A new resting trap to sample fungus-infected mosquitoes, and the pathogenicity of Lecanicillium muscarium to culicid adults

Some soil-dwelling entomopathogenic fungi that are widely used in pest control are also able to reduce the survival of adult mosquito vectors under laboratory conditions. However, there is still little information about the naturally occurring fungal pathogens affecting culicid mosquitoes. As such, we hypothesized that fungi that already kill mosquitoes in realistic domestic environments could be effective against these vectors in human habitations. A simple, inexpensive, handmade, cylindrical kiln-fired clay pot (30 cm height, 24 cm inner diameter, 0.8-1 cm wall thickness) was modified into a trapping device for resting adult mosquitoes and to sample fungus-infected moribund and dead individuals. The entomopathogenic fungus Lecanicillium muscarium was isolated from a dead culicid mosquito collected with this trap in southeastern Tanzania. This isolate is the first L. muscarium reported to occur naturally on adult culicids in Tanzania and was found to be pathogenic also to adults of Aedes aegypti, Anopheles arabiensis and Culex quinquefasciatus under laboratory conditions. The trapping device confirmed its efficacy to sample mosquito-specific fungi in domestic locations and that the isolated fungus might have potential for mosquito control.     

Control of malaria vectors with the insect growth regulator pyriproxyfen in a gem-mining area in Sri Lanka

The study was conducted in eight adjacent villages in central Sri Lanka where there are many shallow pits dug by gem miners that fill with water. These become breeding places of the main malarial vector Anopheles culicifacies, and of the second most important vector Anopheles subpictus, but not of Anopheles varuna, the third most important vector. With the help of local volunteers, data on the adult populations of these three species was collected by various standard methods, and data on the incidence of malaria cases was collected by two clinics set up for the project and through the existing hospitals. Prevalence of malaria infection in symptom-less people was investigated by mass blood surveys. On the basis of a year's pre-intervention data the villages were stratified into four with high levels of malaria transmission and four with lower transmission. Within each stratum two villages were randomly assigned for mosquito control by treating all the gem pits, as well as river bed pools, with a granular formulation of the insect growth regulator pyriproxyfen at a target dose of 0.01 mg a.i./litre. The intervention caused significant reductions in the adult populations of An. culicifacies and An. subpictus. Similarly, incidence of malaria was reduced in the intervention villages to about 24% (95% c.l. 20–29%) of that in the controls. Prevalence of parasitaemia also declined significantly. It is concluded that in this situation where, with active community participation, the breeding sites of the main vectors could be located; vector control by a highly active and persistent insect growth regulator can be a very effective means of malaria control.     

Competitive interactions between larvae of the malaria mosquitoes Anopheles arabiensis and Anopheles gambiae under semi-field conditions in western Kenya

The present paper reports the occurrence of competition between larvae of the malaria mosquito sibling species Anopheles arabiensis and An. gambiae under ambient conditions in western Kenya. Larvae of both species were reared at the same density and under the same food conditions outdoors in single-species and mixed-species populations (species ratio 1:1) in transparent cups that floated in small and large semi-natural pools, which experienced different diurnal variations in water temperature. In a second experiment, both species were reared at similar densities and under the same food conditions in trays in either single-species or mixed-species populations at different proportions (species ratio 1:1, 1:3 or 3:1). Competition affected the development rate of both species in an opposite way: the development time of larvae of An. arabiensis increased whereas the development time of larvae of An. gambiae decreased in the presence of its sibling species. In small pools larvae developing in mixed-species populations experienced a higher mortality than larvae reared in single-species populations, whereas no such effect was observed in the large pools. In both species the time to pupation was longer and emerging females were larger in the small pools. Larval mortality of An. arabiensis was lower in the small pools compared to the large pools, whereas An. gambiae showed the opposite trend. Overall An. arabiensis showed reduced development rates, higher mortality rates and emerged with a larger body size compared to An. gambiae. The implication of these competitive interactions between larvae of An. arabiensis and An. gambiae under semi-filed conditions needs to be considered in the design and implementation of programmes that aim to reduce malaria transmission as competition may alter the species composition in the field.     

