Long-term in vivo imaging of translated RNAs for gene therapy.

To determine the potential of RNA for transient expression, we followed its translational efficiency and expression kinetics in vivo in mouse skin. Three RNA species were delivered in vivo with differing 5' and 3' ends, as well as with different structures that are known to influence their translation fate, such as an internal ribosome entry site (IRES), a cap or a poly(A) tail. RNAs were transferred by electropermeabilization, and each encoded the firefly luciferase enzyme to allow monitoring of translational efficiency by in vivo bioluminescence imaging. We show that all types of naked RNAs delivered into mouse skin are efficient for transient protein expression in vivo. Expression could be achieved with some differences in efficiency and time course, using either capped/polyadenylated RNAs or RNAs containing HCV IRES structures with or without a poly(A) tail. Our data reveal expression occurring up to 2 weeks, suggesting that electroporated RNA has high stability in vivo, particularly capped and polyadenylated RNAs. Our study shows that RNA molecules are efficient tools for the transient expression of proteins in vivo and that they can be used for therapeutic purposes. Changes in RNA features may be used to modulate both expression efficiency and kinetics.

Evidence for regulation of juvenile hormone biosynthesis operating before mevalonate in locust corpora allata.

Exogenous mevalonate poorly stimulated juvenile hormone III (JH-3) biosynthesis by corpora allata from Locusta migratoria. However, mevalonolactone strongly stimulated glands from different physiological states and fully restored JH synthesis in mevinolin-treated glands. Asymmetry in spontaneous rate of JH release was abolished by exogenous mevalonolactone. After transection of nervus corporis allati 1 (NCA-1), the rate of mevalonolactone-stimulated JH synthesis was maintained at the preoperative levels although the spontaneous rate of JH biosynthesis decreased rapidly. These results suggest that the spontaneous asymmetry of JH biosynthesis and the low rate of JH biosynthesis by denervated corpora allata both result from non-stimulation or inhibition acting on the JH pathway before the utilisation of mevalonate.

The pheromone biosynthesis activating neuropeptide (PBAN) of the black cutworm moth, Agrotis ipsilon: immunohistochemistry, molecular characterization and bioassay of its peptide sequence.

PBAN-like immunoreactivity has been detected in the suboesophageal ganglion and the brain (Br-SOG) of larvae and adult males and females of Agrotis ipsilon, using an antiserum against Helicoverpa zea PBAN (Hez-PBAN). The amino acid sequence of A. ipsilon PBAN (Agi-PBAN) was deduced from the cDNA sequence, using both Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and 5' Rapid Amplification of cDNA Ends (RACE). The primers were degenerate sets of oligonucleotides derived from known amino acid sequences of the PBAN precursor. The final cloned fragment contained the complete DNA sequence coding for the putative Agi-PBAN. Based on a comparison with known PBAN processing from the polypeptide precursor, we propose that Agi-PBAN is a 33-amino acid peptide. Agi-PBAN exhibits high sequence homology with Hez-PBAN (88%), Lymantria dispar PBAN (Lyd-PBAN, 88%) and Bombyx mori PBAN (Bom-PBAN, 73%). Agi-PBAN shares the C-terminal hexapeptide sequence (Tyr-Phe-Ser-Pro-Arg-LeuNH2) with all identified PBANs but has only one methionine residue instead of two in Hez-PBAN and Lyd-PBAN, and three in Bom-PBAN. Based on predicted a.a. sequence, Agi-PBAN, with Leu-NH2 as C-terminal motif, has been synthesized and assayed for its ability to promote pheromone production in decapitated females of A. ipsilon. Synthetic Agi-PBAN induced pheromone production in decapitated females as evaluated by the male responsiveness to the pheromonal blend in a wind tunnel.

A cDNA, from Agrotis ipsilon, that encodes the pheromone biosynthesis activating neuropeptide (PBAN) and other FXPRL peptides.

