Activating SRC mutation in a subset of advanced human colon cancers

The discovery of Rous sarcoma virus (RSV) led to the identification of cellular Src (c-Src), a non-receptor tyrosine kinase, which has since been implicated in the development of numerous human cancers. c-Src has been found to be highly activated in colon cancers, particularly in those metastatic to the liver. Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity is downregulated by phosphorylation of a critical carboxy-terminal tyrosine (Tyr 530 in human c-Src, equivalent to Tyr 527 in chicken Src) and have implied the existence of activating mutations in this C-terminal regulatory region. We report here the identification of a truncating mutation in SRC at codon 531 in 12% of cases of advanced human colon cancer tested and demonstrate that the mutation is activating, transforming, tumorigenic and promotes metastasis. These results provide, for the first time, genetic evidence that activating SRC mutations may have a role in the malignant progression of human colon cancer.     

A study of material flow systems (input/output) in high-bay warehouses

A method of planning and designing product sorting systems in warehousing is described. The theoretical background of the analytical models and simulation modelling is given. A calculation of the capacity of elements for joining and dividing for different priorities of material flow is described. New parameters are introduced in the formula for calculating the average number of unit loads in a system for joining, in the direction of the slave flow and for a simple analytical model for joining material flows with two different priorities with an exponential distribution of inter-arrival time. Simulation results are obtained using the GPSS/FON simulation language. Some characteristic results, used in the process of planning and design of two new distribution centres in Belgrade, are shown.     

CO Oxidation over Au/TiO2–Carbon Catalysts: The Effect of Thermal Treatment, Stability and TiO2 Support Structure

The effect of thermal treatment on the catalyst structure and the CO oxidation performance of a Au/TiO₂ catalyst supported on a carbon composite material has been studied. X-ray absorption spectroscopy shows that the carbon composite stabilises the TiO₂ and prevent agglomeration of the particles. The activity measurements show that both Au and TiO₂ need to be present in order to obtain catalytic activity. The catalytic performance was found to be strongly affected by thermal treatments of the active phase prior to the reaction. The thermal treatments have an effect on the ordering of the TiO₂ structure, and on the CO oxidation activity. Heat treatment after Au deposition has a positive effect on the CO oxidation performance. This is attributed to the introduction of a stronger interaction between the oxide and Au which improves the catalytic activity. This also indicates that the TiO₂ support and the Au–TiO₂ interface play important roles in the CO oxidation reaction.     

Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase

The DNA-dependent protein kinase (DNA-PK) plays an important role in mammalian DNA double-strand break repair and immunoglobulin gene rearrangement. The DNA-PK holoenzyme is activated by assembly at DNA ends and is comprised of DNA-PKcs, a 460 kDa protein kinase catalytic subunit, and Ku, a 70 kDa/80 kDa heterodimeric DNA-targeting component. We have solved the three-dimensional structure of DNA-PKcs to approximately 21 A resolution by analytically combining images of nearly 9500 individual particles extracted from cryo-electron micrographs. The DNA-PKcs protein has an open, pseudo 2-fold symmetric structure with a gap separating a crown-shaped top from a rounded base. Columns of density are observed to protrude into the gap from both the crown and the base. Measurements of the enclosed volume indicate that the interior of the protein is largely hollow. The structure of DNA-PKcs suggests that its association with DNA may involve the internalization of double-stranded ends.     

Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer

Adenovirus (Ad) vectors are promising for gene therapy of glioma due to their ability to achieve efficient gene transfer upon intratumoral administration. Yet in this context, Ad mediates widespread gene transfer to both tumor and surrounding parenchyma. Ad entry is dependent upon the expression of fiber receptors, such as coxsackie/adenovirus receptor, and alpha(v) integrins on the target cells for binding and internalization, respectively. We hypothesized that the susceptibility of human gliomas to Ad would likely be heterogeneous due to variable expression of these receptors. It was found that established human glioma cell lines exhibited differential susceptibility to Ad-mediated gene transfer, which correlated directly with the level of radiolabeled Ad binding and with the expression of coxsackie/adenovirus receptor but not with the expression of alpha(v) integrins. To circumvent the lack of fiber receptors and to target Ad gene transfer specifically to tumor cells, we used a bispecific antibody conjugate to ablate Ad binding to fiber receptors and retarget binding to the epidermal growth factor receptor (EGFR), a tumor-associated marker negligibly expressed in normal, mitotically quiescent neural tissues. The results demonstrate that EGFR-targeted Ad gene transfer was EGFR specific and independent of fiber-fiber receptor interactions. Furthermore, EGFR targeting significantly enhanced Ad gene delivery to 7 of 12 established glioma cell lines and to 6 of 8 cultured primary gliomas. Interestingly, EGFR-targeted Ad gene transfer did not correlate with EGFR expression across cell lines, suggesting the importance of other factors. This study establishes that fiber receptor expression limits the utility of Ad vectors for gene transfer to glioma cells and suggests that targeting Ad via EGFR may prove valuable for tumor-specific gene transfer to high-grade gliomas. These findings have key relevance in the context of Ad vector-based approaches for glioma gene therapy.     

Immunoreactivity of PMP-22, P0, and other 19 to 28 kDa glycoproteins in peripheral nerve myelin of mammals and fish with HNK1 and related antibodies

Mammalian peripheral nervous system (PNS) myelin contains several glycoproteins with molecular weights of 19 to 28 kDa, including the major 28 kDa P0 glycoprotein and a recently cloned protein called PMP-22. Some glycoproteins in this M(r) range in humans, cats and some other mammals react with HNK1, a mouse monoclonal antibody that identifies a carbohydrate epitope shared between the immune system and a number of adhesion proteins in the nervous system. A variety of antibodies to P0, PMP-22, and the carbohydrate determinants reacting with HNK1 were used to characterize immunochemically these 19 to 28 kDa glycoproteins of cat PNS myelin. The HNK1-reactive components include P0 and two slightly smaller 23 to 26 kDa proteins that are immunologically related to P0. However, HNK1 reacts most strongly with a lower molecular weight glycoprotein that does not react with the antibodies to P0 and was identified as PMP-22. Since the carbohydrate structure reacting with HNK1 is generally expressed on adhesion molecules, this result suggests that PMP-22 may function in cell-cell or membrane-membrane interactions. Furthermore, the related human anti-MAG monoclonal IgM antibodies from patients with neuropathy also react strongly with PMP-22, suggesting that it may be a target antigen in the pathogenesis of this disease. Purified PNS and CNS myelin from bony fish (toadfish and trout) were also shown to contain major glycoproteins, in the same 19 to 28 kDa M(r) range, that react very strongly with HNK1. It is shown that fish myelin has major proteins of this size that are immunologically and structurally related to mammalian P0, and it is demonstrated here that one of the strongly HNK1-positive proteins reacted well with an antiserum raised to bovine P0. The presence of high levels of the adhesion-related HNK1 epitope on these major myelin proteins of fish suggests that this carbohydrate structure may have played a role in the molecular evolution of myelin.