Specific Inhibition of Tumor Cells by Oncogenic EGFR Specific Silencing by RNA interference

Anticancer agents that have minimal effects on normal cells and tissues are ideal cancer drugs. Here, we show specific inhibition of human cancer cells carrying oncogenic mutations in the epidermal growth factor receptor (EGFR) gene by means of oncogenic allele-specific RNA interference (RNAi), both in vivo and in vitro. The allele-specific RNAi (ASP-RNAi) treatment did not affect normal cells or tissues that had no target oncogenic allele, whereas the suppression of a normal EGFR allele by a conventional in vivo RNAi caused adverse effects, i.e., normal EGFR is vital. Taken together, our current findings suggest that specific inhibition of oncogenic EGFR alleles without affecting the normal EGFR allele may provide a safe treatment approach for cancer patients and that ASP-RNAi treatment may be capable of becoming a safe and effective, anticancer treatment method.     

Specific alteration of NCAM-mediated cell adhesion by an endoneuraminidase

A phage endoneuraminidase that specifically cleaves alpha-2, 8-linked polysialic acid has been found to be a useful probe for examining the biological role of this sugar moiety on the neural cell adhesion molecule (NCAM). The enzyme caused a 3.3-fold increase in the rate of NCAM-dependent aggregation of membrane vesicles from chicken embryonic brain, without the nonspecific effects previously encountered with the use of exoneuraminidases. The enhancement of aggregation was closely correlated with removal of sialic acid as assessed by electrophoretic mobility. Extension of this analysis to cultures of spinal ganglia indicated that removal of sialic acid by the endoneuraminidase results in an increase in the thickness of neurite bundles. This enhancement of fasciculation was reversed by addition of anti-NCAM Fab, suggesting that the enzyme treatment was not toxic and did not produce nonspecific effects on adhesion. Injection of the enzyme into the eyes of 3.5-d chicken embryos consistently produced a striking array of abnormalities in those parts of the neural retina that contained the highest concentrations of NCAM at the time of injection. These perturbations included a dramatic thickening of the neural epithelium in the posterior eye, a failure of cells in this region to elongate radially, formation of an ectopic optic fiber layer, and an incomplete association of the presumptive pigmented epithelium with the neural retina. These results provide the first direct evidence that the polysialic acid on NCAM has a regulatory effect on adhesion between living cells, and that the amount of this carbohydrate is critical for the normal morphogenesis of nerve tissue.     

Specific structural alteration of the influenza haemagglutinin by amantadine.

Amantadine hydrochloride specifically blocks the release of virus particles from H7 influenza virus infected cells. This appears to be the direct consequence of an amantadine induced change in the haemagglutinin (HA) to its low pH conformation. The effect is indirect and mediated via interaction of the drug with the M2 protein since mutants altered in this component alone are insensitive to amantadine. The timing of drug action, some 15-20 min after synthesis, and its coincidence with proteolytic cleavage indicates that the modifications to HA occur late during transport but prior to insertion into the plasma membrane. Reversal by mM concentrations of amines and 0.1 microM monensin indicates that amantadine action causes a reduction in intravesicular pH which triggers the conformational change in HA. We conclude, therefore, that the function of M2 inhibited by amantadine is involved in counteracting the acidity of vesicular compartments of the exocytic pathway in infected cells and is important in protecting the structural integrity of the acid-sensitive glycoprotein.     

Coliphages as indicators of enteroviruses

Coliphages were monitored in conjunction with indicator bacteria and enteroviruses in a drinking-water plant modified to reduce trihalomethane production. Coliphages could be detected in the source water by direct inoculation, and sufficient coliphages were detected in enterovirus concentrates to permit following the coliphage levels through different water treatment processes. The recovery efficiency by different filter types ranged from 1 to 53%. Statistical analysis of the data indicated that enterovirus isolates were better correlated with coliphages than with total coliforms, fecal coliforms, fecal streptococci, or standard plate count organisms. Coliphages were not detected in finished water.     

Specific modifications of hepatoma cell-surface glycoproteins with enzymes : Effects on in vitro growth as investigated by the use of lectins

The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport.     

Coliphages as indicators of enteroviruses.

Coliphages were monitored in conjunction with indicator bacteria and enteroviruses in a drinking-water plant modified to reduce trihalomethane production. Coliphages could be detected in the source water by direct inoculation, and sufficient coliphages were detected in enterovirus concentrates to permit following the coliphage levels through different water treatment processes. The recovery efficiency by different filter types ranged from 1 to 53%. Statistical analysis of the data indicated that enterovirus isolates were better correlated with coliphages than with total coliforms, fecal coliforms, fecal streptococci, or standard plate count organisms. Coliphages were not detected in finished water.     

Bovine enteroviruses as indicators of fecal contamination

Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.     