Field evaluation of an insect growth regulator, pyriproxyfen 0.5% GR against Culex quinquefasciatus, the vector of Bancroftian filariasis in Pondicherry, India

Pyriproxyfen, 0.5% granular formulation (GR), an insect growth regulator (IGR) was tested against Culex quinquefasciatus larvae and pupae in disused wells, cesspits and drains at the dosages of 0.1, 0.25, and 0.5kg(ai)/ha to determine the most appropriate field dosage. The IGR was found to be effective against C. quinquefasciatus larvae and pupae in all the larval habitats tested. In drains with slow moving water, application of pyriproxyfen 0.5% GR at 0.5kg(ai)/ha resulted in >80% emergence inhibition (EI) of adults for 4 weeks. At 0.1 and 0.25kg(ai)/ha, the EI was always less than 80%. In stagnant drains, the IGR yielded >80% EI for 1-week period at 0.1kg(ai)/ha. At 0.25 and 0.5kg(ai)/ha, the efficacy was 5-10 times higher. In cesspits, the EI was >80% for 6 weeks when pyriproxyfen 0.5% GR was applied at 0.1kg(ai)/ha, for 11 weeks at 0.25kg(ai)/ha and 9 weeks at 0.5kg(ai)/ha. In disused wells treated at the dosage 0.1kg(ai)/ha, there was >80% EI for 15 weeks and at 0.25 and 0.5kg(ai)/ha, the effective duration was about 1.6-1.8 times longer (24-28 weeks). Considering the quantum of IGR required and the cost and also for safety reasons, the low dosage 0.1kg(ai)/ha is recommended as the field dosage to be applied at weekly interval in stagnant drains, 6 weeks interval in cesspits and 15 weeks interval in disused wells. Since pyriproxyfen 0.5% GR has a relatively longer residual effect than the other IGRs the operational cost could be minimized. Pyriproxyfen 0.5% GR could be one of the choices in the chemical control strategy in Integrated Vector Control Programmes.     

Synthesis and screening of 1-methyl-4-substituted benzoyl piperazides against adult Setaria digitata for antifilarial activity

There is no safe and effective drug for killing the adult worms for the elimination/control of lymphatic filariasis and research is very much warranted towards the development of a macrofilaricidal drug. Therefore, the synthesis and evaluation of 1-N-methyl-substituted benzoyl/phenyl acetyl piperazides for macrofilaricidal activity were carried out. The title compounds, 1-N-methyl-substituted benzoyl/phenyl acetyl piperazides were synthesized and purified by slightly modifying the reported procedure and characterized by FT-IR, NMR and mass spectral data. The compounds were screened for macrofilaricidal activity against adult Setaria digitata, the bovine filarial worm for motility and MTT reduction assays using the reported procedures. All the compounds synthesized were characterized by spectral data. Out of 26 compounds synthesized and screened for macrofilaricidal activity, six compounds exhibited moderate antifilarial activity. The structure-activity relationships are discussed. In the case of benzoyl piperazides chloro-substitution in the para position enhanced the activity compared to its substitution in meta and ortho positions. In the case of phenyl acetyl piperazides when methyl group was in the meta position it was more active than when it was in para position. In dimethyl substituted compounds, substitutions at 3, 5-positions were more effective than 2, 3 and 3, 4 substitutions.     

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 μg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 μg/ml. The half maximal inhibitory concentration (IC₅₀) of naringenin for motility of adult females was 2.5 μg/ml. Flavone immobilized female adult worms at 31.2 μg/ml (MTT > 80%) and microfilariae at 62.5 μg/ml. Rutin killed microfilariae at 125 μg/ml and inhibited MTT-reduction in female worms for >65% at 500 μg/ml. Naringin had adulticidal effects at 125 μg/ml while chrysin killed microfilariae at 250 μg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin > flavone = hesperetin > rutin > naringin > chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50 mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.     

Anthelmintic activity of benzimidazole derivatives against Toxocara canis second-stage larvae and Hymenolepis nana adults

The anthelmintic activity of 11 benzimidazole derivatives (A1-A11) and 2 thioureides N,N′-disubstituted (B1-B2) was determined. Each compound and albendazole was tested in vitro against Toxocara canis larvae and in vivo against Hymenolepis nana adult. Compounds A1-A6 and B1-B2 were designed as albendazole prodrugs. Compounds A8-A11 were designed as direct analogues of A7, which had previously proved to be an effective agent against Fasciola hepatica. Results of the in vitro screening showed that A6 was more active than albendazole at 0.18 μM (relative mobility 40% and 80%, respectively). Whereas that the in vivo evaluation against H. nana, compounds A7-A11 demonstrated significant activity in terms of removing cestode adults in the range of 88-97%, displaying better efficacy than albendazole (83%).     

Nerve growth factor enhances survival and cytotoxic activity of human eosinophils

Phenotypic change of blood-type eosinophils to tissue-type eosinophils is induced by various cytokines. We examined the effect of nerve growth factor (NGF) as a candidate for a constitutive cytokine which is able to induce the phenotypic change of eosinophils. The viability of human peripheral blood eosinophils cultured for 4 d was increased from a mean of 26% to a mean of 50% ( P  < 0.001) by the addition of NGF (50 ng/ml). Cytotoxic activity of eosinophils determined by larvicidal activity was increased 2–3-fold by the addition of NGF (50 ng/ml) ( P  < 0.05). Furthermore, eosinophil chemotactic activity of NGF was demonstrated by the blind well chamber method. Since NGF is produced constitutively from various kinds of cells in local tissues, it is suggested that NGF might be a cytokine responsible for phenotypic change to tissue type eosinophils in conditions without immune stimuli.     

Cloning, expression, and biological activity of growth hormone in bullfrog (Rana catesbeiana)

The cDNA (GenBank, AY251538) encoding bullfrog growth hormone (fGH) was cloned by RT-PCR from the total RNA of pituitary glands. Its sequence encoded a putative polypeptide of 215 amino acids, including a signal peptide of 25 amino acids with no change to those of other previous reported bullfrog GHs. The fGH precursor shares 98.1, 96.3, and 95.3% homologies to those of other bullfrog GHs (AAB24792, AAB19428, CAA31038) in amino-acid sequence and its nucleotide sequences of the coding region shares 99.1 and 98.5% homologies to those of previous bullfrog GH genes (S52027 and X12520). The fGH cDNA was also efficiently expressed in Escherichia coli carrying a plasmid pGfGH in which the cDNA was under the control of GST promoter of pGEX1-lambdaT. The expressed fusion protein GST-fGH is comprised about 29.3% of the total cellular protein in such bacteria. The purified GST-fGH cannot only showed a obvious dose-response curve when it reacted with the hepatic membrane receptor proteins from bullfrog, but also significantly increased the body weight and length of bullfrog after twice injection and such effects lasted two or three weeks after the last injection with purified GST-fGH.     

Insecticidal and repellence activity of the essential oil of Pogostemon cablin against urban ants species

Ants are highly abundant in neotropical regions, with certain species adapted to the urban environment, where they can cause damage to human health. The main method for controlling ants consists of using organosynthetic insecticides, which are potentially toxic to the environment. Essential plant oils are considered a viable alternative to the use of conventional insecticides. In this study, we analyze the bioinsecticidal activity and repellence of patchouli essential oil (Pogostemon cablin) against three species of urban ants: Camponotus melanoticus, Camponotus novograndensis, and Dorymyrmex thoracicus. The chemical composition of the essential oil was analyzed by GC–MS and GC-FID. The major compounds were patchoulol (36.6%) followed by α-bulnesene (13.95%), and α-guaiene (11.96%). Toxicity and repellency bioassays were performed using the essential oil over the ants, and mortality evaluations were performed at 4, 24, and 48 h after performing the bioassays. Mortality percentage of the ants on 7 μg/mg was on average 84%. The essential oil of P. cablin displayed toxicity against all three species of urban ants, with the lowest LD₅₀ being observed for D. thoracicus (2.02 μg oil/mg insect) after 48 h of exposure compared to C. melanoticus (2.34 μg oil/mg insect) and C. novogranadensis (2.95 μg oil/mg insect). The essential oil of P. cablin was strongly repellent to the three species of ants in all concentrations tested (0.01% and 1% v/v). Considering the potential toxicity and repellency of the P. cablin essential oil to the urban ants, future studies could investigate the practical application of this oil to control of this insects.     

Laboratory evaluation of aqueous leaf extract of Tephrosia vogelii against larvae of Aedes albopictus (Diptera: Culicidae) and non-target aquatic organisms

Mosquito control using insecticides has been the most successful intervention known to reduce malaria prevalence or incidence. However, vector control is facing a threat due to the emergence of resistance to synthetic insecticides. Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. In this research, the leaf aqueous leachate of Tephrosia vogelii was evaluated for its toxicity against larvae of the most invasive mosquito worldwide, Aedes albopictus (Diptera: Culicidae), and toward adults of the water flea, Daphnia magna (Cladocera: Crustacea) and Oreochromis niloticus, two non-target aquatic organisms that share the same ecological niche of A. albopictus. The leaf aqueous leachate of T. vogelii was evaluated against fourth-instar larvae, non-blood fed 3–5 days old laboratory strains of A. albopictus under laboratory condition. In addition, the objective of the present work was to study the environmental safety evaluation for aquatic ecosystem. Mortality was then recorded after 7 d exposure. The leaf aqueous leachate of T. vogelii showed high mosquitocidal activity against larvae of A. albopictus, with a LC₅₀ = 1.18 μg/mL. However, it had a remarkable acute toxicity also toward adults of the non-target arthropod D. magna, with a LC₅₀ = 0.47 μg/L and O. niloticus with a LC₅₀ = 5.31 μg/L. The present findings have important implications in the practical control of mosquito larvae in the aquatic ecosystem, as the medicinal plants studied are commonly available in large quantities. The extract could be used in stagnant water bodies for the control of mosquitoes acting as vector for many communicable diseases.     

A pharmacology-based comparison of the activity of albendazole and flubendazole against Echinococcus granulosus metacestode in sheep

Cyst echinococcosis (CE) is a zoonotic disease caused by the larval stage of the Echinococcus granulosus helminth parasite. The work reported here aimed to compare the efficacy of albendazole (ABZ) and flubendazole (FLBZ) against CE in naturally infected sheep. Additionally, their comparative pharmacokinetic behaviour and the assessment of serum liver enzymes activities were studied. Twelve (12) naturally infected sheep were allocated to the following experimental groups: unmedicated control group, FLBZ-treated and ABZ-treated.     

Activities of naphthoquinones against Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae), vector of dengue and Biomphalaria glabrata (Say, 1818), intermediate host of Schistosoma mansoni

Larvicidal (against Aedes aegypti Linnaeus, 1762) and molluscicidal (against Biomphalaria glabrata Say, 1818) activities of several natural and synthetic naphthoquinones were measured, with significant results. The best larvicidal compound is 3-bromojuglone, while the better molluscicides are 2-bromo- and 3-bromo-5-acetoxy-1,4-naphthoquinones together with the 3-bromo-5-methoxy derivative. The present results reinforce the potential use of substituted hydroxyquinones, their salts and halogenated quinones as very promising compounds against 4th instar larves of Aedes aegypti, the vector of dengue and against adult snail of Biomphalaria glabrata.     

Combinations of ascaridole,carvacrol, and caryophyllene oxide against Leishmania

To date there are no vaccines against Leishmania and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs currently used for leishmaniasis therapy are significantly toxic, expensive, and result in a growing frequency of refractory infections. In this study, we evaluated the effect of combinations of the main components of essential oil from Chenopodium ambrosioides (ascaridole, carvacrol, and caryophyllene oxide) against Leishmaniaamazonensis. Anti-leishmanial effects of combinations of pure compounds were evaluated in vitro and the fractional inhibitory concentration (FIC) indices were calculated. BALB/c mice infected with L. amazonensis were treated with different concentrations of ascaridole–carvacrol combinations by intralesional doses every 4 days. Disease progression and parasite burden in infected tissues were determined. In vitro experiments showed a synergistic effect of the combination of ascaridole–carvacrol against promastigotes of Leishmania with a FIC index of 0.171, while indifferent activities were observed for ascaridole–caryophyllene oxide (FIC index = 3.613) and carvacrol–caryophyllene oxide (FIC index = 2.356) combinations. The fixed ratio method showed that a 1:4 ascaridole–carvacrol ratio produced a better anti-protozoal activity on promastigotes, lower cytotoxicity, and synergistic activity on intracellular amastigotes (FIC index = 0.416). Significant differences (p < 0.05) in lesion size and parasite burden were demonstrated in BALB/c mice experimentally infected and treated with the ascaridole–carvacrol combinations compared with control animals. Carvacrol showed significant higher anti-radical activity in the DPPH assay compared with caryophyllene oxide. Electron spin resonance spectroscopy in combination with spin trapping suggested the presence of carbon-centered radicals after activation of ascaridole by Fe²⁺. The intensity of the signals is preferably decreased upon addition of carvacrol. The ascaridole–carvacrol combination could represent a future alternative to monotherapeutic anti-leishmanial agents.     

