Quantitation of a 55K cellular protein: similar amount and instability in normal and malignant mouse cells.

Quantitative expression of a specific 55,000 (55K)-molecular-weight cellular protein was studied in two groups of mouse embryo fibroblast (clonal) cells originating from two parent clones, one of which possessed high tumorigenicity and the other of which possessed very low tumorigenicity. From the clone with low tumorigenicity, tumor lines and clones were obtained by selecting rare spontaneously transformed highly tumorigenic (mutant) cells. Cells were labeled during exponential growth for 3 h at 37 degrees C, with [35S]methionine, and the cellular 55K protein was immunoprecipitated with a monoclonal antibody and quantitated. There were low and approximately equal amounts of 55K protein in cells (clones) with both low and high tumorigenicity from both groups of cells, and there was no correlation at all between quantitative expression of 55K protein and of cellular tumorigenicity. There was approximately 10- to 20-fold more 55K protein in all simian virus 40-transformed T antigen-positive derivative clones, as shown previously. The T antigen-negative revertant tumor lines and clones obtained by an immunological in vivo selection method had low amounts of 55K protein, similar to the parent cell before simian virus 40 transformation. In all of the T antigen-negative cells, including the highly tumorigenic cells, degradation (turnover?) of the 55K protein was rapid, and a half-life of 15 to 60 min was estimated from pulse-chase experiments. In all of the T antigen-positive cells the 55K protein was stable (half-life greater than 10 h). In primary cells established from the tumors induced by highly tumorigenic cells there was a very low or no detectable amount of the 55K protein. This is in contrast to the primary cells obtained from early murine embryos in which we have reported high amounts of (stable) 55K proteins.     

Mammalian wildlife diseases as hazards to man and livestock in an area of the Llanos Orientales of Colombia

Development of the LLanos Orientales of Colombia, and access to underdeveloped areas in the Llanos, may create disease hazards to man and domestic animals or introduce exotic pathogens, creating reservoirs of infection for domestic animals and acting as limiting factors on the native wild species. A survey of wild animals common to the Llanos revealed a number of parasites indigenous to the area. A total total of 59 mammalian species, representing eight orders were examined. Haematozoa were represented by Trypanosoma cruzi, T. evansi and T. rangeli. Eight species of ticks were found: Amblyomma cajennense, A. auricularium, A. rotundatum, A. maculatum, A. longirostre, A. pacae, Ixodes luciae and Boophilus microplus. Four species of fleas were found: Rhopalopsyllus lugubris lugubris, R. australis tupinus, R. cacicus saevus and Polygenis klagesi samuelis. A species of Echinococcus was commonly found in Cuniculus paca. Serologic titers and/or isolations of pathogenic viral and bacterial agents generally indicated that the wildlife population had not been exposed to the diseases common to the domestic population. A low prevalence of titers to Venezuelan equine encephalomyelitis was found in Cebus apella and Proechimys sp. Neutralizing antibodies to Group B viruses were found in Proechimys sp., Coendor sp. and Nectomys squamipes. Antibodies to Group C viruses were found in Proechimys sp. Serologic titers to Leptospira sejroe and L. tarassovi were found in Proechimys sp. and Didelphis marsupialis. L. tarassovi was isolated from Proechimys sp. Titers to Brucella were not found in 1964 animals. The significance of these findings are discussed.     

Cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulfate with reduced degree of sulfation

Cell lines, selected from two independent clones of an established mouse embryo cell line by their ability to grow as solid tumors in immunocompetent syngeneic hosts, were found to have the same alteration in anion exchange properties as was previously reported for simian virus 40 (SV40)-transformed subclones. One tumor cell line (219CT) and one SV40-transformed subclone (215CSC) were selected for further detailed comparison with their common parent clone (210C). Cellulose acetate electrophoresis at pH 1.0 showed that 215CSC heparan sulfate had a slight overall decrease in sulfation compared with heparan sulfate from 210C; however, no gross difference in sulfation could be detected between heparan sulfate from 219CT and 210C. Analysis of the products of deaminative cleavage of heparan sulfate by nitrous acid under conditions where cleavage occurs quantitatively at N-sulfated glucosamine residues showed that, although heparan sulfate from the three cell lines gave similar yields of O-sulfated disaccharides, both 215CSC and 219CT had only about half as many O-sulfate residues in higher molecular weight oligosaccharides compared to heparan sulfate from 210C. Enzymatic degradation of heparan sulfate with a mixture of enzymes from Flavobacterium heparinum showed that this common alteration in heparan sulfate from both 215CSC and 219CT resulted from a 30% decrease in glucosamine residues bearing 6-O-sulfate groups. As this decrease in 6-O-sulfate glucosamine residues occurs in regions of the chain containing relatively few sulfate groups, it is clear that certain sequences of charged groups present in heparan sulfate frm 210C will be found only rarely in heparan sulfate from 215CSC and 219CT. It is suggested that this will result in alterations of the interaction of heparan sulfate with other molecules in the microenvironment at the cell surface which may be important in the control of such phenomena as cell growth and adhesion.     