Identification of enteroviruses by hemagglutination-inhibition

Kern, Jerome (Pacific Research Section, Honolulu, Hawaii), and Leon Rosen. Identification of enteroviruses by hemagglutination-inhibition. J. Bacteriol. 91:1936-1942. 1966.-Approximately 40% of a group of 906 enterovirus isolates cytopathic for monkey cell cultures were found to possess hemagglutinins for human erythrocytes when tested at temperatures of 4 and 37 C and at pH 5.8 and 7.3. The hemagglutinating isolates could be classified by relatively simple techniques into 18 serotypes. Four of these serotypes, echovirus type 24 and coxsackievirus B types 1, 5, and 6, had not previously been known to include hemagglutinating strains. One serotype, Toluca-3, represented a previously unrecognized enterovirus, and two other serotypes may also represent previously unrecognized enteroviruses.     

Specific interference with gene function by double-stranded RNA in early mouse development

The use of double-stranded (ds) RNA is a powerful way of interfering with gene expression in a range of organisms, but doubts have been raised about whether it could be successful in mammals. Here, we show that dsRNA is effective as a specific inhibitor of the function of three genes in the mouse, namely maternally expressed c-mos in the oocyte and zygotically expressed E-cadherin or a GFP transgene in the preimplantation embryo. The phenotypes observed are the same as those reported for null mutants of the endogenous genes. These findings offer the opportunity to study development and gene regulation in normal and diseased cells.     

Tunable cell-surface mimetics as engineered cell substrates

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed.     

Progression of mammary adenocarcinomas as reflected by nuclear DNA content

In 18 breast cancer patients the DNA histograms observed in the primary tumor at the date of diagnosis were compared with those found in the corresponding local and distant metastases at autopsy up to more than 12 yr later. All patients, except one, exhibited the same type of DNA histogram in both the primary tumor and its metastases. In one patient the DNA histogram changed from an euploid type in the primary breast carcinoma to an aneuploid type in the metastases. The results are interpreted as indicating that mammary adenocarcinoma in general exhibit a high degree of stability of the nuclear DNA content during the history of the disease. It is suggested that in breast cancer progression of the tumor disease is more likely due to a net increase and/or dissemination of tumor cells exhibiting similar genetic properties and malignancy potential than to a progressive dedifferentiation and increase of malignancy of the tumor cells.     

Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression: an RNA interference and structure-based mutagenetic approach

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.     

Protein engineering by cell-surface display

Protein libraries displayed on cell surfaces can be labeled with soluble ligands exhibiting well-characterized binding equilibria and dissociation kinetics, and then quantitatively screened by flow cytometry at a rate of >10(4) clones/second. The promise of cell-surface display for directed evolution is being realized, with significant improvements recently reported in protein ligand binding affinity, stability, expression and enzymatic activity.     

Thermostabilization of enteroviruses by estuarine sediment

The effect of estuarine sediment on the thermoinactivation of poliovirus type 1 and echovirus type 1 was evaluated. Poliovirus survival was prolonged at 24 and 37 degrees C but not at 4 degrees C in the presence of sediment over the time periods observed. Further inactivation studies were performed at 50 and 55 degrees C to maximize the thermal effects, and similar protection was observed. The supernatant fluid from a mixture of seawater and sediment lacked the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediment was due to adsorption to particulates. From these observations, it appears that the adsorption of enteroviruses to estuarine sediments may play a significant role in protecting them against thermoinactivation.     

Specific genetic interference with behavioral rhythms in Drosophila by expression of inverted repeats

We describe a new experimental technique that allows for a tissue-specific reduction of gene activity in the Drosophila nervous system. On the basis of the observation that certain gene functions can be ubiquitously blocked by injecting double-stranded RNA into Drosophila embryos, we employed a method to interfere with an individual gene function permanently in a predetermined cell type. This was achieved by the formation of an inverted-repeat RNA sequence in the tissue of interest under control of the GAL4/UAS binary expression system. As an example, we show that inverted-repeat-mediated interference with the period gene produces a hypomorphic period phenotype. A selective decrease of period RNA appears to be a component of the cellular response.     

Food recognition as reflected in seed handling by Messor arenarius ants

1. This research assessed whether food recognition in Messor arenarius ants is impacted by their food handling.
2. Food choice experiments between whole-wheat seeds and halves of wheat seeds cut longitudinally were conducted in the same nest on two consecutive days.
3. In these experiments, it was found that the proportion of touches of these ants using their antennae on both food items within a collection point during first day of the experiment was significantly higher than the same proportion on the second day of the experiment in the same nest.
4. The proportion of touches by the ants' forelegs was also higher on the first day of the experiment than on the second day in the same nest, but not significantly.
5. These findings seem to account for a possible effect of learning, that is, food recognition.