Mosquito larvicidal and antimicrobial activity of synthesized nano-crystalline silver particles using leaves and green berry extract of Solanum nigrum L. (Solanaceae: Solanales)

Silver nanoparticles (AgNPs) that are synthesized by using aqueous extracts of Solanum nigrum L., is a simple, non-toxic and ecofriendly green material. The present study is based on assessments of the larvicidal and antimicrobial activities of the synthesized AgNPs from fresh leaves, dry leaves and green berries of S. nigrum against larvae of Culex quinquefasciatus and Anopheles stephensi and four human pathogenic and five fish pathogenic bacteria respectively. The synthesized nanoparticles are characterized with UV–vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscope (TEM) analysis. The nanoparticles are spherical to polyhedral in shape with size of 50–100 nm (average size of 56.6 nm). In larvicidal bioassay with synthesized AgNPs, highest mortality are observed at 10 ppm against An. stephensi with LC₅₀ values of 1.33, 1.59, 1.56 ppm and LC₉₀ values of 3.97, 7.31, 4.76 ppm for dry leaves, fresh leaves and berries respectively. Antibacterial activity test reveals better results against fish pathogenic bacteria than human pathogenic bacteria. Non target organism like Toxorhynchites larvae (mosquito predator), Diplonychus annulatum (predatory water-bug) and Chironomus circumdatus larvae (chironomid) are also exposed to respective lethal concentrations (to mosquito larvae) of dry nanoparticles and no abnormality in the non target organisms are recorded. These results suggest that the synthesized AgNPs of S. nigrum have the potential to be used as an ideal eco-friendly compound for the control of the mosquito larvae and harmful bacteria.     

Characterization and regulation of the insulin-like growth factor (IGF) system in the zebrafish (Danio rerio) ovary

Locally produced peptide hormones play an important role in the paracrine/autocrine regulation of ovarian development. The insulin-like growth factor (IGF) system is one family of local factors that has not been well studied in the ovary of fish. This study characterized the zebrafish (Danio rerio) ovarian IGF system, its spatial and temporal expression and regulation by gonadotropins and steroids. Three ligands (igf2a, igf2b, igf3) and two receptors (igf1ra and igf1rb) were demonstrated in the ovary using RT-qPCR. Though it was examined, igf1 expression was not detected in the zebrafish ovary. Igf3 expression significantly decreased in the hours prior to ovulation and was confined to the follicle cells. Igf2a, igf2b and the two receptors were detected in both the follicle cells and the oocyte and were constitutively expressed in ovarian tissue across the daily ovulatory cycle. In vitro addition of human chorionic gonadotropin (hCG; 20 IU/ml) stimulated a significant increase in igf3 expression in both midvitellogenic (MV; 0.45-0.56 mm) and full grown (FG; 0.57-0.65 mm) follicles while igf2b expression increased only in FG follicles. Treatment of follicles in vitro with 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P; 10 ng/ml) significantly decreased igf3 and igf2b expression in both MV and FG follicles. 17β-Estradiol (E₂; 25 ng/ml) had no effect on the expression of igf3 in MV or FG follicles. Igf1rb expression did not change after treatment with hCG, 17,20β-P or E₂. Collectively, these results demonstrate the presence of an ovarian IGF system in zebrafish that is differentially regulated by gonadotropin and steroids.     

The role of insulin-like growth factor-1 (IGF-1) in growth and reproduction in female brown house snakes (Lamprophis fuliginosus)

Insulin-like growth factor-1 (IGF-1) is a peptide hormone critically involved in the regulation of key life-history traits such as growth and reproduction. Its structure and function are well-characterized among diverse mammal, fish, and bird species; however, little is known regarding the activities of IGF-1 in non-avian reptiles, particularly snakes and lizards. Nevertheless, several unique characteristics of reptiles, such as high metabolic flexibility and remarkable diversity in life-history strategy, suggest that they are of great interest in the study of endocrinological mechanisms underlying the regulation and evolution of life-history traits. Here we test for a relationship between IGF-1 and individual feeding rate, growth rate and reproductive stage in lab-reared female offspring of wild-caught oviparous house snakes, Lamprophis fuliginosus. We confirm a positive correlation between IGF-1 and both feeding and growth rates in sexually immature snakes, similar to that reported in other taxa. We also show a family effect on IGF-1, suggesting that IGF-1 levels may be heritable in these snakes, and serve as an important target of selection to produce divergent life-history strategies. Furthermore, we provide evidence that suggests that IGF-1 may peak rapidly after first mating, and subsequently decline prior to egg-laying, a phenomenon not previously reported in other taxa. These findings suggest that further comparative study of IGF-1 in snakes may reveal both the extent to which IGF-1 function is conserved across major taxonomic groups, as well as novel and intriguing roles for IGF-1 in the regulation of reproductive activities.     