A cDNA encoding the prohormone of the pheromone biosynthesis activating neuropeptide (PBAN) in the moth Agrotis ipsilon was isolated. The cDNA contains 834 nucleotides, coding for a 193-amino acid protein that exhibits 89% identity with PBAN prohormones of other moths. The prohormone contains five potential peptides belonging to the FXPRL family. The peptide corresponding to the Bombyx mori diapause hormone exhibits an extra residue, and the C-terminal leucine is replaced by an isoleucine, introducing a new type of variability in this family of peptides. Northern blot analysis revealed expression in suboesophagal ganglion complexes. Constitutive heterologous expression of Agi-PBAN cDNA in yeast, using three different antibodies, did not produce PBAN-immunoreactive material.

Biosynthetic activity of corpora allata, growth of sex accessory glands and mating in the male moth agrotis ipsilon (Hufnagel)

The involvement of both juvenile hormone acid (JHA) and the sex accessory glands (SAGs) in the reproduction of the male moth Agrotis ipsilon was studied as a function of age and mating status. Total protein content analysis followed by gel electrophoresis of the SAGs, radiochemical assay for JHA biosynthesis and surgical and behavioural experiments were performed. Both the protein content of the SAGs and the biosynthetic activity of the corpora allata (CA) increased with age. Allatectomy and JHA/JH treatments showed that the protein content of the SAGs is linked with the activity of the CA. The protein content of the glands, but not the rate of JHA biosynthesis, decreased just after mating, and both increased sharply 24 h later. Injection of fluvastatin, an inhibitor of JH biosynthesis, in males immediately after mating prevented the increase in JHA synthesis and lowered the total protein content of the SAGs. Moreover, fluvastatin disrupted normal spermatophore transfer during the next mating of the injected males. Our results show that JHA controls the reproduction of A. ipsilon males by its separate actions on the sex accessory glands and on sexual behaviour.

[Effect of olive leaves (Olea europea) feeding on the in vitro JH III biosynthesis by the corpora allata in Schistocerca gregaria during vitellogenesis]. / Action d'une alimentation à base de feuilles d'olivier (Olea europea) sur la biosynthèse in vitro de la JH III par les corpora allata chez Schistocerca gregaria au cours de la vitellogenèse.

Per os administration of olive leaves (Olea europea) to females of Schistocerca gregaria results in stopping vitellogenesis. These vitellogenins are not synthesised by the fat body in the heamolymph. The vitellogenin inhibition is induced by the stopping of juvenile hormone JH III by the corpora allata. These corpora allata (Medicago sp.) Synthesise 10 times less JH III than those of alfalfa fed females.

Molecular MR imaging and MR-guided ultrasound therapies in cancer.

Imaging in cancer has moved in the last twenty years from morphological detection of diseases to characterization and categorization of different subtypes of tumors. Functional information, based on dynamic contrast-enhanced imaging of tissue perfusion and evaluation of water diffusion, tissue oxygenation, capillary permeability or lymphatic drainage, plays a major role in that field.The next coming steps will concern the differentiation of biological behaviour of tumors according to their phenotypes by identifying specific surface receptors or products of synthesis.These developments allowing an in vivo identification of the tumor biological singularities is a tremendous progress in the management of cancer at the step of diagnosis but, more importantly, to assess the most appropriated treatment to each tumor type. At the same time, minimally invasive methods of treatment of tumors have also developed, mainly in the field of thermotherapies. Ablation of tumors using radiofrequency is now used in clinics as a new standard within the liver and as a promising additional option in many other organs as kidney, lung and bone. High intensity focused ultrasound (HIFU) showed more restricted developments in clinics, mainly applied to prostatic cancer, because of many technical barriers. We believe that magnetic resonance (MR) imaging and MR-guided HIFU (MRgHIFU) have a great potential in that field due to the capacity of MR imaging to monitor temperature changes for an optimal heat deposition and for an optimal safety. This technique has already gained recognition for the treatment of uterine leiomyomas. But it has still to prove its efficacy in treatment of malignant tumors. This review will focus on some recent developments in molecular characterisation of tumors using MR imaging and in technical improvements necessary for accurate application of MRgHIFU in cancer.

Gas chromatography-mass spectroscopy analysis of juvenile hormone released by insect corpora allata.

Corpora allata incubated in appropriate medium release several compounds including juvenile hormones. Juvenile hormones (14C labeled or unlabeled) were extracted with hexane and directly analyzed by gas chromatography-mass spectroscopy. This method allowed the qualitative and quantitative analysis of total released juvenile hormone. It could also be used as a routine assay for evaluation of corpus allatum activity. Data obtained by this method were compared to those obtained by radiochemical assay.

