During the last decade, it has become apparent that baculoviruses not only represent a powerful expression system for production of recombinant proteins in insect cells but also can be used for transduction of dividing and nondividing mammalian cells and tissues in vitro, ex vivo, and in vivo (49). Advantages of the use of baculoviruses as gene delivery agents include their inability to replicate in mammalian cells, apparent lack of cytotoxicity, capacity to sustain large insertions of foreign DNA, ability to target many different cell types, and superior safety features relative to mammalian virus-based transduction systems (25,37,38,48). Thus, increasing interest for the development of recombinant baculoviruses as gene delivery vectors for use in human gene therapy exists.All baculovirus vectors for gene delivery in mammalian cells reported thus far have been based on the Autographa californica nuclear polyhedrosis virus (AcNPV). This is primarily due to the relatively wide host range of the virus and the multiplicity of lepidopteran cell lines in which it can be grown and propagated effectively as well as the availability of integrated methodologies allowing the rapid generation of recombinant viruses (55, 80). In contrast, there is a paucity of information regarding the suitability of other baculovirus species, particularly species with narrow host ranges to function as transduction vectors for mammalian cells.In this regard, it is also known that AcNPV has a propensity to express endogenous viral genes in nonhost insect species (12, 13). Moreover, studies involving transfection of mammalian cells with gene constructs employing baculovirus gene promoter elements have shown that certain early baculovirus promoters are marginally functional in mammalian cells and can also be activated by mammalian virus regulators (46, 66). These findings raise safety concerns related to the potential for low-level baculovirus gene expression in mammalian cells and the triggering of cellular immune responses against the transduced cells by the recipient host (61).In this work, we have explored the capacity of Bombyx mori NPV (BmNPV), the second best characterized baculovirus species after AcNPV (2,20,21,27,40,56,57,65), which has a very limited host range (58), to function as a transducing vector for mammalian cell lines and primary Schwann cells. The choice for the latter rests with the results of experimental transplantation in rodent and primate models, which has provided substantial evidence that Schwann cells are good candidates for cell therapy in human central nervous system (CNS) demyelinating diseases, such as multiple sclerosis, and trauma (5,6,9,19,26). Schwann cells provide trophic support and remyelinate demyelinated CNS axons. However, their integration in the CNS is limited. Therefore, modifying Schwann cells to express "therapeutic" factors enhancing axonal regeneration and remyelination by ex vivo gene transduction is a promising strategy to improve their capacity to repair the injured or demyelinated nervous ...

Symbiotic nitrogen fixation (SNF) in legume nodules is a highly energy demanding process, fuelled by plant-supplied carbohydrates mainly in the form of sucrose. In this study, we have combined molecular and biochemical approaches in order to study the spatial and temporal organisation of sucrose metabolism in nitrogen-fixing nodules of the model legume Lotus japonicus, with an emphasis on the neglected role of alkaline/neutral invertase. For this purpose, a full-length cDNA clone coding for an alkaline/neutral invertase isoform, termed LjInv1, was identified in a L. japonicus mature nodule cDNA libraries. Alkaline/neutral invertase activity was also found to be the predominant invertase activity in mature nodules. Real-time reverse-transcription polymerase chain reaction analysis was used in order to study the temporal expression patterns of LjInv1 in parallel with genes encoding acid invertase and sucrose synthase (SuSy) isoforms, and enzymes involved in the subsequent hexose partitioning including hexokinase, phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI). The spatial organisation of sucrose metabolism was studied by in situ localisation of LjInv1 transcripts and alkaline/neutral invertase activity, and SuSy protein during nodule development. Furthermore, the spatial organisation of hexose metabolism was investigated by histochemical localisation of hexokinase, PGM and PGI activities in mature nodules. The results considered together indicate that alkaline/neutral invertase could contribute to both the Glc-1-P and Glc-6-P pools in nodules, fuelling both biosynthetic processes and SNF. Furthermore, transcript profiling analysis revealed that genes coding for hexokinase and putative plastidic PGM and PGI isoforms are upregulated during the early stages of nodule development, while the levels of transcripts corresponding to cytosolic PGM and PGI isoforms remained similar to uninfected roots, indicating a possible role of LjInv1 in producing hexoses for starch production and other biosynthetic processes in developing nodules.

The present study demonstrated that species-specific ecdysone-agonist-based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification.

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LjSUT4, encoding a putative sucrose transporter, was identified in a Lotus japonicus nodule cDNA library. The deduced amino acid sequence showed a high degree of identity with sucrose transporters from other plants. Semi-quantitative RT-PCR analysis demonstrated that the L. japonicus SUT4 gene was expressed at high levels in both roots and nodules. In situ hybridization revealed that, in young nodules, SUT4 mRNA transcripts are present in vascular bundles, inner cortex and both infected and uninfected cells while, in mature nodules, accumulation of transcripts was restricted only in vascular bundles and the inner cortex. The results indicated that LjSUT4 codes for a putative sucrose transporter, and its expression pattern suggests a possible shift in the mechanism of sugar transport during nodule development. The role of this polypeptide in sucrose transport and metabolism is discussed.