Genomic Selection Signals in Andean Highlanders Reveal Adaptive Placental Metabolic Phenotypes That Are Disrupted in Preeclampsia.

BACKGROUND: The chronic hypoxia of high-altitude residence poses challenges for tissue oxygen supply and metabolism. Exposure to high altitude during pregnancy increases the incidence of hypertensive disorders of pregnancy and fetal growth restriction and alters placental metabolism. High-altitude ancestry protects against altitude-associated fetal growth restriction, indicating hypoxia tolerance that is genetic in nature. Yet, not all babies are protected and placental pathologies associated with fetal growth restriction occur in some Andean highlanders. METHODS: We examined placental metabolic function in 79 Andeans (18-45 years; 39 preeclamptic and 40 normotensive) living in La Paz, Bolivia (3600-4100 m) delivered by unlabored Cesarean section. Using a selection-nominated approach, we examined links between putatively adaptive genetic variation and phenotypes related to oxygen delivery or placental metabolism. RESULTS: Mitochondrial oxidative capacity was associated with fetal oxygen delivery in normotensive but not preeclamptic placenta and was also suppressed in term preeclamptic pregnancy. Maternal haplotypes in or within 200 kb of selection-nominated genes were associated with lower placental mitochondrial respiratory capacity (PTPRD [protein tyrosine phosphatase receptor-δ]), lower maternal plasma erythropoietin (CPT2 [carnitine palmitoyl transferase 2], proopiomelanocortin, and DNMT3 [DNA methyltransferase 3]), and lower VEGF (vascular endothelial growth factor) in umbilical venous plasma (TBX5 [T-box transcription factor 5]). A fetal haplotype within 200 kb of CPT2 was associated with increased placental mitochondrial complex II capacity, placental nitrotyrosine, and GLUT4 (glucose transporter type 4) protein expression. CONCLUSIONS: Our findings reveal novel associations between putatively adaptive gene regions and phenotypes linked to oxygen delivery and placental metabolic function in highland Andeans, suggesting that such effects may be of genetic origin. Our findings also demonstrate maladaptive metabolic mechanisms in the context of preeclampsia, including dysregulation of placental oxygen consumption.

The metabolic response of human trophoblasts derived from term placentas to metformin.

AIMS/HYPOTHESIS: Metformin is increasingly used therapeutically during pregnancy worldwide, particularly in the treatment of gestational diabetes, which affects a substantial proportion of pregnant women globally. However, the impact on placental metabolism remains unclear. In view of the association between metformin use in pregnancy and decreased birthweight, it is essential to understand how metformin modulates the bioenergetic and anabolic functions of the placenta. METHODS: A cohort of 55 placentas delivered by elective Caesarean section at term was collected from consenting participants. Trophoblasts were isolated from the placental samples and treated in vitro with clinically relevant doses of metformin (0.01 mmol/l or 0.1 mmol/l) or vehicle. Respiratory function was assayed using high-resolution respirometry to measure oxygen concentration and calculated [Formula: see text]. Glycolytic rate and glycolytic stress assays were performed using Agilent Seahorse XF assays. Fatty acid uptake and oxidation measurements were conducted using radioisotope-labelled assays. Lipidomic analysis was conducted using LC-MS. Gene expression and protein analysis were performed using RT-PCR and western blotting, respectively. RESULTS: Complex I-supported oxidative phosphorylation was lower in metformin-treated trophoblasts (0.01 mmol/l metformin, 61.7% of control, p<0.05; 0.1 mmol/l metformin, 43.1% of control, p<0.001). The proton efflux rate arising from glycolysis under physiological conditions was increased following metformin treatment, up to 23±5% above control conditions following treatment with 0.1 mmol/l metformin (p<0.01). There was a significant increase in triglyceride concentrations in trophoblasts treated with 0.1 mmol/l metformin (p<0.05), particularly those of esters of long-chain polyunsaturated fatty acids. Fatty acid oxidation was reduced by ~50% in trophoblasts treated with 0.1 mmol/l metformin compared with controls (p<0.001), with no difference in uptake between treatment groups. CONCLUSIONS/INTERPRETATION: In primary trophoblasts derived from term placentas metformin treatment caused a reduction in oxidative phosphorylation through partial inactivation of complex I and potentially by other mechanisms. Metformin-treated trophoblasts accumulate lipids, particularly long- and very-long-chain polyunsaturated fatty acids. Our findings raise clinically important questions about the balance of risk of metformin use during pregnancy, particularly in situations where the benefits are not clear-cut and alternative therapies are available.

