Identification of tumour-associated and germ line p53 mutations in canine mammary cancer

Mutations of the tumour suppressor p53 gene are found in a number of spontaneous canine cancers and may contribute to increased cytogenetic alterations and tumour formation. Using reverse transcription and DNA amplification, we isolated p53 cDNA from normal and tumour tissue of ten canine mammary cancer patients. DNA sequencing identified p53 mutations in three of the ten patients. These included tumour-associated p53 gene mutations within exons 2 and 5 and a germ line deletion of exons 3 to 7. These results support a role for p53 inactivation in canine mammary tumour formation and breed predisposition to cancer. Such information could prove invaluable in the successful outbreeding of inherited predisposition to cancer in the dog. A putative polymorphism was also identified at codon 69 in exon 4 and we discuss the possibility that similar polymorphisms may be associated with human breast cancer. © 1999 Cancer Research Campaig

Effect of strongly magnetized electrons and Ions on heat flow and symmetry of inertial fusion implosions

This Letter presents the first observation on how a strong, 500 kG, externally applied B field increases the mode-two asymmetry in shock-heated inertial fusion implosions. Using a direct-drive implosion with polar illumination and imposed field, we observed that magnetization produces a significant increase in the implosion oblateness (a 2.5 × larger P 2 amplitude in x-ray self-emission images) compared with reference experiments with identical drive but with no field applied. The implosions produce strongly magnetized electrons ( ω e τ e ≫ 1 ) and ions ( ω i τ i > 1 ) that, as shown using simulations, restrict the cross field heat flow necessary for lateral distribution of the laser and shock heating from the implosion pole to the waist, causing the enhanced mode-two shape

Entry of Human Papillomavirus Type 16 by Actin-Dependent, Clathrin- and Lipid Raft-Independent Endocytosis

Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion

Effects of integrin-mediated cell adhesion on plasma membrane lipid raft components and signaling

Loss of integrin-mediated cell adhesion is known to induce internalization of lipid rafts, which alters of the plasma membrane's physical and signaling properties. Analysis of multiple proteins now shows a wide range of behaviors in which internalization and exit from the ordered to disordered domains are regulated separately

Magnetic Fractionation and Proteomic Dissection of Cellular Organelles Occupied by the Late Replication Complexes of Semliki Forest Virus

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection