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The promyelocytic leukemia gene (PML) encodes a protein which localizes to PML-nuclear bodies (NBs), sub-nuclear multi-protein structures, which have been implicated in diverse biological functions such as apoptosis, cell proliferation and senescence. However, the exact biochemical and molecular basis of PML function up until now has not been defined. Strikingly, over a decade ago, PML-NBs were found to be disrupted in acute promyelocytic leukemia (APL) in which PML is fused to the gene encoding retinoic acid receptor alpha (RARA) due to the t(15;17) chromosomal translocation, generating the PML-RARA chimeric protein. The treatment of APL patients with all-transretinoic acid (ATRA) and arsenic trioxide which target the PML-RARA oncoprotein results in clinical remission, associated with blast cell differentiation and reformation of the PML NBs, thus linking NB integrity with disease status. This review focuses on the current theories for molecular and biochemical functions of the PML-NBs, which would imply a role in the pathogenesis of APL, whilst also discussing the intriguing possibility that their disruption may not be in itself a significant oncogenic event.

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Mouse polyoma virus major coat protein (VP1) expressed from a recombinant baculovirus is efficiently transported to infected cell nuclei and assembles into protein nanospheres morphologically similar to natural capsids. The nanospheres readily combine with plasmid DNA to form a hybrid gene therapy agent known as virus-like particles (VLPs). To facilitate large-scale production of VLPs free from cellular contaminants, the use of stable Drosophila cell lines expressing either wild-type protein, or VP1 tagged with a secretion signal for targeting to the extracellular medium, was investigated. Both wild-type and tagged VP1 expressed at 2-4 mg VP1/litre of culture. As expected, the wild-type protein self-assembled into VLPs. The tagged VP1 was efficiently secreted to the extracellular medium but was also glycosylated, unlike wild-type VP1. Despite this fact, a small fraction of the recombinant secreted protein assembled into VLP-like structures that had altered disulphide bonding, but were still biologically active. These results demonstrate the considerable tolerance in the nanosphere assembly to structural (i.e. aberrant glycosylation) and environmental (i.e. extracellular medium vs. nuclear milieu) changes. Thus, with modifications to improve nanosphere assembly, the secretion method could be adapted to large-scale preparation of VLPs, providing significant advantages over current methods of production of the vector.

RESUMOEmbriões bovinos produzidos in vitro, em estádio de mórula, foram cultivados em meio contendo anticorpos anti H-Y de alto título proveniente de ratos por 24h e, após este tempo, classificados em dois grupos: 1) embriões inibidos em estádio de mórula (classificados como machos) e 2) embriões que se desenvolveram e formaram a blastocele (classificados como fêmeas). O sexo de 311 embriões, distribuídos em três grupos de concentração dos anticorpos, 3%, 5% ou 7%, foi identificado pela reação em cadeia da polimerase. Não houve desvio da proporção entre machos e fêmeas (P>0,05) nos grupos em que se utilizaram os anticorpos anti H-Y, quando comparadas ao grupo-controle, sem adição de anticorpos anti H-Y. Diferentemente dos resultados obtidos utilizando-se embriões bovinos produzidos in vivo, a sexagem com anticorpos anti H-Y de alto título em embriões produzidos in vitro não propiciou sucesso. Palavras-chave: bovino, sexagem de embriões, inibição do desenvolvimento, anticorpos anti H-Y, fecundação in vitro ABSTRACT In vitro produced bovine embryos at morula stage were cultured in medium containing high titer of rat H-Y antisera for 24h. The embryos were classified in two groups: 1) embryos arrested at morula stage (classified as males); and 2) embryos that developed and formed a blastocoele (classified as female