Hypoxia-Inducible Factors as Key Players in the Pathogenesis of Non-alcoholic Fatty Liver Disease and Non-alcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) are a major public health concern with high and increasing global prevalence, and a significant disease burden owing to its progression to more severe forms of liver disease and the associated risk of cardiovascular disease. Treatment options, however, remain scarce, and a better understanding of the pathological and physiological processes involved could enable the development of new therapeutic strategies. One process implicated in the pathology of NAFLD and NASH is cellular oxygen sensing, coordinated largely by the hypoxia-inducible factor (HIF) family of transcription factors. Activation of HIFs has been demonstrated in patients and mouse models of NAFLD and NASH and studies of activation and inhibition of HIFs using pharmacological and genetic tools point toward important roles for these transcription factors in modulating central aspects of the disease. HIFs appear to act in several cell types in the liver to worsen steatosis, inflammation, and fibrosis, but may nevertheless improve insulin sensitivity. Moreover, in liver and other tissues, HIF activation alters mitochondrial respiratory function and metabolism, having an impact on energetic and redox homeostasis. This article aims to provide an overview of current understanding of the roles of HIFs in NAFLD, highlighting areas where further research is needed.

The German trial on Aciclovir and Corticosteroids in Herpes-simplex-virus-Encephalitis (GACHE): a multicenter, randomized, double-blind, placebo-controlled trial.

INTRODUCTION: Comprehensive treatment of Herpes-simplex-virus-encephalitis (HSVE) remains a major clinical challenge. The current therapy gold standard is aciclovir, a drug that inhibits viral replication. Despite antiviral treatment, mortality remains around 20% and a majority of survivors suffer from severe disability. Experimental research and recent retrospective clinical observations suggest a favourable therapy response to adjuvant dexamethasone. Currently there is no randomized clinical trial evidence, however, to support the routine use of adjuvant corticosteroid treatment in HSVE. METHODS: The German trial of Aciclovir and Corticosteroids in Herpes-simplex-virus-Encephalitis (GACHE) studied the effect of adjuvant dexamethasone versus placebo on top of standard aciclovir treatment in adult patients aged 18 up to 85 years with proven HSVE in German academic centers of Neurology in a randomized and double blind fashion. The trial was open from November 2007 to December 2012. The initially planned sample size was 372 patients with the option to increase to up to 450 patients after the second interim analysis. The primary endpoint was a binary functional outcome after 6 months assessed using the modified Rankin scale (mRS 0-2 vs. 3-6). Secondary endpoints included mortality after 6 and 12 months, functional outcome after 6 months measured with the Glasgow outcome scale (GOS), functional outcome after 12 months measured with mRS and GOS, quality of life as measured with the EuroQol 5D instrument after 6 and 12 months, neuropsychological testing after 6 months, cranial magnetic resonance imaging findings after 6 months, seizures up to day of discharge or at the latest at day 30, and after 6 and 12 months. RESULTS: The trial was stopped prematurely for slow recruitment after 41 patients had been randomized, 21 of them treated with dexamethasone and 20 with placebo. No difference was observed in the primary endpoint. In the full analysis set (n = 19 in each group), 12 patients in each treatment arm achieved a mRS of 0-2. Similarly, we did not observe significant differences in the secondary endpoints (GOS, mRS, quality of life, neuropsychological testing). CONCLUSION: GACHE being prematurely terminated demonstrated challenges encountered performing randomized, placebo-controlled trials in rare life threatening neurological diseases. Based upon our trial results the use of adjuvant steroids in addition to antiviral treatment remains experimental and is at the decision of the individual treating physician. Unfortunately, the small number of study participants does not allow firm conclusions. TRIAL REGISTRATION: EudraCT-Nr. 2005-003201-81.

[Mission (im)possible : Setting up a neurological center 12,000 km away with telemedicine]. / "Mission (im)possible" : Aufbau eines neurologischen Zentrums 12.000 km entfernt mittels Telemedizin.

