treatment at 100 °C for 1 min. By gel chromatography on Sephadex the mitogenic activity was found in fractions corresponding to a molecular weight of 40,000–55,000. Moreover, isoelectric focusing indicated an isoelectric point at pH 8.0–8.5. By subjecting MF to CM-32 cellulose ion-exchange chromatography, all activity was detected in the adsorbed fractions. PHA was studied in parallel in some of the experiments. The results clearly showed that MF is distinct from PHA which induces the release of MF. MF was not adsorbed to concanavalin A-Sepharose.     

Reverse signaling through transmembrane TNF confers resistance to lipopolysaccharide in human monocytes and macrophages

We have previously reported that the CD14+ monocytic subpopulation of human PBMC induces programmed cell death (apoptosis) in cocultured endothelial cells (EC) when stimulated by bacterial endotoxin (LPS). Apoptosis is mediated by two routes, first via transmembrane TNF-alpha (mTNF) expressed on PBMC and, in addition, by TNF-independent soluble factors that trigger apoptosis in EC. Neutralizing anti-TNF mAb completely blocked coculture-mediated apoptosis, despite the fact that there should have been additional soluble cell death factors. This led to the hypothesis that a reverse signal is transmitted from the TNF receptor on EC to monocytes (MO) via mTNF that prevents the production of soluble apoptotic factors. Here we have tested this hypothesis. The results support the idea of a bidirectional cross-talk between MO and EC. Peripheral blood MO, MO-derived macrophages (MPhi), or the monocytic cell line Mono Mac 6 were preincubated with human microvascular EC that constitutively express TNF receptor type I (TNF-R1) and subsequently stimulated with LPS. Cell-free supernatants of these preparations no longer induced EC apoptosis. The preincubation of MO/MPhi with TNF-reactive agents, such as mAb and soluble receptors, also blocked the production of death factors, providing further evidence for reverse signaling via mTNF. Finally, we show that reverse signaling through mTNF mediated LPS resistance in MO/MPhi as indicated by the down-regulation of LPS-induced soluble TNF and IL-6 as well as IL-1 and IL-10.