Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng, 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids. glucose and yeastolate ultrafiltrate) into Sf9 cell cultures grown in SF900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 mug/ml of NS1 at a peak cell density of 4.2 x 10(6) cells/ml. In contrast. optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 mug ml of NS1 produced at a peak cell density of 11.3 x 10(6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10(6) cells/ml, unless the cultures were stimulated by the addition of 4 mug/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 mug/ml. which was nearly 5-fold higher than optimised batch cultures. (C) 2002 Elsevier Science B.V. All rights reserved.

Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect`s life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE. (C) 2008 Elsevier Inc. All rights reserved.

Indirect immune detection of ecdysone receptor (EcR) during the formation of DNA puffs in Bradysia hygida (Diptera, Sciaridae)

Gene amplification occurs in Bradysia hygida salivary glands, at the end of the fourth larval instar. The hormone 20-hydroxyecdysone (20E) triggers this process, which results in DNA puff formation. Amplified genes are activated in two distinct groups. The activity of the first group is dependent on high levels of 20E, while the second group needs low hormone levels. Consequently, the salivary glands of B. hygida constitute an interesting biological model to study how 20E, and its receptors, affect gene amplification and activity. We produced polyclonal antibodies against B. hygida EcR (BhEcR). In western blots a polypeptide of about 66 kDa was detected in salivary gland extracts. The antibodies were also used for indirect immune-localization of BhEcR in polytene chromosomes. RNA-polymerase II was also immune-detected. We did not detect the receptor in chromosome C where the first and second groups of DNA puffs form during DNA puff anlage formation, but it was present during puff expansion. During the active phase of both groups of DNA puffs, RNA polymerase II co-localized with BhEcR. After puff regression, these antigens were not detected. Apparently, EcR plays a direct role in the transcription of amplified genes, but its role in gene amplification remains enigmatic.

The vitellogenin gene family of Aedes aegypti

We have been interested in identifying genes that play a role in reproduction of the mosquito Aedes aegypti. Our interests are currently focused on the vitellogenin genes which in the mosquito are expressed only in the fat body in response to the insect steroid hormone, 20-hydroxyecdysone. Four of the five vitellogenin genes in the genome have been cloned. We have examined the relationships between these genes and find that they form a small gene family exhibiting different levels of relationship.

Molecular aspects of Schistosoma mansoni female maturation

Incubation of total protein extracts of Schistosoma mansoni with 3H 17-beta-estradiol and 20-hydroxyecdysone, revealed steroid binding proteins in both, male and female worms. The interaction of nuclear proteins with restriction fragments of the gender and stage-specific gene F-10 was investigated using the "Band-Shift" technique. Distinct male and female nuclear proteins bound to the fragments of this gene. Among the nuclear proteins, only those rich in cysteine residues bound to DNA. In vitro incubation of live worms with the estrogen antagonist Tamoxifen, altered the pattern of the DNA binding proteins, producing in females, a band profile similar to that obtained with male worm protein extracts. When Tamoxifen was injected into schistosome infected mice, the eggs produced by females presented an abnormal morphology, compatible with non-viable eggs. These results suggest that the regulation of transcription of the F-10 gene might involve steroid receptors.

Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis.

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the "black box", remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.

Efeito da adição de colesterol e ecdisona na produção in vitro do baculovírus spodoptera frugiperda MNPV

Among the pests that attack corn crop in Brazil, there is Spodoptera frugiperda (JE Smith, 1797) (Lepidoptera: Noctuidae), known as fall armyworm, which is the major corn pest. Due to genetic instability during serial passage of baculoviruses in insect cell culture, the viral bioinseticides in vitro production development is the greatest challenge for mass production of this bioproduct. Successive passages of virus using extracellular viruses (BVs), necessary during viral bioinseticides production scaling up, leads to the appearance of aberrant forms of virus, a process so called as "passage effect ". The main consequence of passage effect is the production of occlusion bodies (OB) decrease, preventing its production using in vitro process. In this study, it was carried out a serial passage of baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus, isolate 18, using Sf21 cells. A decrease in the production of occlusion bodies from 170 to 92 in the third to fourth passage was observed. A factorial experimental design (22) was employed to verify the influence of two input variables, concentration of the hormone 20 - hydroxyecdysone (CH) and cholesterol (CC) on the values of response variables (volumetric and the specific OB production) of the process, seeking to define the optimum operating ranges trying to reverse or minimize the passage effect. The result indicated a negative influence of the cholesterol addition and positive effect in the hormone supplementation which the optimum range found for the concentrations studied were 8 to 10μg/mL and 5 to 6.5 mg / mL, for cholesterol and hormone concentrations respectively. New experiments were performed with addition of hormone and cholesterol in order to check the influence of these additives on the OB production independently. While the best result obtained from the factorial experiment was 9.4 x 107 OB/mL and 128.4 specific OB/cell, with the addition of only 6μg/mL 20-hydroxyecdysone these concentrations increased to 1.9 x 108 OB/mL and 182.9 OB/cell for volumetric and specific OB production, respectively. This result confirms that the addition of the hormone 20-hydroxyecdysone enhances the SfMNPV in vitro production process performance using Sf21 cells