BACKGROUND: Specialized neurological treatment decreases the mortality and morbidity of stroke patients. In many regions of the world an extensive coverage is not available. The cooperation between the Krankenhaus Nordwest (KHNW, Frankfurt, Germany) and the Government of Brunei Darussalam describes the set-up process of a specialized neurological center, including stroke unit, science and rehabilitation center. AIM: The aim of this project called to teach to treat - to treat to teach was to set up a center of excellence in neurology in Brunei Darussalam over a distance of 12,000 km. Treatment options were elucidated by teaching and taught by case examples. MATERIAL AND METHODS: The construction of the Brunei Neuroscience Stroke and Rehabilitation Center (BNSRC) began in July 2010. To overcome the large distance between the department of neurology and neuroradiology at the KHNW and the BNSRC, a telemedical network was established. We provided daily teleteaching for all professions involved in patient care as well as 24/7 availability of teleneurological services from Germany to support the local team on site. RESULTS: In the BNSRC unit over 1000 patients with ischemic and hemorrhagic stroke and all the various acute neurological conditions were treated from July 2010 until July 2016 as inpatients and over 5000 were treated as outpatients. Since 2010, a total of 52 patients with stroke were treated by thrombolysis within the thrombolytic window and 81 hemicraniectomies were performed. CONCLUSION: The project has shown that it is possible to convey specialized neurological knowledge over large distances to provide significant benefits for patients and caregivers.

Common carotid intima-media thickness does not add to Framingham risk score in individuals with diabetes mellitus: the USE-IMT initiative.

AIMS/HYPOTHESIS: The aim of this work was to investigate whether measurement of the mean common carotid intima-media thickness (CIMT) improves cardiovascular risk prediction in individuals with diabetes. METHODS: We performed a subanalysis among 4,220 individuals with diabetes in a large ongoing individual participant data meta-analysis involving 56,194 subjects from 17 population-based cohorts worldwide. We first refitted the risk factors of the Framingham heart risk score on the individuals without previous cardiovascular disease (baseline model) and then expanded this model with the mean common CIMT (CIMT model). The absolute 10 year risk for developing a myocardial infarction or stroke was estimated from both models. In individuals with diabetes we compared discrimination and calibration of the two models. Reclassification of individuals with diabetes was based on allocation to another cardiovascular risk category when mean common CIMT was added. RESULTS: During a median follow-up of 8.7 years, 684 first-time cardiovascular events occurred among the population with diabetes. The C statistic was 0.67 for the Framingham model and 0.68 for the CIMT model. The absolute 10 year risk for developing a myocardial infarction or stroke was 16% in both models. There was no net reclassification improvement with the addition of mean common CIMT (1.7%; 95% CI -1.8, 3.8). There were no differences in the results between men and women. CONCLUSIONS/INTERPRETATION: There is no improvement in risk prediction in individuals with diabetes when measurement of the mean common CIMT is added to the Framingham risk score. Therefore, this measurement is not recommended for improving individual cardiovascular risk stratification in individuals with diabetes.

Risk of symptomatic intracerebral hemorrhage in patients treated with intra-arterial thrombolysis.

BACKGROUND: In intra-arterial (IA) thrombolysis trials, higher rates of symptomatic intracerebral haemorrhage (sICH) were found than in trials with intravenous (IV) recombinant tissue plasminogen activator (tPA); this observation could have been due to the inclusion of more severely affected patients in IA thrombolysis trials. In the present study, we investigated the rate of sICH in IA and combined IV + IA thrombolysis versus IV thrombolysis after adjusting for differences in clinical and MRI parameters. METHODS: In this multicenter study, we systematically analyzed data from 645 patients with anterior-circulation strokes treated with either IV or IA thrombolysis within 6 h following symptom onset. Thrombolytic regimens included (1) IV tPA treatment (n = 536) and (2) IA treatment with either tPA or urokinase (n = 74) or (3) combined IV + IA treatment with either tPA or urokinase (n = 35). RESULTS: 44 (6.8%) patients developed sICH. sICH patients had significantly higher scores on the National Institutes of Health Stroke Scale (NIHSS) at admission and pretreatment DWI lesions. The sICH risk was 5.2% (n = 28) in IV thrombolysis, which is significantly lower than in IA (12.5%, n = 9) or IV + IA thrombolysis (20%, n = 7). In a binary logistic regression analysis including age, NIHSS score, time to thrombolysis, initial diffusion weighted imaging lesion size, mode of thrombolytic treatment and thrombolytic agent, the mode of thrombolytic treatment remained an independent predictor for sICH. The odds ratio for IA or IV + IA versus IV treatment was 3.466 (1.19-10.01, 95% CI, p < 0.05). CONCLUSION: In this series, IA and IV + IA thrombolysis is associated with an increased sICH risk as compared to IV thrombolysis, and this risk is independent of differences in baseline parameters such as age, initial NIHSS score or pretreatment lesion size.

