The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.     

Germline transmission of genetically modified primordial germ cells

Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.     

DNA trapping electrophoresis

Attempts to improve the size separation of single-stranded DNA in polyacrylamide gels by field-inversion gel electrophoresis (FIGE) have met with limited success. Here we show that attaching a neutral globular protein, streptavidin, to one end of a single-stranded DNA molecule profoundly alters the DNA mobility pattern and increases the band separation manyfold within a size range controlled by voltage and pulse cycle. In constant field, short modified fragments are only slightly retarded but long molecules are retarded dramatically above a 'threshold size' of 0.6 kilobases at 60 V per cm. At this voltage, molecules above a 1.2-kilobase 'cut-off' do not enter the gel. Both the threshold and the cut-off sizes decrease as the voltage increases. In FIGE, the longer the reverse pulse, the larger the modified fragments that enter the gel. We interpret these results as the trapping by the gel matrix of the protein attached to the DNA. The probability of release then depends on the balance between the electric field and thermal motion: the larger the DNA and the higher the voltage, the harder it is to release.     

Morphology and intracortical projections of functionally characterised neurones in the cat visual cortex

The neuronal structure and connectivity underlying receptive field organisation of cells in the cat visual cortex have been investigated. Intracellular recordings were made using a micropipette filled with a histochemical marker, which was injected into the cells after their receptive fields had been characterised. This allowed visualisation of the dendritic and axonal arborisations of functionally identified neurones.     

The structures of five newAspidosperma alkaloids related to uleine

Zusammenfassung Aus der Rinde des brasilianischen BaumesAspidosperma dasycarpon A. DC. wurden fünf neue Alkaloide (II–VI) isoliert, die das selten vorkommende Uleinskelett besitzen. Die Strukturen wurden durch chemische Korrelation mit Ulein (I) sichergestellt.

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Paper XLVII in the seriesAlkaloid Studies. For paper XLVI seeK. S. Brown Jr. andC. Djerassi, J. Am. chem. Soc.86, in press (1964).     

Pore-forming toxins

Pore-forming toxins are widely distributed proteins which form lesions in biological membranes. In this review, bacterial pore-forming toxins are treated as a paradigm and discussed in terms of the structural principles on which they work. Then, a large family of bacterial toxins, the cholesterol-binding toxins, are analyzed in depth to provide an overview of the processes involved in pore formation. The ways in which the cholesterol-binding toxins (cholesterol-dependent cytolysins) interact with membranes and form pores, the structure of the monomeric soluble and oligomeric pore-forming states, and the effects of the toxin on membrane structure are discussed. By surveying the range of work which has been done on cholesterol-binding toxins, a working model is elaborated which reconciles two current, apparently diametrically opposed, models for their mechanism. Received 7 September 2001; received after revision 12 November 2001; accepted 5 December 2001