Large-scale preparation and in vitro characterization of biologically active human placental (20 and 22K) and pituitary (20K) growth hormones: Placental growth hormones have no lactogenic activity in humans

Expression plasmids containing DNA sequences optimized for expression in Escherichia coli were prepared encoding human pituitary (hGH-N 20K) and placental (hGH-V 20 and 22K) growth hormones. The proteins were expressed in bacteria, refolded and purified to homogeneity by anion-exchange chromatography on Q-Sepharose according to a unique protocol developed for each protein. The yields from 5l of fermentation culture varied between 400 and 700mg of electrophoretically pure, over 95% monomeric protein. Circular dichroism (CD) analysis revealed similarity of the purified hGHs' secondary structure to that of the pituitary hGH-N 22K, except for hGH-V 20K, in which the alpha-helix content was lower. The purified proteins were stable as a 0.1% sterile solution held at pH 10-11 at 4 degrees C for at least one month. All three purified hGH molecules formed a 1:2 complex with hGH receptor extracellular domain (hGHR-ECD), similar to hGH-N 22K. Binding experiments using hGHR-ECD revealed that the differences between the two 22K variants or between the two 20K variants were not significant, except that hGH-V 20K exhibited slightly lower affinity. Somatogenic activity was tested in vitro using FDC-P1 cell lines. Whereas the bioactivity of 22K hGHs and hGH-N 20K in FDC-P1-9D11 cells stably transfected with hGHR was almost equal and two to threefold higher than that of hGH-V 20K, in FDC-P1 3B9 cells stably transfected with rabbit (rb) GHR, the bioactivity of both 20K analogues was significantly (five to ninefold) lower than that of the 22K hormones. The lactogenic activity measured in heterologous assays (Nb2-11C cells and Baf/3 cells stably transfected with the long form of rabbit prolactin receptor) revealed that the activity of hGH-N 20K was close to that of hGH-N 22K in the Baf/3 cells, but 4.5-fold lower in the Nb2 cells. The activity of hGH-V 22K was ninefold less in Nb2 cells and 55-fold less in Baf/3 cells, whereas hGH-V 20K had no lactogenic activity in either bioassay. In contrast, in a homologous lactogenic assay using Baf/3 LP cells stably transfected with hPRLR, the activity of both placental hGHs was nil and the activity of hGH-N 20K was 4.3-fold lower than that of hGH-N 22K. The latter finding raises the question of whether the lack of intrinsic lactogenic activity in the placental hGHs that dominate during pregnancy has any physiological relevance.     

Differential involvement of signaling pathways in the regulation of growth hormone release by somatostatin and growth hormone-releasing hormone in orange-spotted grouper (Epinephelus coioides)

Somatostatin is the most effective inhibitor of GH release, and GHRH was recently identified as one of the primary GH-releasing factors in teleosts. In this study, we analyzed the possible intracellular transduction pathways that are involved in the mechanisms induced by SRIF and GHRH to regulate GH release. Using a pharmacological approach, the blockade of the PLC/IP/PKC pathway reversed the SRIF-induced inhibition of GH release but did not affect the GHRH-induced stimulation of GH release. Furthermore, SRIF reduced the GH release induced by two PKC activators. Inhibitors of the AC/cAMP/PKA pathway reversed both the SRIF- and GHRH-induced effects on GH release. Moreover, the GH release evoked by forskolin and 8-Br-cAMP were completely abolished by SRIF. The blockade of the NOS/NO pathway attenuated the GHRH-induced GH release but had minimal effects on the inhibitory actions of SRIF. In addition, inhibitors of the sGC/cGMP pathway did not modify the SRIF- or GHRH-induced regulation of GH release. Taken together, these findings indicate that the SRIF-induced inhibition of GH release is mediated by both the PLC/IP/PKC and the AC/cAMP/PKA pathways and not by the NOS/NO/sGC/cGMP pathway. In contrast, the GHRH-induced stimulation of GH secretion is mediated by both the AC/cAMP/PKA and the NOS/NO pathways and is independent of the sGC/cGMP pathway and the PLC/IP/PKC system.