Both long-term HIV infection and highly active antiretroviral therapy are independent risk factors for early carotid atherosclerosis.

OBJECTIVE: There is controversy over whether or not chronic HIV infection contributes to atherosclerosis. We investigated the relationship between HIV infection, antiretroviral medication and ultrasound evidence of early atherosclerosis in the context of vascular risk factors. DESIGN: A case-control design with 292 HIV-positive subjects and 1168 age- and sex-matched controls. METHODS: We assessed vascular risk factors, blood pressure, serum lipids and carotid intima media thickness (IMT) in cases and controls. With multivariate regression models, we investigated the effects of HIV status and antiretroviral medication on IMT. RESULTS: The common carotid artery (CCA) IMT value was 5.70% (95% confidence interval [3.08-8.38%], p<0.0001) or 0.044 mm [0.021-0.066 mm] (p=0.0001) higher in HIV-positives, adjusted for multiple risk factors. In the carotid bifurcation (BIF), the IMT values were 24.4% [19.5-29.4%] or 0.250 mm [0.198-0.303 mm] higher in HIV patients (p<0.0001). An investigation of antiretroviral substances revealed higher CCA- and BIF-IMT values in patients receiving combination antiretroviral therapy (HAART). CONCLUSIONS: HIV infection and HAART are independent risk factors for early carotid atherosclerosis. Assuming a risk ratio similar to that in large population-based cohorts, the observed IMT elevation suggests that vascular risk is 4-14% greater and the "vascular age" 4-5 years higher in HIV-positive subjects. The underlying mechanisms remain to be clarified.

Anthropometric approximation of body weight in unresponsive stroke patients.

BACKGROUND AND PURPOSE: Thrombolysis of acute ischaemic stroke is based strictly on body weight to ensure efficacy and to prevent bleeding complications. Many candidate stroke patients are unable to communicate their body weight, and there is often neither the means nor the time to weigh the patient. Instead, weight is estimated visually by the attending physician, but this is known to be inaccurate. METHODS: Based on a large general population sample of nearly 7000 subjects, we constructed approximation formulae for estimating body weight from simple anthropometric measurements (body height, and waist and hip circumference). These formulae were validated in a sample of 178 consecutive inpatients admitted to our stroke unit, and their accuracy was compared with the best visual estimation of two experienced physicians. RESULTS: The simplest formula gave the most accurate approximation (mean absolute difference 3.1 (2.6) kg), which was considerably better than the best visual estimation (physician 1: 6.5 (5.2) kg; physician 2: 7.4 (5.7) kg). It reduced the proportion of weight approximations mismatched by >10% from 31.5% and 40.4% (physicians 1 and 2, respectively) to 6.2% (anthropometric approximation). Only the patient's own estimation was more accurate (mean absolute difference 2.7 (2.4) kg). CONCLUSIONS: By using an approximation formula based on simple anthropometric measurements (body height, and waist and hip circumference), it is possible to obtain a quick and accurate approximation of body weight. In situations where the exact weight of unresponsive patients cannot be ascertained quickly, we recommend using this approximation method rather than visual estimation.

Predicting creatinine clearance by a simple formula following live-donor kidney transplantation.

The outcome after live-donor kidney transplantation is influenced by many parameters. The aim of our study was to establish a multivariate prognostic model for calculating the recipient's creatinine clearance after transplantation. Basic immunological, donor-, recipient- and process-related variables were assessed in a series of 18 live-donor kidney transplant patients with an uncomplicated postoperative course. Multivariate analysis was carried out with automated forward and backward selection. The following four parameters were included in the predictive model: recipient age, recipient BMI, graft clearance and degree of relationship. The coefficient of determination (R(2)) was 0.67. It could be shown that a significant prediction of creatinine clearance after living related kidney transplantation can be made, based on simple variables. Therefore, this formula could help to detect early complications in the post-transplantation course if the recipient's creatinine clearance drops below the predicted result.