The capybara (Hydrochoerus hydrochaeris) as a reservoir host for Trypanosoma evansi

Discovery of two ill horses and three dogs naturally infected with Trypanosoma evansi near an experimental station in the Eastern Plains of Colombia led to a search for reservoir hosts of the parasite. Infection was detected in 8/33 healthy capybaras (Hydrochoerus hydrochaeris), none of the remaining 14 horses, and none of 32 Zebu cattle (Bos indicus), 18 paca (Cuniculus paca) and 20 spiny rats (Proechimys sp.). Contrary to common opinion, the results indicated a carrier state in the capybara. Diagnosis was based on morphology, behaviour in albino rats, and pathogenicity and host range in domestic animals.     

Conjugation of chemical handles and functional moieties to DNA during solid phase synthesis with sulfonyl azides

Labelling of oligonucleotides with dyes, targeting ligands, and other moieties has become ever more essential in life-sciences. Conventionally, modifications are introduced to oligonucleotides during solid phase synthesis by special phosphoramidites functionalised with a chemical handle or the desired functional group. In this work, we present a facile and inexpensive method to introduce modifications to oligonucleotides without the need for special phosphoramidites. Sulfonyl azides are applied to react with one or more selected phosphite intermediates during solid phase synthesis. We have prepared 11 sulfonyl azides with different chemical handles such as amine, azide, alkyne, and thiol, and we have further introduced functionalities such as pyrene, other dyes, photo-switchable azobenzenes, and a steroid. The method is compatible with current phosphoramidite-based automated oligonucleotide synthesis and serves as a simple alternative to the unstable and expensive special phosphoramidites currently used for conjugation to oligonucleotides.     

TBX2 acts as a potent transcriptional silencer of tumour suppressor genes through interaction with the CoREST complex to sustain the proliferation of breast cancers

Chromosome 17q23 amplification occurs in 20% of primary breast tumours and is associated with poor outcome. The TBX2 gene is located on 17q23 and is often over-expressed in this breast tumour subset. TBX2 is an anti-senescence gene, promoting cell growth and survival through repression of Tumour Suppressor Genes (TSGs), such as NDRG1 and CST6. Previously we found that TBX2 cooperates with the PRC2 complex to repress several TSGs, and that PRC2 inhibition restored NDRG1 expression to impede cellular proliferation. Here, we now identify CoREST proteins, LSD1 and ZNF217, as novel interactors of TBX2. Genetic or pharmacological targeting of CoREST emulated TBX2 loss, inducing NDRG1 expression and abolishing breast cancer growth in vitro and in vivo. Furthermore, we uncover that TBX2/CoREST targeting of NDRG1 is achieved by recruitment of TBX2 to the NDRG1 promoter by Sp1, the abolishment of which resulted in NDRG1 upregulation and diminished cancer cell proliferation. Through ChIP-seq we reveal that 30% of TBX2-bound promoters are shared with ZNF217 and identify novel targets repressed by TBX2/CoREST; of these targets a lncRNA, LINC00111, behaves as a negative regulator of cell proliferation. Overall, these data indicate that inhibition of CoREST proteins represents a promising therapeutic intervention for TBX2-addicted breast tumours.     

The Crystal Structure and Small-Angle X-Ray Analysis of CsdL/TcdA Reveal a New tRNA Binding Motif in the MoeB/E1 Superfamily

Cyclic N⁶-threonylcarbamoyladenosine (‘cyclic t⁶A’, ct⁶A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct⁶A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct⁶A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t⁶A to form ct⁶A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K⁺ and Na⁺ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t⁶A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct⁶A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.