Selectivity of diacylhydrazine insecticides to the predatory bug Orius laevigatus: In vivo and modeling/docking experiments

BACKGROUND: Knowledge of pesticide selectivity to natural enemies is necessary for a successful implementation of biological and chemical control methods in integrated pest management (IPM) programs. Diacylhydrazine (DAH)-based ecdysone agonists also known as molting-accelerating compounds (MACs) are considered a selective group of insecticides, and their compatibility with predatory Heteroptera, which are used as biological control agents, is known. However, their molecular mode of action has not been explored in beneficial insects such as Orius laevigatus (Fieber) (Hemiptera: Anthocoridae). RESULTS: In this project in vivo toxicity assays demonstrated that the DAH-based RH-5849, tebufenozide and methoxyfenozide have no toxic effect against O. laevigatus. The ligand-binding domain (LBD) of the ecdysone receptor (EcR) of O. laevigatus was sequenced and a homology protein model was constructed which confirmed a cavity structure with 12 ?-helixes, harboring the natural insect molting hormone 20-hydroxyecdysone. However, docking studies showed that a steric clash occurred for the DAH-based insecticides due to a restricted extent of the ligand-binding cavity of the EcR of O. laevigatus. CONCLUSIONS: The insect toxicity assays demonstrated that MACs are selective for O. laevigatus. The modeling/docking experiments are indications that these pesticides do not bind with the LBD-EcR of O. laevigatus and support that they show no biological effects in the predatory bug. These data help in explaining the compatible use of MACs together with predatory bugs in IPM programs. Keywords: Orius laevigatus, selectivity, diacylhydrazine insecticides, ecdysone receptor, homology modelling, docking studies.

Nonsteroidal moulting hormone agonists: effects on protein synthesis and cuticle formation in colorado potato beetle larvae.

Larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say), that were orally treated with RH-0345 at 0.1 mg l?1, RH-5849 at 10 and 50 mg l?1, tebufenozide at 2 g l?1, and 20-hydroxyecdysone at 2 g l?1, showed symptoms of prematuremoulting, followed by inhibition of ecdysis. In addition, fresh weight gain and total protein content were blocked. The effects on haemolymphal and cuticular polypeptides after PAGE were linked with premature, new epicuticle deposition as was observed under the electron microscope. These observations support the concept that the ecdysteroid-mimicking action of the three nonsteroidal molecules is specific

Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used Anopheles gambiae, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5′ flanking sequence (−855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5′ flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the Aedes aegypti ecdysone receptor/Ultraspiracle nuclear receptor complex, and, seemingly, the endogenous Anopheles gambiae nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an A. gambiae cell line.

Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members.

We have designed a rapid cloning and screening strategy to identify new members of the nuclear hormone receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Using this approach, we isolated three Drosophila genes, designated DHR38, DHR78, and DHR96. All three genes are expressed throughout third-instar larval and prepupal development. DHR38 is the Drosophila homolog of NGFI-B and binds specifically to an NGFI-B response element. DHR78 and DHR96 are orphan receptor genes. DHR78 is induced by 20-hydroxyecdysone (20E) in cultured larval organs, and its encoded protein binds to two AGGTCA half-sites arranged as either direct or palindromic repeats. DHR96 is also 20E-inducible, and its encoded protein binds selectively to the hsp27 20E response element. The 20E receptor can bind to each of the sequences recognized by DHR78 and DHR96, indicating that these proteins may compete with the receptor for binding to a common set of target sequences.

Six new cases confirm the clinical molecular profile of complete combined 17α-hydroxylase/ 17,20-lyase deficiency in Brazil

In 2004, Costa-Santos and cols. reported 24 patients from 19 Brazilian families with 17&#945;-hydroxylase deficiency and showed that p.W406R and p.R362C corresponded to 50% and 32% of CYP17A1 mutant alleles, respectively. The present report describes clinical and molecular data of six patients from three inbred Brazilian families with 17&#945;-hydroxlyse deficiency. All patients had hypogonadism, amenorrhea and hypertension at diagnosis. Two sisters were found to be 46,XY with both gonads palpable in the inguinal region. All patients presented hypergonadotrophic hypogonadism, with high levels of ACTH (&gt; 104 ng/mL), suppressed plasmatic renin activity, low levels of potassium (< 2.8 mEq/L) and elevated progesterone levels (&gt; 4.4 ng/mL). Three of them, including two sisters, were homozygous for p.W406R mutation and the other three (two sisters and one cousin) were homozygous for p.R362C. The finding of p.W406R and p.R362C in the CYP17A1 gene here reported in additional families, confirms them as the most frequent mutations causing complete combined 17&#945;-hydroxylase/17,20-lyase deficiency in Brazilian patients.

A ginastica em Campinas : suas formas de expressão da decada de 20 a decada de 70

Universidade Estadual de Campinas. Faculdade de Educação Física

Teste "W" 20 metros : proposta computadorizada para analise da performance especifica em atletas de voleibol

Universidade Estadual de Campinas . Faculdade de Educação Física

Avaliação isocinetica de joelho em jogadores de futebol categoria sub-20

Universidade Estadual de Campinas . Faculdade de Educação Física