Synthesis of lipids in the fat body of Gryllus bimaculatus: age-dependency and regulation by adipokinetic hormone.

The free abdominal fat body of adult female Mediterranean field crickets, Gryllus bimaculatus, synthesizes lipids from [1-(14)C]-acetate in vitro. Up to an age of 12h, the incorporation of acetate into lipids is very low and then rises to a maximum 24h after adult emergence. Thereafter, the incorporation of acetate decreases to moderate levels at day 2 and then slowly decreases until day 30. The adipokinetic hormone of G. bimaculatus (Grb-AKH) significantly inhibits the incorporation of acetate at a concentration of 10(-11) M; maximum inhibition (approximately 95%) is reached at 10(-8) M. The inhibiting effect of Grb-AKH is fast, dose-dependent, and reversible. The periovaric fat body shows a similar pattern of acetate incorporation, although rates of incorporation are lower; the incorporation can be inhibited by Grb-AKH as well. The segmental abdominal fat body and the fat body from the head both incorporate acetate into lipids at low rates that cannot be inhibited significantly by AKH. Prepurified brain extracts significantly inhibit acetate incorporation by free abdominal fat bodies at a concentration of 0.1 brain equivalent. Allatostatins and crustacean cardioactive peptide, which are both present in cricket brains, are not responsible for this inhibiting effect. Octopamine causes a dose-dependent inhibition of acetate incorporation whereas synephrine had no such effect. The inhibiting effect of Grb-AKH on the formation of lipid stores in the fat body and its consequences for reproductive processes are discussed.

A comparative study on hypertrehalosaemic hormones in the Hymenoptera: sequence determination, physiological actions and biological significance.

A new hypertrehalosaemic peptide (Tea-HrTH; pQLNFSTGWGG-NH(2)) was isolated from the corpora cardiaca (CC) of the sawfly Tenthredo arcuata. The hypertrehalosaemic peptides found in the CC of five Bombus species and the paper wasp Polistes fuscata were identical to the adipokinetic hormone II of the desert locust, Schistocerca gregaria (Scg-AKH-II). The hypertrehalosaemic peptides found in the yellowjacket Vespula vulgaris and the hornet Vespa crabro were identical to the adipokinetic hormone of the cricket, Gryllus bimaculatus (Grb-AKH).All species examined had a large storage crop which, when filled with honey, held up to one-third of their total body weight. Overwintering queens of P. fuscata had large stores of carbohydrates and lipids in the abdomen, and were able to survive months of fasting. Workers of Bombus hortorum (bumble-bee), Apis mellifera (honey-bee) and V. vulgaris had little or no fat body. These species could fly as long as sugar was present in their crops, but they stopped flying as the carbohydrates in the crop disappeared. There was no significant increase in the haemolymph carbohydrate titres after injections of CC extracts or corresponding synthetic peptides into workers of B. hortorum or into males and females of T. arcuata. There was a moderate increase in haemolymph carbohydrate titres when these peptides were injected into overwintering queens of P. fuscata and into workers of V. crabro, both with significant amounts of fat body. However, well-fed V. vulgaris workers, with very little fat body, also responded to their own hypertrehalosaemic peptide.

Structure elucidation and biological activity of an unusual adipokinetic hormone from corpora cardiaca of the butterfly, Vanessa cardui.

A structurally unusual member of the adipokinetic hormone/red pigment-concentrating hormone peptide family was isolated from corpora cardiaca of the painted lady butterfly, Vanessa cardui. Its primary structure was assigned by Edman degradation and nano-electrospray-time-of-flight mass spectrometry as pQLTFTSSWGGK (Vac-AKH). Vac-AKH represents the first 11mer and the first nonamidated peptide in this family. The peptide shows significant adipokinetic activity in adult specimens of V. cardui. Injection of 10 pmol of synthetic Vac-AKH into 4-day-old decapitated males resulted in an approximately 150% increase of hemolymph lipids after 90 min. Half maximal adipokinetic activity was achieved with about 0. 1 pmol of Vac-AKH. During a 2-h incubation of corpora cardiaca/corpora allata complexes in medium containing 50 mM KCl, significant amounts of Vac-AKH were released from the glands.

Interaction between Manduca sexta allatotropin and manduca sexta allatostatin in the fall armyworm Spodoptera frugiperda.