Juvenile-hormone-mediated plasticity of aggregation behaviour and olfactory processing in adult desert locusts.

In desert locusts Schistocerca gregaria, aggregation behaviour is elicited by aggregation pheromones. In this study, we show that the behavioural response to the major and most potent adult aggregation pheromone component, phenylacetonitrile, is age- and juvenile-hormone-dependent. Furthermore, we show that juvenile hormone influences the responsiveness of olfactory interneurons in the antennal lobe to aggregation pheromone, whereas the responsiveness of antennal receptor neurons is not changed. Old locusts and locusts injected with juvenile hormone, in contrast to young locusts and locusts deprived of juvenile hormone through allatectomy, i.e. after surgical removal of the gland producing this hormone, do not display any aggregation behaviour, as indicated by long-term behavioural observations. The lack of positive olfactory-guided behaviour coincides with an impairment of the central olfactory system, which displays a lower number of neurons responding to aggregation pheromone. Indirect and direct actions of juvenile hormone at different levels of the central nervous system may thus contribute to the regulation and modulation of behavioural responsiveness in the locust.

The HMG-CoA reductase inhibitor fluvastatin inhibits insect juvenile hormone biosynthesis.

Fluvastatin (Sandoz Compound XU 62-320), a synthetic HMG-CoA reductase inhibitor, was assayed in vitro and in vivo for its ability to suppress juvenile hormone (JH) biosynthesis by corpora allata of Locusta migratoria migratorioides. Fluvastatin inhibited JH biosynthesis by corpora allata in vitro. Exogenous mevalonic acid lactone restored JH biosynthesis in corpora allata inhibited by fluvastatin. Fluvastatin injected into locusts in vivo inhibited JH biosynthesis, but maximal inhibition lasted for only 6 hr. There were no discernible effects on either JH-regulated metamorphosis or oocyte maturation. Lengthening of the fourth larval stadium was observed and increased doses (single or repeated injections) were fatal.

The Release of Isoprenoids by Locust Corpora Allata in vitro.

Corpora allata of the African locust Locusta migratoria, incubated in vitro, biosynthesized together with juvenile hormone III (JH-III), several molecules labelled by both [2-(14)C] sodium acetate and L-[methyl-(3)H] methionine. By a combination of chromatographic procedures including reverse phase-high-performance liquid chromatography (HPLC), normal phase HPLC and thin layer chromatography (TLC), four labelled compounds were separated. They were isoprenoids, as revealed by inhibition of their synthesis by the hydroxy-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor fluvastatin and restoration by exogenous mevalonolactone. They could be produced by incubating corpora allata with JH-III, suggesting that they were JH-III metabolites. They were produced by the corpora allata from both males and females and released into the incubation medium. Their rate of synthesis changed considerably depending on the sample, and in some cases they were the major isoprenoic products of the corpora allata.

Molecular cloning and tissue expression of an insect farnesyl diphosphate synthase.

The enzyme farnesyl-diphosphate synthase (FPS, EC2.5.1.1/EC2.5.1.10), which has been shown to play a key role in isoprenoid biosynthesis, catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and di-methylallyl diphosphate. Insects do not synthesize cholesterol de novo, rather farnesyl diphosphate leads to the formation of nonsterol isoprenoids, which are essential for insect development and reproduction. In this paper, we describe the characterization of one FPS from the moth Agrotis ipsilon, the first insect FPS to be reported. An homologous probe was obtained through a nested PCR strategy using degenerate primers designed from the conserved domains of FPS from other organisms. The complete cDNA clone was isolated by PCR screening of a brain cDNA library by using homologous primers deduced from the probe. Analysis of the nucleotide sequence revealed that the cDNA encodes a polypeptide of 412 amino acids (Mr = 47 170), which shares regions similar to the FPS of other organisms, but exhibits singularities such as an extra N-terminal extension of approximately 70 amino acid residues. Using an RNase protection assay, a protected fragment corresponding to the region encoding the FPS catalytic site was found in brain, ovary, fat body and corpora allata samples, but not in muscle. FPS is overexpressed in the corpora allata, the endocrine gland that produces the juvenile hormones. These hormones are specific to insects and play a crucial role in regulating insect physiology.