A peptide that strongly stimulates juvenile hormone (JH) biosynthesis in vitro by the corpora allata (CA) was purified from methanolic brain extracts of adult Spodoptera frugiperda. Using HPLC separation followed by Edman degradation and mass spectrometry, the peptide was identified as Manduca sexta allatotropin (Mas-AT). Treating the CA from adult S. frugiperda with synthetic Mas-AT (at 10(-6) M) caused an up to sevenfold increase in JH biosynthesis. The stimulation of JH synthesis was dose-dependent and reversible. Synthetic M. sexta allatostatin (Mas-AS) (10(-6) M) did not affect the spontaneous rate of JH secretion from CA of adult S. frugiperda, nor did any of the allatostatins of the Phe-Gly-Leu-amide peptide family tested. However, when CA had been activated by Mas-AT (10(-6) M), addition of synthetic Mas-AS (10(-6) M) reduced JH synthesis by about 70%. This allatostatic effect of Mas-AS on allatotropin-activated glands was also reversible. When CA were incubated in the presence of both Mas-AT (10(-6) M) and various concentrations of Mas-AS (from 10(-8) to 10(-5) M), the stimulation of JH-biosynthesis observed was inhibited in a dose-dependent manner. The experiments demonstrate a novel mechanism of allatostatin action. In S. frugiperda JH synthesis was inhibited only in those glands which had previously been activated by an allatotropin.

Identification of multiple peptides homologous to cockroach and cricket allatostatins in the stick insect Carausius morosus.

Eighteen peptides were isolated from brain extracts of the stick insect Carausius morosus. The peptides were purified in four steps by high-performance liquid chromatography, monitored by their ability to inhibit juvenile hormone biosynthesis by corpora allata of the cricket Gryllus bimaculatus in vitro, and chemically characterised by Edman degradation and mass spectrometry. We obtained complete primary-structure information for nine peptides, four of which belong to the peptide family characterised by a common C-terminal pentapeptide sequence -YXFGLamide. The remaining five belong to the W(2)W(9)amide peptide family, nonapeptides characterised by having the amino acid tryptophan in positions 2 and 9. The amino-acid sequence of two other peptides could not be completely resolved by means of Edman degradation; however, these peptides could be allocated to the -YXFGLamide and the W(2)W(9)amide family, respectively, by comparison of retention times, co-elution and mass spectrometry. Both classes of neuropeptides strongly inhibit juvenile hormone biosynthesis in crickets but show no inhibiting effect on the corpora allata of the stick insect.

Juvenile hormone biosynthesis in larval and adult stick insects, Carausius morosus.

Corpora allata (CA) from adult egg-carrying Indian stick insects, Carausius morosus, synthesise and release juvenile hormone (JH) III in vitro. No JH biosynthesis was observed in larvae, young adults, and old adult females that do not carry sclerotised eggs. In females, which bear sclerotised eggs, a consistent JH biosynthesis was observed. Supplementation of precursors of JH biosynthesis (farnesol, mevalonic acid lactone) greatly enhanced JH biosynthesis in a stage-, age-, and dose-dependent manner, but CA from the last larval instar retained the biosynthesised JH within the gland. Elevated calcium concentration in the incubation medium stimulated JH biosynthesis by CA from older adults but had either no or a poor effect on CA from young adults and larvae. The results obtained with farnesol, mevalonic acid lactone, and calcium indicate that the rate-limiting steps of JH biosynthesis very likely occur before the formation of mevalonic acid and that these early steps cannot be stimulated by elevated calcium concentrations in larvae and young adults. In older adults, in which spontaneous JH biosynthesis occurs, elevated calcium concentration can markedly stimulate JH biosynthesis. A pre-purified extract from brains of adult females had a stimulating effect on JH biosynthesis by CA from adult females. The results indicate that JH biosynthesis in C. morosus may require food-derived farnesol and may be regulated by allatotropic signals from the brain, possibly triggered by sclerotised oocytes in the ovary.

Hypertrehalosaemic peptides in the honeybee (Apis mellifera): purification, identification and function.

The corpora cardiaca (CC) of the Italian race (including also the africanised variety) of the honeybee (Apis mellifera ligustica) contain approximately 3 pmol of a hypertrehalosaemic peptide. This peptide is identical in structure to the adipokinetic hormone (AKH) found in Manduca sexta, Mas-AKH. The CC of the dark European race of the honeybee (Apis mellifera carnica) contain no detectable Mas-AKH or any other adipokinetic/hypertrehalosaemic peptide. This is the first report of the occurrence of this peptide in a non-lepidopteran insect and of an intraspecific variation with regards to the presence or absence of a hypertrehalosaemic peptide in the CC of an insect. Extracts of A. m. ligustica CC elicit a strong adipokinetic/hypertrehalosaemic response when injected into crickets and cockroaches but extracts of A. m. carnica CC elicit no such responses when injected into crickets, cockroaches and butterflies. A weak hypertrehalosaemic response to injected Mas-AKH was observed in winter bees of both races, but there was no response in spring/summer bees. However, if a seasonal difference exists, it is at best minimal. Honeybees always have access to a more than adequate supply of high energy food in the form of nectar or honey stored in the hive. Thus, though A. m. ligustica CC contain a hypertrehalosaemic peptide, there is neither a glycogen-mobilising function of this hormone nor an adequate glycogen store in their fat body for its effective utilisation.

Ecdysteroid release by the prothoracic gland of Gryllus bimaculatus (Ensifera: Gryllidae) during larval-adult development.

The in vitro secretion of ecdysteroids from the prothoracic glands of larvae of Gryllus bimaculatus was analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (65-93%), with lesser amounts of ecdysone (7-35%). Production and release of ecdysteroids from the prothoracic glands are calcium-dependent. The rate of ecdysteroid release was low during the beginning and the end of the last two larval stages and high in between. Prothoracic glands from young adult females produced only minor amounts of ecdysteroids and ceased hormone production around day 4 after the moult.

A family of neuropeptides that inhibit juvenile hormone biosynthesis in the cricket, Gryllus bimaculatus.

Four nonapeptides that inhibit juvenile hormone synthesis have been isolated by four high performance liquid chromatographic steps from extracts of the brain of the field cricket, Gryllus bimaculatus. The primary structures of these peptides were assigned by Edman degradation and mass spectrometry as Gly-Trp-Gln-Asp-Leu-Asn-Gly-Gly-Trp-NH2 (Grb-AST B1), Gly-Trp-Arg-Asp-Leu-Asn-Gly-Gly-Trp-NH2 (Grb-AST B2), Ala-Trp-Arg-Asp-Leu-Ser-Gly-Gly-Trp-NH2 (Grb-AST B3), and Ala-Trp-Glu-Arg-Phe-His-Gly-Ser-Trp-NH2 (Grb-AST B4). Each of the peptides shows high sequence similarity to the locustamyoinhibiting peptide (Lom-MIP), but is structurally different from all the allatostatins so far identified. The synthetic allatostatins Grb-AST B1-4 are potent inhibitors (50% inhibition at 10(-8) to 7 x 10(-8) M) of juvenile hormone III biosynthesis by corpora allata from 3-day-old virgin females of G. bimaculatus using an in vitro bioassay. At 10(-7) M, Grb-AST B1 also strongly inhibits juvenile hormone III biosynthesis by corpora allata from 2-day-old adult males and 1-day-old (males and females) and 4-day-old (females) last instar larvae of G. bimaculatus. The inhibitory effect of Grb-AST B1 was also evident on corpora allata from a related species, Acheta domesticus. Inhibition of juvenile hormone synthesis by Grb-AST B1-4 is reversible.

Identification of two allatostatins from the cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae): additional members of a family of neuropeptides inhibiting juvenile hormone biosynthesis.

Two peptide inhibitors of juvenile hormone biosynthesis, designated G. bimaculatus allatostatins A1 and A2, have been purified from extracts of the brain of the field cricket Gryllus bimaculatus. The primary structures of these peptides were assigned as Ala-Gln-His-Gln-Tyr-Ser-Phe-Gly-Leu-NH2 (Grb-AST A1) and Ala-Gly-Gly-Arg-Gln-Tyr-Gly-Phe-Gly-Leu-NH2 (Grb-AST A2). Each of the peptides shows C-terminal amino acid sequence similarity to cockroach allatostatins and blowfly callatostatins. The two peptides are potent inhibitors of in vitro juvenile hormone production by corpora allata from virgin females of G. bimaculatus.