Molecular cloning and structural analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase of the moth Agrotis ipsilon.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which plays a key role in isoprenoid biosynthesis, catalyses the synthesis of mevalonate from HMG-CoA. Insects do not synthesize cholesterol de novo, rather mevalonate derivatives lead to non-sterol isoprenoids which are essential for development and reproduction. In this paper, we describe an HMG-CoA reductase of the moth Agrotis ipsilon and we report its expression in fat body, ovary, muscle, brain and corpora allata tissues of adult specimens. The analysis of the cDNA reveals that it encodes a polypeptide of 833 amino acids (Mr = 89785). Alignments of this HMG-CoA reductase from A. ipsilon with the homologous sequences of other eukaryotes shows a high degree of conservation in all species studied. Parsimony analysis based on these alignments produced dendrograms congruent with the current systematic schemes. This suggests that, during eukaryote evolution, HMG-CoA reductase diversified in parallel with taxonomic splitting.

Hydroxy juvenile hormones: new putative juvenile hormones biosynthesized by locust corpora allata in vitro.

The in vitro production of sesquiterpenoids was investigated by using corpora allata (CA) of the African locust Locusta migratoria migratorioides. Labeled products from unstimulated biosynthesis were extracted, purified by normal phase HPLC, and derivatized to determine the functional groups present. An extra hydroxyl group was detected in each of two juvenile hormone (JH) biosynthetic products. One compound, NP-8, was found to co-migrate with a chemically-synthesized (Z)-hydroxymethyl isomer, 12'-OH JH-III, but not with the (E)-hydroxymethyl isomer, 12-OH JH III. Mass spectral analyses further supported the identity of the synthetic material with that biosynthesized by the corpora allata. A second compound was identified as the 8'-OH JH-III based on spectroscopic analyses. 12'-OH JH-III exhibited morphogenetic activity when tested on the heterospecific Tenebrio test. These data suggest that 12'-OH JH-III and 8'-OH JH-III are additional biosynthetically-produced and biologically-active juvenile hormones, and constitute the first known members of the class of hydroxy juvenile hormones (HJHs).

Immunological evidence for an allatostatin-like neuropeptide in the central nervous system of Schistocerca gregaria, Locusta migratoria and Neobellieria bullata.

Methanolic brain extracts of Locusta migratoria inhibit in vitro juvenile hormone biosynthesis in both the locust L. migratoria and the cockroach Diploptera punctata. A polyclonal antibody against allatostatin-5 (AST-5) (dipstatin-2) of this cockroach was used to immunolocalize allatostatin-5-like peptides in the central nervous system of the locusts Schistocerca gregaria and L. migratoria and of the fleshfly Neobellieria bullata. In both locust species, immunoreactivity was found in many cells and axons of the brain-retrocerebral complex, the thoracic and the abdominal ganglia. Strongly immunoreactive cells were stained in the pars lateralis of the brain with axons (NCC II and NCA I) extending to and arborizing in the corpus cardiacum and the corpora allata. Although many neurosecretory cells of the pars intercerebralis project into the corpus cardiacum, only 12 of them were immunoreactive and the nervi corporis cardiaci I (NCC I) and fibers in the nervi corporis allati II (NCA II) connecting the corpora allata to the suboesophageal ganglion remained unstained. S. gregaria and L. migratoria seem to have an allatostatin-like neuropeptide present in axons of the NCC II and the NCA I leading to the corpus cardiacum and the corpora allata. All these data suggest that in locusts allatostatin-like neuropeptides might be involved in controlling the production of juvenile hormone by the corpora allata and, perhaps, some aspects of the functioning of the corpus cardiacum as well. However, when tested in a L. migratoria in-vitro juvenile hormone-biosynthesis assay, allatostatin-5 did not yield an inhibitory or stimulatory effect. There is abundant AST-5 immunoreactivity in cell bodies of the fleshfly N. bullata, but none in the CA-CC complexes. Apparently, factors that are immunologically related to AST-5 do occur in locusts and fleshflies but, the active portion of the peptide required to inhibit JH biosynthesis in locusts is probably different from that of AST-5.